Chimeric molecules for complement immune recruitment

Abstract

Applicants have synthesized and evaluated several bifunctional molecules targeting both the complement system and surface proteins of the opportunistic bacterial pathogen Pseudomonas aeruginosa (P. aeruginosa), which poses significant health threats to humans. From this, Applicants isolated a compound that effectively activated complement immunity to inhibit bacterial growth. Furthermore, Applicants developed a robust testing platform for complement-recruiting compounds, allowing the streamlined assessment of ternary complex formatoon and the efficacy of novel bifunctional molecules for recruiting the complement system and inhibiting bacterial growth. These findings demonstrate the potential of this approach in combating antibiotic-resistant P. aeruginosa infections, with implications for other bacterial pathogens or diseases.

Description

CHIMERIC MOLECULES FOR COMPLEMENT IMMUNE RECRUITMENTCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 538,274, filed September 13, 2023; U.S. Provisional Application No. 63 / 538,275, filed September 13, 2023; U.S. Provisional Application No. 63 / 538,272, filed September 13, 2023; U.S. Provisional Application No. 63 / 544,075, filed October 13, 2023; U.S. Provisional Application No. 63 / 544,090, filed October 13, 2023; U.S. Provisional Application No. 63 / 544,097, filed October 13, 2023; and U.S. Provisional Application No. 63 / 623,588, filed January 22, 2024. The entire contents of the above-identified applications are hereby fully incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant No. HR0011-21-2- 0010, awarded by the Defense Advanced Research Projects Agency. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The subject matter disclosed herein is generally directed to multifunctional small molecules for alternative therapeutic strategies. In particular, the multifunctional molecules described herein combat infections by recruiting the complement immune system to targeted pathogens.BACKGROUND

[0004] The increasing prevalence of antibiotic resistance and immune evasion necessitates the development of alternative therapeutic strategies. Here, Applicants propose the employment of bifunctional molecules targeting the extracellular components of bacteria to recruit the complement immune system to the site of infection, enhancing bacterial cell death.

[0005] Accounting for thousands of deaths and hundreds of millions of dollars in healthcare costs per year in the US alone, Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic gramnegative bacterial pathogen that is intrinsically resistant to numerous antibiotics and systematically evades the immune system1’2’3 4. These resistance features of P. aeruginosa and other similarpathogens complicate treatment strategies and necessitate innovative drug discovery approaches5’6. Previous work in re-engaging the complement immune system7, the body’s natural and broad spectrum clearance pathway for microbes, has shown promise in reducing model bacterial infections using bifunctional compounds that tag the cell surface with complement-recruiting factors89 10 11. Combined with the high abundance of complement proteins in bloodstream, this strategy led to significantly higher than normal local concentrations of immune cells near the sight of the infection for effective cell killing7 12,13. However, this emerging field has been explored mainly through model compounds and organisms without focusing on the exact complement immune pathway. As such, there are also no readily available assay platforms for effective compound development for the complement immune recruitment and no systematic studies of the key components of these bifunctional compounds nor of the downstream effects of antibody recruitment in the case of P. aeruginosa.

[0006] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention.SUMMARY

[0007] In one aspect, the present disclosure provides complement recruiting chimeric (CRC) molecule according to the formula R1-L-R2, wherein Ri is a pathogen binding moiety; R2 is a complement recruiting moiety; and L is a linker, wherein the linker may optionally comprise an electrophilic reactive group. In example embodiments, the complement recruiting moiety is an antibody binding moiety that binds an antibody that recruits one or more components of the complement system. In example embodiments, the compliment recruiting moiety binds to the light chain or the heavy chain of the antibody. In example embodiments, the compliment recruiting moiety binds to the antigen binding site of the light chain.

[0008] In example embodiments, the antibody binding moiety is a small molecule, a polypeptide, or a hapten. In example embodiments, the hapten is 2,4-dinitrophenyl (DNP),N02 , or an analog thereof, and wherein the hapten is bound by an anti-DNP antibody.

[0009] In example embodiments, the antibody binding moiety comprises rhamnose,or an analog or derivative thereof.

[0010] In example embodiments, the antibody binding moiety comprisesor an analog or derivative thereof.

[0011] In example embodiments, the antibody binding moiety comprises of; or an analog or derivative thereof.

[0012] In example embodiments, the pathogen is a virus, bacteria, fungi, or protozoa. In example embodiments, the bacteria is a gram-negative bacteria. In example embodiments, the gram-negative bacteria pathogen is Pseudomonas Aeruginosa (PsA). In example embodiments, the pathogen binding moiety binds a protein, lipopolysaccharide, or a polysaccharide. In example embodiments, the protein, lipopolysaccharide, or polysaccharide is pathogen-specific. In example embodiments, the pathogen binding moiety is a peptidomimetic antibiotic macrocycle, or a lectin binder. In example embodiments, the lectin binder is LecA (PA-IL) or LecB (PA-IIL). In example embodiments, wherein the pathogen binding moiety comprises of glucopyranoside, fucopyranoside, or an analog or derivative thereof. In example embodiments, the pathogen binding moiety comprises ofanalog or derivative thereof.

[0013] In example embodiments, the pathogen binding moiety is a colistin-targeting liposaccharide. In example embodiments, wherein the pathogen binding moiety comprises:

[0014] In example embodiments, the pathogen binding moiety comprises:or an analog or derivative thereof.

[0015] In example embodiments, the pathogen binding moiety is murepavadin, polymyxin, vancomycin, or an analog or derivative thereof. In example embodiments, L comprises alkane; alkene; alkyne; amine; ether; thiol; sulfone; carbonyl; acyl; ketone; carboxylate ester; amide; enone; anhydride; imide; PEG; or any combination thereof. In example embodiments, L comprises:o o is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0016] In example embodiments, L comprises:wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14; or an analog or derivative thereof.In example embodiments, L comprises:O CN OI ,nand n is 1, 2, 3, 4, or 5;o 0H N N H iii) n and n is between 0 and 6.

[0017] In example embodiments, L is selected from:

[0018] In example embodiments, the CRC molecule is according to the formula:O— s. ,1 R2R’ ■ " o y — ' , wherein R1is the pathogen binding moiety and R2is the antibody binding moiety. In example embodiments, R1is galactopyranoside and R2is DNP.

[0019] In example embodiments, the CRC molecule is according to the formula: ..N 7 ° 2N, wherein R1is the pathogen binding moiety and R2is the antibody binding moiety. In example embodiments, R1is fucopyranoside and R2is DNP.

[0020] In example embodiments, the CRC molecule is according to the formula:NN' O• ■*, HN . o . • N . . R2’ •?. f ?0 20R’, \ ', wherein the top R1and bottom R1are independently selected pathogen binding moieties and R2is the antibody binding moiety. In example embodiments, the top R1and bottom R1are fucopyranoside and R2is DNP.

[0021] In example embodiments, the CRC molecule is according to the formula:OR?R1.0v, wherein R1is the pathogen binding moiety and R2is the antibody binding moiety. In example embodiments, R1is polymyxin B and R2is DNP.

[0022] In example embodiments, the CRC molecule is according to the formula: the triangle comprises of any of the hexagon comprises ofand optionally comprise an electrophilic reactive group.

[0023] In example embodiments, the CRC molecule is according to the formula:HO wherein the triangle comprises of the complement recruiting moiety.

[0024] In example embodiments, the complement recruiting moiety is a FcIII-peptide.

[0025] In example embodiments, the CRC molecule is according to the formula:zI

[0026] In example embodiments, the CRC molecule is according to the formula: owherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0027] In example embodiments, the CRC molecule is according to the formula:

[0028] In one aspect, the present disclosure provides a method of targeted activation of the complement immune system against pathogens comprising administering to a cell or cell population a chimeric molecule as described herein, wherein binding of the complement recruiting moiety to an antibody activates the complement immune system against a target pathogen.

[0029] In one aspect, the present disclosure provides a method of treating pathogenic infections comprising administering to a cell or cell population a chimeric molecule as described herein, wherein binding of the complement recruiting moiety to an antibody activates the complement immune system against a target pathogen.

[0030] In example embodiments, the pathogen is a virus, bacteria, fungi, or protozoa. In example embodiments, the gram-negative bacteria pathogen is Pseudomonas aeruginosa (PsA).

[0031] In one aspect, the present disclosure provides a complement recruiting chimeric (CRC) molecule comprising an antibody fragment bound to one or more pathogen binding moieties via one or more linkers, wherein the linker may optionally comprise an electrophilic reactive group. In example embodiments, the antibody fragment is an Fc fragment. In example embodiments, the Fc fragment is an IgG Fc fragment.

[0032] In one aspect, the present disclosure provides a CRC molecule according to the formula R1-L-R2, wherein Ri is a pathogen binding moiety; R2 is a complement recruiting moiety; and L is a linker, wherein the linker may optionally comprise an electrophilic reactive group. In example embodiments, the complement recruiting moiety binds one or more Clq polypeptides. In example embodiments, the complement recruiting moiety binds a Clq A-chain, B-chain, C-chain, or a combination thereof. In example embodiments, the complement recruiting moiety is a small molecule, a polypeptide, or a hapten.

[0033] In example embodiments, the compliment recruiting moiety comprises of:wherein R1 comprises ofR2 comprises of

[0034] In example embodiments, the pathogen is a virus, bacteria, fungi, or protozoa. In example embodiments, the bacteria is a gram-negative bacteria. In example embodiments, the gram-negative bacteria pathogen is Pseudomonas aeruginosa (PsA). In example embodiments, the pathogen binding moiety binds a protein, lipopolysaccharide, or a polysaccharide. In example embodiments, the protein, lipopolysaccharide, or polysaccharide is pathogen-specific. In example embodiments, the pathogen binding moiety is a peptidomimetic antibiotic macrocycle, or a lectin binder. In example embodiments, the lectin binder is LecA (PA-IL) or LecB (PA-IIL). In example embodiments, the pathogen binding moiety comprises of glucopyranoside, fucopyranoside, or an analog or derivative thereof.

[0035] In example embodiments, the pathogen binding moiety comprises of analog or derivative thereof.

[0036] In example embodiments, the pathogen binding moiety is a colistin-targeting liposaccharide. In example embodiments, the pathogen binding moiety comprises:

[0037] In example embodiments, the pathogen binding moiety comprises:; or an analog or derivative thereof.

[0038] In example embodiments, the pathogen binding moiety is murepavadin, polymyxin, vancomycin, or an analog or derivative thereof. In example embodiments, the linker(s) are individually selected from: alkane; alkene; alkyne; amine; ether; thiol; sulfone; carbonyl; acyl; ketone; carboxylate ester; amide; enone; anhydride; imide; PEG, or any combination thereof. In example embodiments, linker(s) are selected from:o o is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0039] In example embodiments, the linker(s) are selected from:or derivative thereof.

[0040] In example embodiments, the linker(s) are selected from:O O i) the group consisting of

[0041] In example embodiments, the linker(s) are selected from: or .

[0042] In one aspect, the present disclosure provides a method of targeted activation of the complement immune system against pathogens comprising administering to a cell or cell population a chimeric molecule as described herein, wherein the antibody fragment induces recruitment of the complement immune system upon binding of the one or more pathogen moi eties to a target pathogen.

[0043] In one aspect, the present disclosure provides a method of targeted activation of the complement immune system against pathogens comprising administering to a cell or cell population a chimeric molecule as described herein, wherein the complement recruiting moiety induces recruitment of the complement immune system upon binding of the pathogen binding moiety to a target pathogen. In example embodiments, the pathogen is a virus, bacteria, fungi, or protozoa. In example embodiments, the bacteria is a gram-negative bacteria. In example embodiments, the gram-negative bacteria pathogen is Pseudomonas aeruginosa (PsA).

[0044] In one aspect, the present disclosure provides a method of treating pathogenic infections comprising administering to a cell or cell population a chimeric molecule as described herein, wherein the antibody fragment induces recruitment of the complement immune system upon binding of the one or more pathogen moieties to a target pathogen.

[0045] In one aspect, the present disclosure provides a method of treating pathogenic infections comprising administering to a cell or cell population a chimeric molecule as described herein, wherein the complement recruiting moiety induces recruitment of the complement immune system upon binding of the pathogen binding moiety to a target pathogen. In example embodiments, the pathogen is a virus, bacteria, fungi, or protozoa. In example embodiments, the gram-negative bacteria pathogen is Pseudomonas aeruginosa (PsA).

[0046] In one aspect, the present disclosure provides a complement recruiting chimeric (CRC) molecule according to the formula R1-L-R2, wherein Ri is a pathogen binding moiety; R2 is a complement recruiting moiety; and L is a linker, wherein the linker may optionally comprise an electrophilic reactive group. In example embodiments, the complement recruiting moiety binds to C3 or C5. In example embodiments, the complement recruiting moiety is a small molecule, a polypeptide, or a hapten. In example embodiments, the complement recruiting moiety is, , or an analog or derivative thereof.

[0047] In example embodiments, the complement recruiting moiety comprises of:OH wherein R1comprises of wherein R2comprises ofL x cr V or .

[0048] In example embodiments, the pathogen binding moiety binds a virus, bacteria, fungi, or protozoa. In example embodiments, the bacteria is a gram-negative bacteria. In example embodiments, the gram-negative bacteria pathogen is Pseudomonas Aeruginosa (PsA). In example embodiments, the pathogen binding moiety binds a protein, lipopolysaccharide, or a polysaccharide. In example embodiments, the protein, lipopolysaccharide, or polysaccharide is pathogen-specific. In example embodiments, the pathogen binding moiety is a peptidomimetic antibiotic macrocycle, or a lectin binder. In example embodiments, the lectin binder is LecA (PAIL) or LecB (PA-IIL). In example embodiments, the pathogen binding moiety comprises of glucopyranoside, fucopyranoside, or an analog or derivative thereof. In example embodiments, the pathogen binding moiety comprises: analog or derivative thereof.

[0049] In example embodiments, the pathogen binding moiety is a colistin-targeting liposaccharide. In example embodiments, the pathogen binding moiety comprises:or an analog or derivative thereof.

[0051] In example embodiments, the pathogen binding moiety is murepavadin, polymyxin, vancomycin, or an analog or derivative thereof. In example embodiments, the linker is selected from: alkane; alkene; alkyne; amine; ether; thiol; sulfone; carbonyl; acyl; ketone; carboxylate ester; amide; enone; anhydride; imide; PEG, or any combination thereof. In example embodiments, the linker is selected from:o o is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0052] In example embodiments, the linker is selected from:wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14; or an analog or derivative thereof.

[0053] In example embodiments, the linker is selected from:2, 3, 4, or 5;

[0054] In example embodiments, L is selected from:

[0055] In example embodiments, the CRC molecule is according to the formula: wherein 2, 5, and 6 are independently selected from R or 5;R1comprises: andR2comprises: an analog or derivative thereof.

[0056] In example embodiments, the CRC molecule is according to the formula:wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and positions 2, 5, and 6 are independently selected from R and S.

[0057] In one aspect, the present disclosure provides a method of targeted activation of the complement immune system against pathogens comprising administering to a cell or cell population a chimeric molecule as described herein, wherein binding of C3 or C5 activates the complement system upon binding of the pathogen binding moiety to a target pathogen.

[0058] In one aspect, the present disclosure provides a method of treating pathogenic infections comprising administering to a cell or cell population a chimeric molecule as described herein, wherein binding of C3 or C5 activates the complement system upon binding of the pathogen binding moiety to a target pathogen. In example embodiments, the pathogen is a virus, bacteria, fungi, or protozoa. In example embodiments, the gram-negative bacteria pathogen is Pseudomonas aeruginosa (PsA).

[0059] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0060] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which:

[0061] FTG. 1A-1B - Recruitment of the complement immunity to the P. aeruginosa using chimeric molecules. (1 A) The scheme of the approach; (IB) Structures of chimeric molecules and controls used in this work.

[0062] FIG. 2A-2E - Ternary complex formation between P. aeruginosa, chimeric molecules and antiDNP. (2 A) Flow Cytometry approach; (2B) Mean Fluorescence Intensity (MFI) measurement for complexes between antiDNP, bacterial cells and compounds 1, 2, 3 or 4; (2C) % of cells positive for ternary complex formation; (2D) ternary complex formation assessment between bacterial cells, antiDNP and compounds 1, 2, 3 or 4 by Biolayer Interferometry (BLI); (2E) disruption of ternary complex formation in the presence of lOx of compound 5 and 6.

[0063] FIG. 3A-3B - Complement-dependent toxicity of 4. (3A) ODeoo Growth of P. aeruginosa treated with 4 in the presence or absence of human pooled serum. (3B) ODeoo Growth of P. aeruginosa in the presence of serum and compound 4, 5 or DMSO only.

[0064] FIG. 4A-4D - Compound 4 effect on P. aeruginosa through the recruitment of complement immunity. (4A) ODeooGrowth of P. aeruginosa treated with 4 at 0.625 pM in the presence of serum, heat-inactivated serum or C3 inhibitor-treated serum. (4B) Deposition of C3b on the P. aeruginosa in the presence of 20% serum alone or 50 pM of 4 and 20% serum. (4C) P. aeruginosa cells stained with Syto BC in the presence of 10 pM of compound 4 in the presence of 0% serum, 2.5% serum or 5% serum. (4D) Viability of HEK293T cells after incubation with compound 4 at variety of concentrations for 72 h incubation.

[0065] FIG. 5A-5H - Analysis of Ternary Complex Formation in P. aeruginosa Grown in Different Media by Flow Cytometry. (5A-5C) Bacterial cells cultured in LB media. (5 A) Gating strategy. (5B) Fluorescence intensity distribution of DY-649 from conjugated antiDNP, interacting with P. aeruginosa through chimeric molecules. (5C) Percentage of cells exhibiting ternary complex formation with high FITC and DY-649 intensities (single replicate for all samples is shown for the representation). (5D-5H) Bacterial cells cultured in M9 media. (5D) Gating strategy. (5E) Fluorescence intensity distribution of DY-649 bound to antiDNP, interacting with P. aeruginosa through chimeric molecules. (5F) Percentage of cells showing ternary complex formation with high FITC and DY-649 intensities. (5G) Mean Fluorescence Intensity (MFI) across samples. (5H) The overall percentage of cells positive for ternary complex formation.

[0066] FTG. 6A-6F - P. aeruginosa growth in LB media in the presence of (6 A) 4 at 0.3125 pM-20pM (6B) 4 at 0.3125 pM-20pM and 10 % serum; (6C) 5 at 0.3125 pM-20pM; (6D) 5 at 0.3125 pM-20pM and 10 % serum; (6E) 4 at 0.3125 pM-20pM and 10 % serum, treated with 1 pM of C3 inhibitor (AMY101); (6F) 4 at 0.3125 pM-20pM and 10 % of heat-inactivated serum.

[0067] FIG. 7 - Normalized Area Under the Curve (AUCnorm) analysis of compound 4 dosedependent growth inhibition responses of P. aeruginosa in LB media under varied conditions.

[0068] FIG. 8A-8F - P. aeruginosa growth in M9 media in the presence of (8A) 4 at 0.3125 pM-20pM (8B) 4 at 0.3125 pM-20pM and 10 % serum; (8C) 5 at 0.3125 pM-20pM; (8D) 5 at 0.3125 pM-20pM and 10 % serum; (8E) 4 at 0.3125 pM-20pM and 10 % serum, treated with 1 pM of C3 inhibitor (AMY101); (8F) 4 at 0.3125 pM-20pM and 10 % of heat-inactivated serum.

[0069] FIG. 9 - Normalized Area Under the Curve (AUCnorm) analysis of compound 4 dosedependent growth inhibition responses of P. aeruginosa in M9 media under varied conditions.

[0070] FIG. 10 - Normalized Area Under the Curve (AUCnorm) analysis of compound 4 dose-dependent growth inhibition responses of P. aeruginosa in LB media in the presence of regular serum, AMY101 -treated serum serum or Heat-inactivated (HI) serum.

[0071] FIG. 11 - Normalized Area Under the Curve (AUCnorm) analysis of compound 4 dose-dependent growth inhibition responses of P. aeruginosa in M9 media in the presence of regular serum, AMY101 -treated serum serum or Heat-inactivated (HI) serum.

[0072] FIG. 12A-12E - (12A-12C) Cultured bacterial cells. Gating strategy and fluorescence intensity distribution, interacting with P. aeruginosa through chimeric molecules. (12D-12E) Experimental results for chimeric molecules.

[0073] FIG. 13 - Complement recruitment chimeras via specific IgG recruitment.

[0074] FIG. 14 - PMB-DNP analogs via click chemistry (for gram negative bacteria).

[0075] FIG. 15 - PMB-DNP analogs via amide chemistry (for gram negative bacteria)

[0076] FIG. 16 - PMB-Rhamnose / Galactose analogs via click chemistry (for gram negative bacteria).

[0077] FIG. 17 - PMB (SAR)-DNP analogs via click chemistry (for gram negative bacteria).

[0078] FIG. 18 - PMB (SAR)-DNP analogs via click chemistry (for gram negative bacteria).

[0079] FIG. 19 - Murepavadin-DNP analogs via click chemistry (for gram negative bacteria)

[0080] FTG. 20 - Murepavadin (SAR)-DNP analogs via click chemistry (for gram negative bacteria).

[0081] FIG. 21 - Murepavadin (SAR)-DNP analogs via click chemistry (for gram negative bacteria).

[0082] FIG. 22 - Murepavadin (SAR)-DNP analogs via click chemistry (for gram negative bacteria).

[0083] FIG. 23 - Murepavadin (SAR)-DNP analogs via click chemistry (for gram negative bacteria).

[0084] FIG. 24 - Murepavadin (SAR)-DNP analogs via click chemistry (for gram negative bacteria).

[0085] FIG. 25 - Murepavadin (SAR)-DNP analogs via click chemistry (for gram negative bacteria).

[0086] FIG. 26 - Murepavadin (SAR)-DNP analogs via click chemistry (for gram negative bacteria).

[0087] FIG. 27 - Murepavadin (SAR)-DNP analogs via click chemistry (for gram negative bacteria).

[0088] FIG. 28 - Murepavadin (SAR)-DNP analogs via click chemistry (for gram negative bacteria).

[0089] FIG. 29 - Murepavadin (SAR)-DNP analogs via click chemistry (for gram negative bacteria).

[0090] FIG. 30 - Vancomycin-PEGn-DNP analogs via click chemistry (for gram positive bacteria).

[0091] FIG. 31 - Vancomycin-PEGn-DNP analogs via click chemistry (for gram positive bacteria).

[0092] FIG. 32 - Complement recruitment chimeras via whole IgG recruitment.

[0093] FIG. 33 - PMB-FCIII analogs via click chemistry (for gram negative bacteria).

[0094] FIG. 34 - PMB-FCIII analogs via click chemistry (for gram negative bacteria).

[0095] FIG. 35 - Murepavadin-FCIII analogs via click chemistry (for gram negative bacteria).

[0096] FIG. 36 - Vancomycin-FCIII analogs via click chemistry (for gram positive bacteria).

[0097] FIG. 37 - Development of complement-recruiting chimeras for pathogenic bacteria.

[0098] FTG. 38 - Complement recruitment chimeric molecules for infectious disease.

[0099] FIG. 39 - Bacterial killing: Bac Titer-Gio assay.

[0100] FIG. 40 - Complement dependent killing: Head-to-head comparison E.coli vs PsA.

[0101] FIG. 41 - Complement dependency: Head-to-head comparison E.coli vs PsA.

[0102] FIG. 42 - Ternary complex formation.

[0103] FIG. 43 - Bacterial killing: Head-to-head comparison E.coli vs PsA.

[0104] FIG. 44A-44F - (44A) A ternary complex between bacteria, chimera, and complement pathway effector can trigger cytolysis, phagocytosis and chemotaxis. (44B) Class I chimeras (Aims 1 & 3) for targeted activation of classical pathway via IgG recruitment. (44C) An approach to prioritize chimeras by rank ordering activities in ternary complex and cytolysis (or phagocytosis / chemotaxis) assays. (44D) A MicroMap platform to capture enzymatic cascade upon complement activation. (44E-44F) Proposed cytotoxicity studies and in vivo studies in lung infection model on prioritized chimeras.

[0105] FIG. 45A-45C - (45A) An enzyme inhibitor operates by “occupancy- driven pharmacology” that is stoichiometric (lacks turnover), (45B) An example of chimera that operates by “event-driven pharmacology” that is sub-stoichiometric (exhibits turnover). (45C) Complement proteins are present at high concentrations in human serum.

[0106] FIG. 46A-46F - (46A) Structure of chimera 1 and controls. (46B-46C) Outline of the BLI experiment (46B) and ternary complex data (46C). (46D) Demonstration of ternary complex using flow cytometry. (46E-46F) Clinical isolates (46E) from various sites and ternary complex formation using clinical isolates of PsA (46F).

[0107] Fig. 47A-47I - (47A) Dose-dependent growth inhibition of 1 is contingent on the presence of complement-preserved serum. (47B-47C) 1 is more potent than control compound 3 that lacks DNP (47C) Heat-inactivated serum or complement inhibitor lowers the activity of 1. (47D) 1 enhances deposition of complement protein C3 on PsA. (47E) HEK 293T viability is unaffected even at 80 pM of 1 (>130-fold selectivity for PsA over human kidney cell). (47F) Structureofmurepavidin-DNPchimera4.(47G) Ternary complex between anti-DNP:4:PsA. (47H) Dose-dependent growth inhibition by 4. (471) Selectivity of growth inhibition of 4 for PsA over E. coli as 4 does not bind to E. coli.

[0108] Fig. 48A-48C (48A) Structure of chimera 5. (48B-C) Ternary complex formation (48B) and growth inhibition (48C) by 5.

[0109] Fig. 49A-49E - (49A-49B) ATP-glo assay principle. (49B) ATP-glo assay has high Z’ pointing to robustness. (49C) Complement-dependent cytotoxicity of PMBN conjugates. (49D) Ternary complex formation using BLI. (49E) / <on / L>rr values derived.

[0110] Fig. 50A-50D - (50A) Design of Class la-b chimeras. (50B) Linkers, (50C) IgG binders, and (50D) pathogen surface binders used for Class la-b chimeras.

[0111] Fig. 51A-51C. (51A) Workflow to determine on-target CDC chimeras (51B-C) uMap platform for proximity labeling on cell surface conjugation.

[0112] Fig. 52A-52B - ATP-glo viability of (52A) WT and MCR-1 E.coli with polymyxin B (PMB) of chimera 5 or (52B) B.subtilis with a vancomycin-based Class lb chimera.

[0113] Fig. 53 - Complement is a natural defense mechanism against bacterial infections.

[0114] Fig. 54 - High-throughput assay for measuring complement-dependent cytolysis (CDC). Assay is robust and general for different bacterial strains. Compatible with 384-well plates. Test different chimeras and serum conditions in parallel.

[0115] Fig. 55 - Chimeras recruiting IgG via Fab-domain.

[0116] Fig. 56 - Fab-recruiting chimeras induce CDC (E.coli). Short PEG linkers enhance passive membrane disruption. Longer PEG linkers selectively induce CDC.

[0117] Fig. 57A-57C - Fab-recruiting chimeras induce CDC (E.coli). (57A) Growth inhibition (P. aeruginosa / PA14). (57B) C3 deposition on (P. aeruginosa / PA14). (57C) HEK293T cytotoxicity. Chimeras inhibit growth of P. aeruginosa and deposits C3 on the bacterial surface. Chimeras are highly selective for bacterial cells (> 120-fold).

[0118] Fig. 58 - Ternary complex formation (BLI). Chimera forms a ternary complex between bacteria and antibody. Chimera recruits antibodies to different clinical PA isolates.

[0119] Fig. 59 - Chimeras recruiting IgG via Fc-domain. Fc-recruiting chimera forms a ternary complex and selectively induces CDC. Fc-recruiting chimera may cause a more uniform patient response.

[0120] FIG. 60 - Generalization of HaC platform. T cell activation assay by HaCs that target FGFR, BTK, JAK3, ITK, and EGFRT790 / L858R. EGFR (T790M / L858R) targeting HaC 11selectively activates T-cell only with the double mutated cell line (Hl 975) and not with the wild type (A431).

[0121] FIG. 61 - Mapping of displayed peptides for BTK, EGFR, FGFR4, ITK. Grey bars show detected peptides, while purple bars show labeled peptides. No overlap exists between the detected and labeled peptides, suggesting haptenization alters MHC-I processing. Data obtained from the cancer antigen atlas (caAtlas).

[0122] FIG. 62A-62G - (62A) A ternary complex between bacteria, chimera, and complement pathway effector can trigger cytolysis, phagocytosis and chemotaxis. (62B) Class I chimeras (Aims 1 & 3) for targeted activation of classical pathway via IgG recruitment. (62C) Class II chimeras (Aims 2 & 3) for targeted activation of classical (Class Ila). (62D) An approach to prioritize chimeras by rank ordering activities in ternary complex and cytolysis (or phagocytosis / chemotaxis) assays. (62E) A MicroMap platform to capture enzymatic cascade upon complement activation. (62F-62G) Proposed cytotoxicity studies and in vivo studies in lung infection model on prioritized chimeras.

[0123] FIG. 63A-63C. (63A) Structure of Class 1c chimera. (63B) BLI studies confirms engineered Fc’s affinity and specificity for Clq over wt Fc. (63C) Growth inhibition of Class Ic chimera.

[0124] FIG. 64 - Design of Class Ic chimeras.

[0125] FIG. 65A-65F - (65A) 3D box plot of enriched compounds in the Clq DEL screen illustrating SAR convergence of preferred building blocks (synthon) for binding. (65B) Highly enriched scaffolds that emerged from the Clq screen. (65C) SPR data for binding validation of Clq DEL hits and screening of azetidine-RCM for Cl binding. (65D) Structures of libraries enriched for Clq binding. (65E) Determination of Ad for azetidine hit (BRD5001 and BRD7021). (65F) Workflow to develop complement-recruiting chimeras.

[0126] FIG. 66 - ATP-glo viability of WT and MCR-1 E.coli with polymyxin B (PMB) of chimera 6.

[0127] FIG. 67A-67F - (67A) A ternary complex between bacteria, chimera, and complement pathway effector can trigger cytolysis, phagocytosis and chemotaxis. (67B) Class II chimeras (Aims 2 & 3) for targeted activation of alternate (Class lib) and lectin pathways (Class lie). (67C) An approach to prioritize chimeras by rank ordering activities in ternary complex and cytolysis (orphagocytosis / chemotaxis) assays. (67D) A MicroMap platform to capture enzymatic cascade upon complement activation. (67E-67F) Proposed cytotoxicity studies and in vivo studies in lung infection model on prioritized chimeras.

[0128] FIG. 68A-68H - (68A) 3D box plot of enriched compounds in the C3 DEL screen illustrating SAR convergence of preferred building blocks (cycles) for target binding. (68B) Highly enriched sultam library that emerged from the C3 screen. (68C) BLI data for binding validation of C3-DEL-sultam compound. (68D) SPR screening of sultams for C3 binding. (68E) Structures of C3 binding sultams (BRD6432 and BRD8499). (68F) Determination of C3- binding affinity constants for BRD6432 and BRD8499 stereoisomers (1R2R3R) using SPR. (68G) SPR- RU of C3-sultams (BRD6432 and BRD8499) shows stereochemical dependence and is highest for ( \R2R3R) isomer. (68H) Determination of C3-binding affinity constants for BRD5877.

[0129] FIG. 69A-69I - (69A) Distributions of distances of lysines from the ligands bound in binding pockets of proteins (PDBbind analysis). (69B) A binding pocket ID workflow using a lysine reactive warhead. (69C) Dose dependent labeling C3 was observed with DEL hit bearing lysine-reactive warhead (69D) Chain-specific labelling of C3 using lysine-reactive probe (R=reduced; NR=not reduced). (69E) Concentration-dependent labelling of Lysine-1436 detected by mass spectrometer. (69F) MS / MS spectra of lysine 1436 in C3-peptide (YELDKAFSDR). (69G) Docking studies preliminarily identify binding pocket. Here, the tighter binder (1R2R3R) fits in the pocket while the weak binder (1S2R3S) does not. (69H) C3-recruting chimera 6 prepared by conjugating C3-binder BRD6432 and PMBN. (691) C3-recruting chimera 6 induces dosedependent growth inhibition of PsA with MIC of - 150-300 nM.

[0130] FIG. 70 - Bead saturation experiment for MBL protein (M=marker, IN = input, U = unbound, B = bound). Workflow to develop complement-recruiting chimeras.

[0131] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTSGeneral Definitions

[0132] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosurepertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2ndedition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4thedition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2ndedition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew el al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2ndedition (2011).

[0133] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0134] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0135] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0136] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / - 10% or less, + / -5% or less, + / - 1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0137] As used herein, a “biological sample” may contain whole cells and / or live cells and / or cell debris. The biological sample may contain (or be derived from) a “bodily fluid”. The presentinvention encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from a mammal organism, for example by puncture, or other collecting or sampling procedures.

[0138] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0139] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0140] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication,published patent document, or patent application was specifically and individually indicated as being incorporated by reference.OVERVIEW

[0141] Embodiments disclosed herein provide compositions and methods for eliciting complement immune recruiting against pathogens. Compositions comprising a chimeric molecule (referred to herein “complement immune recruiting chimeric molecule”, “complement recruiting chimeric molecule”, or “recruiting chimeric molecule”) that can bind the pathogen via a pathogen protein binding moiety and recruit a complement immune system. The pathogen binding moiety and the compliment recruiting moiety are connected by a linker, in some instances a linker comprising one or more electrophilic reactive groups that can facilitate transfer of either the compliment recruiting moiety to the pathogen protein or vice versa. The complement recruiting chimeric (CRC) molecule may also be used for targeted activation of the complement immune system against pathogens. Accordingly, the complement recruiting chimeric (CRC) molecule may also be used for treating pathogenic infections.

[0142] The complement recruiting chimeric molecule is modular in design and can be rapidly adapted to new pathogens without requiring building of a new molecule from the ground up. For example, the same recruiting moiety may be used with any pathogen binding moiety requiring only a change in the pathogen binding moiety to re-purpose the molecule for binding a different pathogen. The ability to keep the compliment recruiting moiety constant for a given molecule design also allows for the design of a single complement immune recruiting molecule that can be used with multiple chimeric molecules each comprising different pathogen binding moieties but having the same recruiting moiety. The small molecule nature of the complement immune recruiting molecules also increases cell penetration and delivery. The designs of the complement immune recruiting chimeric molecules also allow targeting of intracellular pathogens and are not limited to extracellular pathogens as are some current therapeutic modalities.Complement Recruiting Chimeric Molecule

[0143] In example embodiments, a complement recruiting chimeric molecule according to the formula: R1-L-R2, wherein Ri is a pathogen binding moiety; R2 is a complement recruiting moiety; and L is a linker, wherein the linker may optionally comprise an electrophilic reactive group. Inexample embodiments, the complement recruiting moiety binds an antibody that recruits one or more components of the complement system.Complement System

[0144] The complement system (i.e., complement immune system - “CIS”) participates in the innate immune defense against pathogens. Recognition molecular complexes initiate the CIS when they undergo conformational changes upon detection of a pathogen. The CIS comprises cell surface membrane proteins and / or plasma proteins operating in tissue, plasma, or cells. There are three CIS pathways: 1) classical, 2) lectin, and 3) alternative. A core aspect of the CIS is the C3 protein. Upon activation of the CIS, inactive C3 proteins are cleaved into C3a and C3b, which are inflammation mediators and opsonin respectively. Inflammatory mechanisms of the complement pathway include recruitment of phagocytes to the site of infection and activation of leukocytes, endothelial cells, or platelets. Terminal complement components have direct lytic capacity to kill pathogens.

[0145] In particular, under the classical CIS pathway (“CP”), the pathogen recognition molecule is Clq. It comprises 18 polypeptide chains consisting of three types, A, B, and C. These 18 polypeptide chains form six globular target recognition domains (gClq) and are attached to a collagen-like region (CLR). Clq is capable of binding to more than 100 different molecules, including IgG and IgM containing immune complexes. The CP can be activated in either an immune complex-dependent or -independent manner. Clq recognizes pathogen-associated molecules such as lipopolysaccharide and bacterial porins. Clq associates with SP complexes, consisting of Clr and Cis. Upon activation, SPs cleave complement components C4 and C2 and the resulting complex, C4b2a, is the C3 convertase for the CP. This C3 convertase comprises of enzymatic activity and can cleave the central complement component C3 to bioactive fragments C3a and C3b thereby eliciting an immune response. See e.g., Merle, N. S.; et al. Complement System Part I - Molecular Mechanisms of Activation and Regulation. Frontiers in Immunology, 2015, 6.Complement Recruiting Moiety

[0146] In example embodiments, R2 is a recruiting moiety and the recruiting moiety can comprise of a complement recruiting moiety. In an example embodiment, the complement recruiting moiety binds an antibody that recruits one or more components of the complementsystem. In certain example embodiments discussed below, the complement recruiting moiety may also be referred to as an antibody binding moiety. The complement recruiting moiety may be chosen to harness the immune effector functions of natural antibodies, and the complement recruiting moiety comprises a hapten that binds endogenous antibodies. In an aspect, the complement recruiting moiety binds IgG, IgM or both IgG and IgM antibodies. Exemplary IgG and IgM antibody isotypes may comprise anti-DNP, anti-Gal or anti-Rhamnose antibodies. The chimeric molecule comprising the complement recruiting moiety can be provided at a ratio of 2 to 250 times the amount of antibody.

[0147] In example embodiments, the compliment recruiting moiety binds to the light chain or the heavy chain of the antibody. In example embodiments, the compliment recruiting moiety binds to the antigen binding site of the light chain. In example embodiments, the complement recruiting moiety is a small molecule, a polypeptide, or a hapten. In example embodiments, the hapten is 2,4- dinitrophenyl (DNP), , , or an analog thereof, and whei in the hapten is bound by an anti -DNP antibody.

[0148] In example embodiments, the complement recruiting moiety comprisesHQ OH

[0149] In example embodiments, the complement recruiting moiety comprises, or an analog or derivative thereof.

[0150] In example embodiments, the complement recruiting moiety comprises of

[0151] In example embodiments, the complement recruiting moiety is an anti-rhamnose binding moiety is rhamnose (Rha), OH OH or a derivative thereof.

[0152] In one embodiment, the complement recruiting moiety is an anti -dinitrophenyl binding moiety is dinitrophenyl (DNP). Dinitrophenyl can be utilized in a small chimeric molecule, withthe antibody target an anti-DNP TgG. In an example embodiment, the compound is a dinitrophenyl,, or a derivative thereof.

[0153] An exemplary anti-gal binding moiety is galactose-a-l,3-galactose (otGal),

[0154] In example embodiments, the compliment recruiting moiety binds one or more Clq polypeptides. In example embodiments, the compliment recruiting moiety binds a Clq A-chain, B-chain, C-chain, or a combination thereof. In example embodiments, the compliment recruiting moiety is a small molecule, a polypeptide, or a hapten.

[0155] In example embodiments, the compliment recruiting moiety comprises ofR1wherein R1 comprises of andR2 comprises ofN; or ' .

[0156] In example embodiments, the compliment recruiting moiety binds to C3 or C5. In example embodiments, the compliment recruiting moiety is a small molecule, a polypeptide, or a hapten. In example embodiments, the complement recruiting moiety iso, , or an analog or derivative thereof.

[0157] In example embodiments, the complement recruiting moiety comprises of:wherein R1comprises ofwherein R2comprises ofPathogen Binding Moiety

[0158] The pathogen binding moiety equips the chimeric molecule with a mechanism to bind or associate with a target pathogen. The pathogen binding moiety of the chimeric molecule binds the pathogen enabling recruitment of the complement system to the bound pathogen via the complement recruiting moiety.Oligosaccharide Bindins Moieties

[0159] In one example embodiment, the pathogen binding moiety may bind to pathogenspecific oligosaccharides. Oligosaccharide binding moieties include small molecules. For example, small molecules that include boronic acid are typically used to bind to oligosaccharides. See e.g. Jin S., et al. “Carbohydrate Recognition by Boronolectins, Small Molecules, and Lectins. Med. Res. Rev. 2009, herein incorporated by reference. Other oligosaccharide binding moieties include carbohydrate binding proteins, are important targets when considering antiviral and anticancer drugs. The localizing moiety can be, for example, a lectin, facilitating interaction sites for carbohydrates. Exemplary molecules include small molecule boronolectins, nucleic acid-based boronolectins, and peptidoboronolectins. See, e.g. Jin et al., Med. Res Rev. 2010 March; 30(2): 171-257; doi: 10.1002 / med.20155, incorporated herein by reference, specifically Figures 1-50 for binding molecules and the complexes formed. Publicly available computational methods are available using developed bioinformatics to select small molecules capable of binding carbohydrates, see, e.g. , Zhao et al., Current Protocols in Protein Science 94: 1 10.1002 / cpps.75; Shionyu-Mitsuyama C, Shirai T, Ishida H, Yamane T (2003) Protein Eng 16: 467-478; and Kulharia M, Bridgett SJ, Goody RS, Jackson RM (2009) InCa-SiteFinder: a method for structurebased prediction of inositol and carbohydrate binding sites on proteins. J Mol Graph Model 28: 297-303.

[0160] Analysis of binding site residues along with stabilizing residues in protein- carbohydrate complexes can allow for identification of folding and binding of the complexes to understand interactions in addition to non-covalent interactions of hydrogen bonding and nonpolar interactions. See, e.g., Shanmugam et al., doi.: 10.2174 / 0929866525666180221122529. Utilizing publicly available tools, carbohydrate binding moieties, including binding sites and predicted folding can be used for the design of multifunctional molecules comprising such a carbohydrate binding moiety.Lipid Bindins Moieties

[0161] In one example embodiment, the pathogen binding moiety may bind pathogen-specific lipids. Lipids include charged lipids, e.g. phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), and the PLphosphate, -bisphosphate, and -trisphosphate (PIPs - a family of seven anionic charged lipids), and ganglioside (GM). Zwitterionic lipids, e.g.,phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM) lipids, Ceramides (CER), diacylglycerol (DAG), and lysophosphatidylcholine (LPC) lipids, sphingolipids, glycerophospholipids, cholesterol, phosphatidylglycerols.

[0162] Lipid binding moieties may be incorporated into chimeric molecules. For example, certain steroids are capable of targeting and binding to lipids. Other lipid binding moieties such as proteins can either bind lipids specifically, where a clear binding site for a given lipid can be identified, or nonspecifically, where lipids act as a medium, and physical properties like thickness, fluidity, or curvature regulate the protein function. Phosphoinositide binding domains such as FYVE or PX, or the FRRG motif in the 0-propeller of PROPPINs are more common domains that can be used to identify lipid binding proteins. The FYVE domain, named after the first four proteins to contain the motif (Fabl, YOTB, Vacl EEA1) contains several conserved regions, which can also be utilized to identify related domains. See, e.g., A.H. Lystad, A. Simonsen Phosphoinositide-binding proteins in autophagy, FEBS Lett., 590 (2016), pp. 2454-2468, 10.1002 / 1873-3468.12286. Additional FYVE domain-containing proteins include SARA, FRABIN, DFCP1 FGD1, ANKFY1, EEA1 FGD1, FGD2, FGD3, FGD4, FGD5, FGD6, FYCO1, HGS MTMR3, MTMR4, PIKFYVE, PLEKHF1, PLEKHF2, RUFY1, RUFY2, WDF3, WDFY1, WDFY2, WDFY3, ZFYVE1, ZFYVE16, ZFYVE19, ZFYVE20, ZFYVE21, ZFYVE26, ZFYVE27, ZFYVE28, ZFYVE9.

[0163] Autophagy related (ATG) proteins may be utilized as lipid binding moiety in the present invention, including LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2. De la Ballina (2019), doi.org / 10.1016 / j.jmb.2019.05.051. Lipid-binding proteins include protein HCLS1 binding protein 3 (HS1BP3) that is able to negatively regulate the activity of phospholipase DI (PLD1). Dikic et al., Mechanism and medical implications of mammalian autophagy. Nat. Rev. Mol. Cell Biol., 19 (2018), pp. 349-364, doi: 10.1038 / s41580-018-0003-4.Protein Bindins Moiety

[0164] In one example embodiment, the pathogen binding moiety may bind pathogen-specific proteins. The following provide examples of further, non-limiting, target protein binding moieties to various target proteins of interest in oncology and infectious disease contexts and the use of which will discussed further in the Methods of Use section below.

[0165] In example embodiments, the pathogen binding moiety is murepavadin, polymyxin, vancomycin, or an analog or derivative thereof.Polymyxin

[0166] In preferred embodiments, the PsA associated target protein binding moiety is a polymyxin or an analog or derivative thereof. In an embodiment, the polymyxin is polymyxin B or polymyxin E (Colistin), which has the formula:

[0167] In an embodiment, the polymyxin moiety is according to:Murepavadin

[0168] In an example embodiment, the chimeric molecule comprises one, two or three murepavadin moieties, or an analog or derivative thereof. In an embodiment, the murepavadin moiety is according to:Vancomycin

[0169] In an example embodiment, the chimeric molecule comprises one, two or three Vancomycin moieties, or an analog or derivative thereof. In an embodiment, the Vancomycin moiety is according to:OH OHLectin Binders

[0170] In an example embodiment, the pathogen binding moiety may bind to pathogenspecific lectins. In example embodiments, the pathogen binding moiety comprises galactopyranoside, fucopyranoside, or a derivative thereof. In example embodiments, the lectin comprises of: or an analog or derivative thereof.

[0171] In example embodiments, the lectin binder is LecA (PA-IL) or LecB (PA-IIL). In example embodiments, the lectin binder comprises ofor an analog or derivative thereof.PtpA, PtpB

[0172] In one example embodiment, target protein binding moiety is a PtpA binding moiety is derivatives thereof.

[0173] In preferred embodiments, the PtpB binding moiety is according to the formulaor any derivatives thereof.SayM

[0174] In one example embodiment, the target protein binding moiety is a SapM binding moiety. In an example embodiment, the SapM binding moiety contains a trihydroxy-benzene group. In an example embodiment, the SapM binding moiety comprises of a benzylidenemalononitrile scaffold. In one example embodiment, the SapM binding moiety has the formula:OH O OH OOH OH OHOHOH or any derivatives thereof. In one example embodiment the SapM binding moiety is L-ascorbic acid (L-AC) and 2-phospho-L-ascorbic acid (2P-AC).Linker

[0175] A linker or linking moiety is a bifunctional or multifunctional moiety that can be used to link one or more of pathogen binding moiety and one or more of the compliment recruiting moiety. In some embodiments, the linker has a functionality capable of reacting with the moieties for covalent attachment. The linker moiety is preferably a chemical linker moiety and is represented in the formulas of the present invention as L. In an embodiment, the linker moiety may preferably comprise one or more repeats, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more repeats, which may be utilized to facilitate or improve spacing, conformation, and / or performance of the molecules. The linker described herein may refer to L, both LI and L2, or LI and L2 are different linkers described herein.

[0176] A linker or linking moiety can be used to link a recruiting moiety to the pathogen binding moiety, and / or the electrophilic reactive group to either the compliment recruiting binder,pathogen binding moiety, or both. When more than one linker molecule is used in a molecule, the linkers may be the same or different from each other.

[0177] In example embodiments, the molecule of any of the previous claims, wherein Li and L2 are individually selected from: alkane; alkene; alkyne; amine; ether; thiol; sulfone; carbonyl; acyl; ketone; carboxylate ester; amide; enone; anhydride; imide; PEG, or any combination thereof.

[0178] In example embodiments, L comprises: is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0179] In example embodiments, L comprises:Owherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14; or an analog or derivative thereof.

[0180] In example embodiments, Li and L2 are individually comprise:o o or an analog or derivative thereof, wherein n is 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, or 15.

[0181] In example embodiments, Li and L2 are individually comprise.o 5-

[0182] In example embodiments, Li and L2 are individually comprise:O i) the group consisting of

[0183] In example embodiments, L comprises of: or .

[0184] In an embodiment, L is a rigid linker, which may be selected from the group consisting any combination thereof; and wherein any atom in within a ring may substituted for C, N O, S; the linkers may bond to one or more PEG molecules before bonding to A and optionally B; and m and n may be independently selected from 0 to 6.

[0185] In preferred example embodiments, the linker L has one covalent attachment point to a kinase binding molecule and two covalent attachment points to the other kinase bindingmolecule. A covalent attachment point may be any single, double, triple, or quadruple bond between one component of the BFM / chimeric molecule and another. In preferred example embodiments, the linker is attached to one kinase binding molecule, i.e. A, and the other, i.e. B, according to the formula

[0186] In one example embodiment, the PEG compounds in the previously mentioned linker can be substituted for any linker mentioned herein. In One example embodiment, the previously mentioned linker is optimized for physiochemical properties, such as solubility and / or permeability, and / or pharmacokinetic properties, such as microsomal stability or target binding.

[0187] In one example embodiment, the complement recruiting chimeric molecule has an adapter or reactive handle, both used herein interchangeably. The reactive handle comprises the group on the complement recruiting chimeric molecule that attaches to the linker. In one example embodiment, the reactive handle can perform click chemistry, amide coupling chemistry, crosslinking chemistry, alkylation, or sulfonation chemistry. (See e.g. I, K.; Brechbiel, M. W. Growing Applications of “Click Chemistry” for Bioconjugation in Contemporary Biomedical Research. Cancer Biotherapy and Radiopharmaceuticals, 2009, 24, 289-302.)Electrophilically Reactive Linkers

[0188] In another aspect, embodiments disclosed herein are directed to complement recruiting chimeric molecules comprising an electrophilic linker having enhanced stability, reactivity, and tuneability. In one example embodiment the linker molecule has the formula Li-El, EL-Li, or Li- EI-L2, wherein Li and L2 are independently selected from alkane, alkene, amine, either, thiol, sulfone, carbonyl, acyl, ketone, carboxylate ester, amide, enone, anhydride, imide, and PEG, and wherein EL is an electrophilic reactive group. The electrophilically reactive linkers may be used to attach all or a portion of Li or L2 to a target polypeptide. The El group of the linker molecule may be selected based on an ability to covalent attach all or a portion of Li or L2 to a cysteine, alysine, a methionine, or a tyrosine residue on the target polypeptide. In one example embodiment Li and L2may independently be any of the linkers described above.Cysteine Reactive Electrophilic Linkers

[0189] In one example embodiment, the linker molecule comprises and El group suitable for attachment of all or a portion of Li or L2 to a cysteine, and wherein the El is selected from the group consisting of:Lysine Reactive Electrophilic Linkers

[0190] In one example embodiment, the linker molecule comprises and El group suitable for f attachment of all or a portion of Li or L2 to a lysine, and wherein El isO a 2R< N MeR2wherein Ri is selected from the group consisting of;AcN NPh; and wherein R2 is selected from the group consisting of

[0191] In another example embodiment, the linker molecule comprises an El group suitable for attachment of all or a portion all or a portion of Li or L2 to a lysine, wherein the El group is selected from;Methionine Reactive Electrophilic Linkers

[0192] In another example embodiment, the linker molecules comprise an El group suitable for attachment of all or a portion all or a portion of Li or L2 to a methionine, wherein the El group is selected from;Use of Electrophilically Reactive Linker Groups

[0193] The electrophilicly reactive linkers disclosed in this section may be used in complement recruiting chimeric molecules disclosed in this application. For example, a target polypeptide binding moiety may be attached to one of the linkers and recruiting moiety may be attached to the other end of the linker. The target polypeptide binding moiety may be selected to selectively bind a target polypeptide, such as a pathogen, at a catalytical or allosteric site to allow for the electrophilically reactive linkers disclosed herein to covalently attach the compliment recruiting moiety to the target polypeptide at cysteine, lysine, methionine, or tyrosine.Example Recruiting Chimeric Molecules

[0194] Recruiting chimeric molecules may be assembled using any combination of the above recruiting binding moieties, linkers, electrophilic reactive groups, and pathogen binding moieties.The following description provides, by way of reference only, certain chimeric molecules that can be generated according to the design principles and examples moieties provided above.

[0195] In example embodiments, the compliment recruiting chimeric molecule is according to the formula: o— .J. R2K’ O ‘,-• — ' , wherein R1is the pathogen binding moiety and R2is the complement recruiting moiety. In example embodiments, R1is galactopyranoside and R2is DNP.

[0196] In example embodiments, the compliment recruiting chimeric molecules is according to the formula:R< N 7 °2N, wherein R1is the pathogen binding moiety and R2is the complement recruiting moiety. In example embodiments, R1is fucopyranoside and R2is DNP.

[0197] In example embodiments, the compliment recruiting chimeric molecules is according to the formula:' O■ \ HN . 0 . ■ N . • ■ . . R2’ ’ ’ 5 f ‘ 0 / 0 20\ N, wherein the top R1and bottom R1are independently selected target binding moieties and R2is the complement recruiting moiety. In example embodiments, the top R1and bottom R1are fucopyranoside and R2is DNP.

[0198] In example embodiments, the compliment recruiting chimeric molecules is according to the formula:OR?R1.0v, wherein R1is the pathogen binding moiety and R2is the complement recruiting moiety. In example embodiments, R1is polymyxin B and R2is DNP.

[0199] In example embodiments, the CRC molecule is according to the formula: the triangle comprises of any of the hexagon comprises ofand optionally comprise an electrophilic reactive group.

[0200] In example embodiments, the CRC molecule is according to the formula:HO wherein the triangle comprises of the complement recruiting moiety.

[0201] In example embodiments, the complement recruiting moiety is a FcIII-peptide.

[0202] In example embodiments, the compliment recruiting chimeric molecules are accordingo the formula:.wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0203] In an example embodiment, the compliment recruiting moiety comprises of: wherein R1 comprises ofR2 comprises of

[0204] In example embodiments, the chimeric molecule is according to the formula: wherein 2, 5, and 6 are independently selected from R or 5;R1comprises: oHO o ci o O , Br, or an analog or derivative thereof; andR2comprises:H N O an analog or derivative thereof.

[0205] In example embodiments, the compliment recruiting chimeric molecule is according to the formula:wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and positions 2, 5, and 6 are independently selected from R and S.Methods of UsePathogenic Applications

[0206] The chimeric molecules disclosed herein can be utilized in methods of targeted activation of the complement system against and treating infection by a pathogen. Methods of treating infection by a pathogen can include generating a repurposed / reprogrammed cellular protein by administering a chimeric molecule, as described herein. In one aspect, as described herein, are methods of targeted activation of the complement immune system against pathogens comprising administering to a cell or cell population any chimeric molecule as described herein, wherein binding of the complement recruiting moiety to an antibody activates the complement immune system against a target pathogen.

[0207] Methods for treating infection by a pathogen are provided. The method comprises, administering to a cell or cell population a chimeric molecule as described herein, wherein binding of the complement recruiting moiety to an antibody activates the complement immune system against a target pathogen. The chimeric molecule labels the complement immune system compound with the pathogen binding moiety for the target substrate; and modifying the pathogen protein by binding of the repurposed / reprogrammed complement immune system compound to the pathogen via the pathogen binding moiety, whereby the repurposed / reprogrammed complement immune system compound introduces one or more modifications to the target substrate. In one example embodiment, the complement immune system compound to be reprogrammed is an antibody. In one example embodiment, the pathogen is a viruses, bacteria, fungi, or protozoa. In one example embodiment, the bacteria is Mycobacterium tuberculosis (Mtb) or Pseudomonas aeruginosa (PsA). In one example embodiment, the pathogen is Mtb and the pathogen protein is one or more of PtpA, PtpB, SapM, ESAT-6, and Rv2966c. In one example embodiment, the pathogen is (PsA) and the pathogen binding moiety is Colistin. In one example embodiment, the kinase binding moiety is a kinase inhibitor. In one example embodiment, the kinase inhibitor is a promiscuous inhibitor. In one example embodiment, a step of administering a coupling molecule thereby quenching the inhibitor activity of the kinase inhibitor is provided.

[0208] A pathogen may include viruses, bacteria, fungi, and protozoa. In one example embodiment, the pathogen is a pathogenic bacterium and may include: spirochetes; spirilla; vibrios; gram-negative aerobic rods and cocci; enterics; pyogenic cocci; and endospore-formingbacteria; actinomycetes and related bacteria; rickettsias and chlamydiae; mycoplasmas, which are groups defined by some bacteriological criteria. A pathogenic bacteria may include: Escherichia coli, Salmonella enterica, Salmonella typhi, Shigella dysenteriae, Yersina pestis, Pseudomonas aeruginosa, Vibrio cholerae, Bordetella pertussis, Haemophilus influenza, Helicobacter pylori, Campylobacter jejuni, Neisseria gonorrhoeae, Neisseria meningitidis, Brucella abortus, Bacteroides fragilis, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Bacillus anthracis, Bacillus cereus, Clostridium tetani, Clostridium perfringens, Clostridium botulinum, Clostridium difficile, Corynebacterium diphtheriae, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium leprae, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Rickettisas, Treponema pallidum, Borrelia burgdorferi, or a variant thereof. (Todar, K. Textbook of Bacteriology (2020) Online)

[0209] A target virus may belong to any morphological category including helical, envelope, or icosahedral. A target virus may comprise of DNA or RNA, may be single stranded or double stranded, and may be linear or circular. The genome of the virus may be one nucleic acid molecule or several nucleic acid segments. A target virus may belong to the family: Adenoviridae, Papovaviridae, Parvoviridae, Herpesviridae, Poxviridae, Anelloviridae, Pleolipoviridae, Reoviridae, Picornaviridae, Caliciviridae, Togaviridae, Arenaviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Rhabdoviridae, Filoviridae, Astroviridae, Bornaviridae, Arteriviridae, Hepeviridae, Retroviridae, Caulimoviridae, Hepadnaviridae, Coronaviridae. In one example embodiment, the virus is SARS-CoV-2. (Gelderblom HR. Structure and Classification of Viruses. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 41).

[0210] In example embodiments, the pathogen is a pathogenic fungus and may include: Aspergillus; Blastomyces; Candida; Coccidioides; Cryptococcus; Fusarium; Microsporum; Epidermophyton; Trichophyton; Histoplasma; Rhizopus; Mucor; Rhizomucor; Syncephalastrum; Cunninghamella; Apophysomyces; Lichtheimia (formerly Absidia); Eumycetoma; Pneumocystis; Trichophyton; Microsporum; Epidermophyton; Sporothrix; Paracoccidioides; Talaromyces or a variant or species thereof. (CDC)

[0211] In an exemplary embodiment, the pathogen is a pathogenic protozoa belonging to the group: Sarcodina; Mastigophora; Ciliophora; or Sporozoa defined by their mode of movement.(CDC) Tn one example embodiment, the pathogenic protozoa may include: Entamoeba; Trichomonas; Leishmania; Chilomonas; Giardia; Isopora; Sarcocystis; Nosema; Balantidium; Eimeria; Histomonas; Trypanosoma; Plasmodium; Babesia; or Haemoproteus or a variant or species thereof.

[0212] In one aspect, the method comprises treating infection by a pathogen as a result of M. tb. In an example embodiment, the pathogenic target is Mycobacterium tuberculosis (M. tb). Successful infection of host macrophages by M. tb hinges on the weakening of the diverse microbicidal responses by the host cell. M. tb furthers infection by impeding the host cellular signaling machinery. Therefore, infection can be inhibited by phosphorylating proteins associated with M. tb, which promotes increased signaling to the immune system. In one example embodiment, the chimeric molecule pathogen binding moiety targets the pathogen is M.tb and the pathogen protein is one or more of PtpA, PtpB, SapM, ESAT-6, and Rv2966c.

[0213] In a preferred embodiment, the target protein is Mtb protein tyrosine phosphatases (Ptp). Ptps belong to a large family of signaling enzymes and are required for optimal bacillary survival. PTPs, with protein tyrosine kinases, regulate numerous cellular functions, such as cell growth, proliferation, differentiation, metabolism, and immune response. M.tb encodes PtpA and PtpB and secretes them into the cytoplasm of host macrophages. PtpA prevents phagolysosome acidification by dephosphorylation of its substrate, Human Vacuolar Protein Sorting 33B. This results in the exclusion of the macrophage vacuolar-H+-ATPase (V-ATPase) from the vesicle. Once inside the macrophage, mPTPB activates Akt signaling and simultaneously blocks ERK1 / 2 and p38 activation thereby preventing host macrophage apoptosis and cytokine production. Inhibition of PtpA and PtpB decreases Mtb survival. See e.g. Dutta N.K. et al. Mycobacterial Protein Tyrosine Phosphatases A and B Inhibitors Augment the Bactericidal Activity of the Standard Anti -tuberculosis Regimen. ACS Infect Dis. 2016; 2(3):231-239 and Ruddraraju, K.V. et al. Therapeutic Targeting of Protein Tyrosine Phosphatases from Mycobacterium tuberculosis. Microorganisms 2021, 9(1), 14 incorporated herein by reference.

[0214] In an example embodiment, a method of treating Mycobacterium tuberculosis in macrophages is provided, comprising administering chimeric molecule of the present invention. In one example the small molecule comprises a MAPK kinase binder, a linker, an electrophilic reactive group, and a Mycobacterium pathogen binding moiety. In one example embodiment, themolecule comprises a binder of MAPK p38a based on the inhibitor SB203580 that further comprises an azide bio-orthogonal group. The binding moiety comprises a linker connected to the electrophilic reactive group N-acyl-N-alkyl sulfonamide. The electrophilic reactive group, in turn, is connected to the Mtb protein pathogen binding moiety for PtpA via a linker. Upon binding to the MAPK kinase via SB203580, proximal lysines of the binding pocket of p38a MAPK, KI 5, K54, K66, KI 52, KI 65, will react with the lysine-reactive group (e.g., N-acyl-N-alkyl sulfonamide) and expel the kinase inhibitor, leaving the MAPK tagged with the PtpA binder. The kinase which is covalently labeled with the PtpA binder can then hyper and / or neo-phosphorylate the Mtb PtpA proteins, which can lead to HLA-display and generation of a strong immune response against the pathogen-specific phosphopeptides, allowing deactivation and elimination of infected macrophages by the immune system. Administration of a cyclooctyne coupling molecule can quench the azide biorthogonal group displayed on the kinase binding molecule and deactivate the expelled kinase binding moiety.

[0215] In one preferred embodiment, the target protein is SapM. M. tb produces SapM, an alkaline phosphatase, for survival as it inhibits phagosome maturation in host macrophages. See e.g., Fernandez- Soto, P., et. al. Mechanism of catalysis and inhibition of Mycobacterium tuberculosis SapM, implications for the development of novel antivirulence drugs. Sci Rep 2019, 9, 10315 and Fernandez-Soto, P., et al. Discovery of uncompetitive inhibitors of SapM that compromise intracellular survival of Mycobacterium tuberculosis. Sci Rep 2021, 11, 7667. incorporated herein by reference.

[0216] In one example embodiment, the target protein is ESAT-6. M tb. produces ESAT-6 to mediate regulation of host immune responses. ESAT-6 directly inhibits T cell IFN-y production by activation of p38 MAPK and indirectly through reprogramming of antigen presenting cells to produce less IL-12p70, an essential IFN-y stimulating cytokine. Jung, BG , et al. Early Secreted Antigenic Target of 6-kDa of Mycobacterium tuberculosis Stimulates IL-6 Production by Macrophages through Activation of STAT3. Sci Rep 2017, 7, 40984.

[0217] In one example embodiment, the target protein is Rv2966c. Rv2966c is a RsmD-like methyltransferase, which carries out the transferase function through its N-terminal domain. This protein has been identified as a potential therapeutic target for its role in the function of M. tb and a conserved subunit similar to that of ATP binding pocket of kinases. The highly conserved subunitis called S-adenosyl -methionine (SAM). In one example embodiment, the Rv2966c binding moiety targets a SAM subunit. See e.g. Kumar A., et al. “Structural and Functional Characterization of Rv2966c Protein Reveals an RsmD-like Methyltransferase from Mycobacterium tuberculosis and the Role of Its N-terminal Domain in Target Recognition” J Biol Chem. 2011, 286(22): 19652-19661 and Copeland, R., et al. “Protein methyltransferases as a target class for drug discovery.” Nat Rev Drug Discov 2009, 8, 724-732, incorporated herein by reference.

[0218] Uridine 5 '-monophosphate kinase (UMPK) catalyzes the reversible transfer of the y- phosphoryl group from ATP to UMP in the presence of a divalent cation, usually Mg2+. Sequence comparisons of bacterial UMPKs show they do not resemble UMPKs from other organisms. Furthermore, bacterial UMPKs are specific for UMP and exist in solution as stable homohexamers. See e.g., RostirollaD. C.^ etal. “UMP Kinase from Mycobacterium Tuberculosis: Mode of Action and Allosteric Interactions, and Their Likely Role in Pyrimidine Metabolism Regulation.” Archives of Biochemistry and Biophysics 2011, 505 (2), 202-212.

[0219] In one aspect, the method comprises treating infection by a pathogen as a result of PsA. PsA is a gram-negative, rod-shaped bacterium that commonly causes nosocomial pneumonia. In an example embodiment, the chimeric molecule pathogen binding moiety targets the pathogen PsA and is Colistin. Colistin, a polymyxin, is sulfo-methylated and naturally ineffective antibiotic. Within the body at physiological temperature and pH, Colistin is hydrolyzed and becomes active. Colistin binds to phospholipids and disrupts the bacterial cell membrane. See e.g. Levin A. S., et al. Intravenous Colistin as Therapy for Nosocomial Infections Caused by Multidrug-Resistant Pseudomonas aeruginosa and Acinetobacterbaumannii, Clinical Infectious Diseases 1999, 28, (5), 1008-1011. Colistin binds to lipopolysaccharide in the bacterial outer membrane, and also targets lipopolysaccharide in the cytoplasmic membrane. See, e.g., Sabnis et al., 2019, Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane, eLife doi: 10.7554 / eLife.65836.Pharmaceutical Formulations

[0220] Also described herein are pharmaceutical formulations that can contain an amount, effective amount, and / or least effective amount, and / or therapeutically effective amount of one or more compounds, molecules, compositions, vectors, vector systems, cells, or a combinationthereof (which are also referred to as the primary active agent or ingredient elsewhere herein) described in greater detail elsewhere herein a pharmaceutically acceptable carrier or excipient. As used herein, “pharmaceutical formulation” refers to the combination of an active agent, compound, or ingredient with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo. As used herein, “pharmaceutically acceptable carrier or excipient” refers to a carrier or excipient that is useful in preparing a pharmaceutical formulation that is generally safe, non-toxic, and is neither biologically or otherwise undesirable, and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable carrier or excipient” as used in the specification and claims includes both one and more than one such carrier or excipient. When present, the compound can optionally be present in the pharmaceutical formulation as a pharmaceutically acceptable salt. In some embodiments, the pharmaceutical formulation can include, such as an active ingredient, a CRISPR-Cas system or component thereof described in greater detail elsewhere herein. In some embodiments, the pharmaceutical formulation can include, such as an active ingredient, a CRISPR-Cas polynucleotide described in greater detail elsewhere herein. In some embodiments, the pharmaceutical formulation can include, such as an active ingredient one or more modified cells, such as one or more modified cells described in greater detail elsewhere herein.

[0221] In some embodiments, the active ingredient is present as a pharmaceutically acceptable salt of the active ingredient. As used herein, “pharmaceutically acceptable salt” refers to any acid or base addition salt whose counter-ions are non-toxic to the subj ect to which they are administered in pharmaceutical doses of the salts. Suitable salts include, hydrobromide, iodide, nitrate, bisulfate, phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, camphorsulfonate, napthalenesulfonate, propionate, malonate, mandelate, malate, phthalate, and pamoate.

[0222] The pharmaceutical formulations described herein can be administered to a subject in need thereof via any suitable method or route to a subject in need thereof. Suitable administration routes can include, but are not limited to auricular (otic), buccal, conjunctival, cutaneous, dental,electro-osmosis, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intraarterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracistemal, intracorneal, intracoronal (dental), intracoronary, intracorporus cavernosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, occlusive dressing technique, ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, and / or vaginal administration, and / or any combination of the above administration routes, which typically depends on the disease to be treated and / or the active ingredient(s).

[0223] Where appropriate, compounds, molecules, compositions, vectors, vector systems, cells, or a combination thereof described in greater detail elsewhere herein can be provided to a subject in need thereof as an ingredient, such as an active ingredient or agent, in a pharmaceutical formulation. As such, also described are pharmaceutical formulations containing one or more of the compounds and salts thereof, or pharmaceutically acceptable salts thereof described herein. Suitable salts include, hydrobromide, iodide, nitrate, bisulfate, phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, napthalenesulfonate, propionate, malonate, mandelate, malate, phthalate, and pamoate.

[0224] In some embodiments, the subject in need thereof has or is suspected of having a cancer or a symptom thereof. In some embodiments, the subject in need thereof has or is suspected ofhaving, a neurobi ologi cal disease or disorder, a psychiatric disease or disorder, a cancer, an autoimmune disease or disorder, a thrombosis disease, a heart disease, a kidney disease, a lung disease, or a blood vessel disease, or a combination thereof. As used herein, “agent” refers to any substance, compound, molecule, and the like, which can be biologically active or otherwise can induce a biological and / or physiological effect on a subject to which it is administered to. As used herein, “active agent” or “active ingredient” refers to a substance, compound, or molecule, which is biologically active or otherwise, induces a biological or physiological effect on a subject to which it is administered to. In other words, “active agent” or “active ingredient” refers to a component or components of a composition to which the whole or part of the effect of the composition is attributed. An agent can be a primary active agent, or in other words, the component(s) of a composition to which the whole or part of the effect of the composition is attributed. An agent can be a secondary agent, or in other words, the component(s) of a composition to which an additional part and / or other effect of the composition is attributed.

[0225] Further embodiments are illustrated in the following Examples which are given for illustrative purposes only and are not intended to limit the scope of the invention.EXAMPLESExample 1 - Bifunctional Molecules for Recruiting the Complement System to Microbial Pathogens

[0226] In this work, Applicants study the effects of a series of bifunctional molecules that Applicants envisioned to specifically recruit the host’s complement immune system to the site of P. aeruginosa infection (Fig. 1 A) using a series of assays developed for this purpose. To design a molecule that recruits the complement system in the majority of people, Applicants employed the small-molecule hapten 2,4-dinitrophenyl (DNP) (Fig. IB), which is recognized by antibodies (antiDNP) that are present endogenously in a high percentage of the population14 15. This hapten has been previously shown to recruit the immune system to an infection via the formation of a hapten-pathogen complex in the presence of a bifunctional compound. The hapten-pathogen complex is then bound by the Clq protein to activate the complement cascade16,17, which ultimately leads to the formation of a membrane attack complex (MAC) that creates pores in the bacterial membrane, causing osmolysis and eventually bacterial cell lysis18,19. Here, Applicants specifically investigated the use of DNP to recruit the complement system to the site of P.aeruginosa infection. Applicants developed two assays for measuring the formation of the ternary complex between P. aeruginosa, the bifunctional compound, and anti-DNP antibodies and show that the complement system is required for bacterial cell killing using these compounds. The innovative approach thus holds therapeutic promise and offers a powerful tool for studying the complement system in the context of specific pathogens.

[0227] To determine the best agents for targeting the bacteria, Applicants synthesized two categories of bifunctional compounds that differed in their bacteria-targeting moieties while all sharing the same antibody-targeting moiety, DNP, which was used before to recruit antibodies to microorganisms (bacteria10,11, viruses20and fungi21) and cancer cells22,23,24. The first category of bifunctional compounds (compounds 1, 2, and 3) contain sugar-based ligands designed to target lectin A / lectin B P. aeruginosa surface proteins, which play a crucial role in bacterial cell adhesion and biofilm formation. For compound 1, Applicants employed an a-D-galactose sulfonamide derivative that was shown to bind to LecA with a Ki ~ 4.16 pM, authors also employed this derivative as part of bifunctional molecules.25To investigate a second ligand as well as the effects of multivalency, compounds 2 and 3 used one or two units of methyl-a-L-fucoside analogues26, respectively, which binds to LecB with a Ka of 430 nM27. As the second class of molecule tested here, bifunctional compound 4 contains a derivative of colistin-targeting liposaccharide (LPS) termed polymyxin B28, which was studied previously in the context of antibody-recruiting molecules.11Applicants used design of the compound developed by Pires and colleagues, where they showed that it is able to recruit antiDNP antibodies to E.Coli and P. aeruginosa, and had some complement-dependent effect on E.Coli, mostly focusing on the investigation of the linker optimizations to improve binary and ternary binding. In this work, Applicants take this approach further and focus on several compounds that are different in their bacterial-targeting moieties. Applicants developed two orthogonal ternary complex assays that allowed Applicants to prioritize compounds for further complement immunity-focused biological evaluation. In addition, Applicants also provide a framework for functional biological antibacterial evaluation focusing on the complement immunity recruitment and Complement-Dependent Cytotoxicity (CDC). To determine the best groups and attachment points for compounds 1, 2, 3 in this work, Applicants extensively researched the structure-activity relationship (SAR) of the ligands in bifunctionalconjugate25,26’27’29. To determine the specific linker for this work, Applicants referenced previous research on lectin probe-antibiotic conjugate generation, lectin location, and synthetic feasibility.

[0228] Effective immune recruitment to bacteria using bifunctional molecules requires successful target engagement and robust, sustained interactions between P. aeruginosa, the bifunctional molecule, and the anti-DNP antibody, which altogether is termed the ternary complex30’31’32. Most proximity-induced molecules operate within cells, bringing together two proteins or intricate protein systems, which has led to the establishment of comprehensive biophysical33-34’35and cellular36,37workflows for characterizing ternary complexes between bifunctional molecules and proteins of interest. Applicants research aimed to investigate the interaction between biomolecules on the bacterial surface, bifunctional molecules, and antibodies present in extracellular environments. Applicants evaluated ternary complex formation using two techniques: 1) flow cytometry (Fig. 2A) and 2) bio-layer interferometry (2) (Fig. 2D). Using flow cytometry to measure the binding and ternary complex formation, Applicants demonstrated superior P. aeruginosa binding with compound 4 as compared to the lectin-targeting compounds (1, 2, and 3), which did not form strong ternary complexes (Fig. 2B, 2C). As a complementary approach, Applicants employed a BLI assay to assess real-time complex formation between anti- DNP, bifunctional molecules, and whole bacterial cells38. The flow cytometry method enabled Applicants to assess the 'snapshots' of ternary complex formation, thereby facilitating the capture of only robust complexes with slow off-rates. However, this approach potentially overlooks complexes with short off-rates, typically invisible in the flow cytometry format. Consequently, Applicants chose to utilize Bio-layer Interferometry for ternary complex formation. This technique provides real-time monitoring of association and dissociation, thereby enabling the capture of transient interactions as well. Compound 4 showed clear association and dissociation phases, indicating strong ternary complex formation, while compounds 1, 2, and 3 did not exhibit observable binding (Fig. 2D). As a control, excess of the monofunctional analogs containing only the bacteria-binding moiety (5), or only DNP -binding moiety (6) interfered with ternary complex formation (Fig. 2E), emphasizing the importance of both moieties of the bifunctional molecule. These findings contribute to developing optimized bifunctional molecules, including colistin derivatives, for bacterial immune system recruitment.

[0229] As it induced the best ternary complex formation of the compounds tested, compound 4 was thus the only one carried forward for additional efficacy testing (Fig. 3). To do this, Applicants assessed the growth of P. aeruginosa in different concentrations of compound 4 (Fig. 3A, Fig. S3), with and without complement-preserved human pooled serum. As illustrated in Figure 3A, Applicants observed both serum-dependent and concentration-dependent inhibition of bacterial growth in the presence of compound 4 and complement-preserved human pooled serum. The normalized area under the curve analysis shows the reduction of growth of P. aeruginosa by more than 80 % when treated with 4 in the presence of serum compared to the absence of serum in tested conditions. As a control, the removal of the complement-binding moiety in compound 5 reduced but not completely abolish the activity, underscoring the significance of both chemical moieties for inhibiting bacterial growth (Fig. 3B). Compound 5 still maintains the antibacterial activity only in the presence of serum at 0.63 pM that might highlight the disruptive nature of colistin binding to membrane, so making it easier for the complement attack on its own39’40.

[0230] To confirm that the observed growth inhibition is mediated through the complement immunity, Applicants examined the efficacy of compound 4 at varying concentrations in the presence of serum in which the complement immunity has been heat inactivated (Fig. 4A). In the presence of this heat-inactivated serum, compound 4 did not inhibit bacterial growth at lower concentrations as was seen in complement-preserved serum, thus emphasizing the necessity of complement immunity for the toxicity of compound 4. Furthermore, compound 4 did not inhibit bacterial growth when treated with 1 pM of AMY101, an inhibitor of C3, in complement- preserved serum, indicating the essential role of C3 in the complement-mediated toxicity of compound 4 (Fig. 4A).

[0231] Applicants next examined the effect of the complement-recruiting compounds on a highly virulent reference strain of P. aeruginosa that has been useful in developing therapeutics, termed P. aeruginosa PA14, by measuring markers of complement immunity. Following a 20- minute treatment of P. aeruginosa with 50 pM of compound 4, Applicants observed the deposition of complement-marker C3b (Fig. 4B), which leads to the formation of the Membrane Attack Complex (MAC) and eventual bacterial cell lysis. Figure 4C presents fluorescence images of P. aeruginosa cells stained with Syto BC, a nucleic acid stain that highlights bacterial DNA. In the presence of compound 4 but without co-treatment with complement-containing serum, the cellularDNA remained highly concentrated within individual cells. In contrast, in the presence of both serum and compound 4, bacterial clumps exhibited diffused green fluorescence, indicative of DNA release from lysed cells (Fig. 4C). As a control, compound 4 showed no toxicity against HEK293T cells lacking LPS target (Fig. 4D). As such, the serum-mediated bacterial killing of compound 4 depends on both the presence of the colistin-targeting target and complement system recruitment.

[0232] In conclusion, this study demonstrates the potential use of bifunctional molecules to recruit the complement immune system as a promising therapeutic strategy to combat antibioticresistant and immune-evading bacterial infections. Though tested specifically against P. aeruginosa infections here, this therapeutic strategy is modular and thus easily adaptable to any pathogen with a known binding ligand. The testing platforms established herein should accelerate the discovery of complement-recruiting compounds by simplifying and streamlining the screening and testing process and will serve as valuable tools for studying the interactions between the complement system and specific pathogens. Overall, utilizing the natural defense mechanisms of the complement system and recruiting it to the site of infection has generated a novel strategy with the potential for treating any number of pathogen-related infections.References for Example 1 and Background Section(1) Poole, K. Bacterial Multidrug Resistance— Emphasis on Efflux Mechanisms andPseudomonas Aeruginosa. J Antimicrob Chemother 1994, 34 (4), 453-456. doi.org / 10.1093 / jac / 34.4.453.(2) Livermore, D. M. Multiple Mechanisms of Antimicrobial Resistance in Pseudomonas Aeruginosa: Our Worst Nightmare? Clinical Infectious Diseases 2002, 34 (5), 634-640. doi.org / 10.1086 / 338782.(3) Thakur, M.; Khushboo, null; Kumar, Y.; Yadav, V.; Pramanik, A.; Kumar Dubey, K.Understanding Resistance Acquisition by Pseudomonas Aeruginosa and Possible Pharmacological Approaches in Palliating Its Pathogenesis. Biochem Pharmacol 2023, 115689. doi.org / 10.1016 / j.bcp.2023.115689.(4) Tamma, P. D.; Aitken, S. L.; Bonomo, R. A.; Mathers, A. J.; van Duin, D.; Clancy, C. J. Infectious Diseases Society of America 2023 Guidance on the Treatment of Antimicrobial Resistant Gram-Negative Infections. Clin Infect Dis 2023, ciad428. doi.org / 10.1093 / cid / ciad428.(5) Burrows, L. L. The Therapeutic Pipeline for Pseudomonas Aeruginosa Infections. ACS Infect Dis 2018, 4 (7), 1041-1047. doi.org / 10.1021 / acsinfecdis.8b00112.(6) Soukarieh, F.; Williams, P.; Stocks, M. J.; Camara, M. Pseudomonas Aeruginosa Quorum Sensing Systems as Drug Discovery Targets: Current Position and Future Perspectives. J Med Chem 2018, 61 (23), 10385-10402. doi.org / 10.1021 / acs.jmedchem.8b00540.(7) Dunkelberger, J. R.; Song, W.-C. Complement and Its Role in Innate and Adaptive Immune Responses. Cell Res 2010, 20 (1), 34-50. doi.org / 10.1038 / cr.2009.139.(8) Bertozzi, C. R.; Bednarski, M. D. Antibody Targeting to Bacterial Cells UsingReceptor-Specific Ligands. J. Am. Chem. Soc. 1992, 114 (6), 2242-2245. doi.org / 10.1021 / ja00032a046.(9) Bertozzi, C. R.; Bednarski, M. D. A Receptor-Mediated Immune Response UsingSynthetic Glycoconjugates. J. Am. Chem. Soc. 1992, 114 (14), 5543-5546. doi.org / 10.1021 / ja00040a009.(10) Fura, J. M.; Pidgeon, S. E.; Birabaharan, M.; Pires, M. M. Dipeptide-Based Metabolic Labeling of Bacterial Cells for Endogenous Antibody Recruitment. ACS Infect Dis 2016, 2 (4), 302-309. doi . org / 10.1021 / acsinfecdis.6b00007.(11) Feigman, M. S.; Kim, S.; Pidgeon, S. E.; Yu, Y.; Ongwae, G. M.; Patel, D. S.; Regen, S.; Im, W .; Pires, M. M. Synthetic Immunotherapeutics against Gram -Negative Pathogens. Cell Chem Biol 2018, 25 (10), 1185-1194.e5. doi. org / 10.1016 / j.chembiol.2018.05.019.(12) Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The Immune System in Health and Disease, 5th ed.; New York: Garland Science, 2001.(13) Heesterbeek, D. A. C.; Angelier, M. L.; Harrison, R. A.; Rooijakkers, S. H. M. Complement and Bacterial Infections: From Molecular Mechanisms to Therapeutic Applications. J Innate Immun 2018, 10 (5-6), 455-464. doi.org / 10.1159 / 000491439.(14) Jakobsche, C. E.; Parker, C. G.; Tao, R. N.; Kolesnikova, M. D.; Douglass, E. F.;Spiegel, D. A. Exploring Binding and Effector Functions of Natural Human Antibodies Using Synthetic Immunomodulators. ACS Chem Biol 2013, 8 (11), 2404-2411. doi.org / 10.1021 / cb4004942.(15) Sheridan, R. T. C.; Hudon, J.; Hank, J. A.; Sondel, P. M.; Kiessling, L. L. Rhamnose Glycoconjugates for the Recruitment of Endogenous Anti-Carbohydrate Antibodies to Tumor Cells. Chembiochem 2014, 15 (10), 1393-1398. doi.org / 10.1002 / cbic.201402019.(16) Stokol, T.; O’Donnell, P.; Xiao, L.; Knight, S.; Stavrakis, G.; Botto, M.; von Andrian, U. H.; Mayadas, T. N. Clq Governs Deposition of Circulating Immune Complexes and Leukocyte Fcgamma Receptors Mediate Subsequent Neutrophil Recruitment. J Exp Med 2004, 200 (7), 835— 846. doi.org / 10.1084 / jem.20040501.(17) Venkatraman Girija, U.; Gingras, A. R.; Marshall, J. E.; Panchai, R.; Sheikh, M. A.; Harper, J. A. J.; Gal, P.; Schwaeble, W. J.; Mitchell, D. A.; Moody, P. C. E.; Wallis, R. Structural Basis of the Clq / Cls Interaction and Its Central Role in Assembly of the Cl Complex of Complement Activation. Proc Natl Acad Sci U S A 2013, 110 (34), 13916-13920. doi.org / 10.1073 / pnas.1311113110.(18) Miiller-Eberhard, H. J. The Killer Molecule of Complement. J Invest Dermatol 1985, 55 (1 Suppl), 47s-52s. doi.org / 10.l l l l / 1523-1747.epl2275445.(19) Menny, A.; Serna, M.; Boyd, C. M.; Gardner, S.; Joseph, A. P.; Morgan, B. P.; Topf, M.; Brooks, N. J.; Bubeck, D. Cry oEM Reveals How the Complement Membrane Attack Complex Ruptures Lipid Bilayers. Nat Commun 2018, 9 (1), 5316. doi.org / 10.1038 / s41467-018-07653-5.(20) Parker, C. G.; Domaoal, R. A.; Anderson, K. S.; Spiegel, D. A. An Antibody-Recruiting Small Molecule That Targets HIV Gpl20. J Am Chem Soc 2009, 131 (45), 16392-16394. doi.org / 10.1021 / ja9057647.(21) Chirkin, E.; Muthusamy, V.; Mann, P.; Roemer, T.; Nantermet, P. G.; Spiegel, D. A. Neutralization of Pathogenic Fungi with Small-Molecule Immunotherapeutics. Angew Chem Int Ed Engl 2017, 56 (42), 13036-13040. doi.org / 10.1002 / anie.201707536.(22) Jakobsche, C. E.; McEnaney, P. J.; Zhang, A. X.; Spiegel, D. A. Reprogramming Urokinase into an Antibody -Recruiting Anticancer Agent. ACS Chem Biol 2012, 7 (2), 316-321. doi.org / 10.1021 / cb200374e.(23) Rullo, A. F.; Fitzgerald, K. J.; Muthusamy, V.; Liu, M.; Yuan, C.; Huang, M.; Kim, M.; Cho, A. E.; Spiegel, D. A. Re-Engineering the Immune Response to Metastatic Cancer: Antibody-Recruiting Small Molecules Targeting the Urokinase Receptor. Angew Chem Int Ed Engl 2016, 55 (11), 3642-3646. doi.org / 10.1002 / anie.201510866.(24) Schrand, B.; Clark, E.; Levay, A.; Capote, A. R.; Martinez, O ; Brenneman, R ; Castro,I.; Gilboa, E. Hapten-Mediated Recruitment of Polyclonal Antibodies to Tumors Engenders Antitumor Immunity. Nat Commun 2018, 9 (1), 3348. doi.org / 10.1038 / s41467-018-05566-x.(25) Meiers, J.; Zahorska, E.; Rbhrig, T.; Hauck, D.; Wagner, S.; Titz, A. Directing Drugs to Bugs: Antibiotic-Carbohydrate Conjugates Targeting Biofilm-Associated Lectins of Pseudomonas Aeruginosa. J Med Chem 2020, 63 (20), 11707-11724. doi.org / 10.1021 / acs.jmedchem.0c00856.(26) Sommer, R.; Rox, K.; Wagner, S.; Hauck, D.; Henrikus, S. S.; Newsad, S.; Arnold, T.;Ryckmans, T.; Brbnstrup, M.; Imberty, A.; Varrot, A.; Hartmann, R. W .; Titz, A. Anti-Biofilm Agents against Pseudomonas Aeruginosa: A Structure-Activity Relationship Study of C- Glycosidic LecB Inhibitors. J Med Chem 2019, 62 (20), 9201-9216. doi . org / 10.1021 / acs.j medchem ,9b01120.(27) Sommer, R.; Exner, T. E.; Titz, A. A Biophysical Study with Carbohydrate Derivatives Explains the Molecular Basis of Monosaccharide Selectivity of the Pseudomonas Aeruginosa Lectin LecB. PLOS ONE 2014, 9 (11), el 12822. doi.org / 10.1371 / journal.pone.0112822.(28) Domingues, M. M.; Inacio, R. G.; Raimundo, J. M.; Martins, M.; Castanho, M. A. R. B.; Santos, N. C. Biophysical Characterization of Polymyxin B Interaction with LPS Aggregates and Membrane Model Systems. Biopolymers 2012, 98 (4), 338-344. doi. org / 10.1002 / bip.22095.(29) Sommer, R.; Wagner, S.; Rox, K.; Varrot, A.; Hauck, D.; Wamhoff, E.-C.; Schreiber,J.; Ryckmans, T.; Brunner, T.; Rademacher, C.; Hartmann, R. W.; Bronstrup, M.; Imberty, A.;Titz, A. Glycomimetic, Orally Bioavailable LecB Inhibitors Block Biofilm Formation of Pseudomonas Aeruginosa. J Am Chem Soc 2018, 140 (7), 2537-2545. doi.org / 10.1021 / jacs.7bl 1133.(30) Modell, A. E.; Lai, S.; Nguyen, T. M.; Choudhary, A. Bifunctional Modalities forRepurposing Protein Function. Cell Chem Biol 2021, 28 (7), 1081-1089. doi.org / 10.1016 / j.chembiol.2021.06.005.(31) Singh, S.; Tian, W .; Severance, Z. C.; Chaudhary, S. K.; Anokhina, V.; Mondal, B.; Pergu, R.; Singh, P.; Dhawa, U.; Singha, S.; Choudhary, A. Proximity-Inducing Modalities: The Past, Present, and Future. Chem Soc Rev 2023. doi.org / 10.1039 / d2cs00943a.(32) Wurz, R. P.; Rui, H.; Dellamaggiore, K.; Ghimire-Rijal, S.; Choi, K.; Smither, K.; Amegadzie, A.; Chen, N.; Li, X.; Banerjee, A.; Chen, Q.; Mohl, D.; Vaish, A. Affinity and Cooperativity Modulate Ternary Complex Formation to Drive Targeted Protein Degradation. Nat Comnnrn 2023, 14 (1), 4177. doi.org / 10.1038 / s41467-023-39904-5.(33) Roy, M. J.; Winkler, S.; Hughes, S. J.; Whitworth, C.; Galant, M.; Famaby, W .; Rumpel, K.; Ciulli, A. SPR-Measured Dissociation Kinetics of PROTAC Ternary Complexes Influence Target Degradation Rate. ACS Chem Biol 2019, 14 (3), 361-368. doi . org / 10.1021 / acschembio.9b00092.(34) Schiemer, J.; Horst, R.; Meng, Y.; Montgomery, J. I.; Xu, Y.; Feng, X.; Borzilleri, K.; Uccello, D. P.; Leverett, C.; Brown, S.; Che, Y.; Brown, M. F.; Hayward, M. M.; Gilbert, A. M.; Noe, M. C.; Calabrese, M. F. Snapshots and Ensembles of BTK and CIAP1 Protein Degrader Ternary Complexes. Nat Chem Biol 2021, 17 (2), 152-160. doi.org / 10.1038 / s41589-020-00686- 2.(35) Przytulski, K.; Glaza, P.; Brach, K.; Sagan, M.; Statkiewicz, G.; Klajn, J.; Walczak, M. J. Comparative Analysis of Biophysical Methods for Monitoring Protein Proximity Induction in the Development of Small Molecule Degraders. Biochim Biophys Acta Gen Subj 2023, 1867 (9), 130398. doi. org / 10.1016 / j.bbagen.2023.130398.(36) Cotnoir-White, D.; El Ezzy, M.; Boulay, P.-L.; Rozendaal, M.; Bouvier, M.; Gagnon, E.; Mader, S. Monitoring Ligand-Dependent Assembly of Receptor Ternary Complexes in Live Cells by BRETFect. Proc Natl Acad Sci U S A 2018, 115 (11), E2653-E2662. doi . org / 10.1073 / pnas.1716224115.(37) Shyu, Y. J.; Suarez, C. D.; Hu, C.-D. Visualization of Ternary Complexes in Living Cells by Using a BiFC-Based FRET Assay. Nat Protoc 2008, 3 (11), 1693-1702. doi.org / 10.1038 / nprot.2008.157.(38) Verzijl, D.; Riedl, T.; Parren, P. W. H. I.; Gerritsen, A. F. A Novel Label-Free Cell- Based Assay Technology Using Biolayer Interferometry. Biosens Bioelectron 2017, 57, 388-395. doi. org / 10.1016 / j. bios.2016.08.095.(39) Ontong, J. C.; Ozioma, N. F.; Voravuthikunchai, S. P.; Chusri, S. Synergistic Antibacterial Effects of Colistin in Combination with Aminoglycoside, Carbapenems, Cephalosporins, Fluoroquinolones, Tetracyclines, Fosfomycin, and Piperacillin on MultidrugResistant Klebsiella Pneumoniae Isolates. PLoS One 2021, 16 (1), e0244673. doi.org / 10.1371 / journal.pone.0244673.(40) Weber, B. S.; De Jong, A. M.; Guo, A. B. Y.; Dharavath, S.; French, S.; Fiebig-Comyn, A. A.; Coombes, B. K.; Magolan, J.; Brown, E. D. Genetic and Chemical Screening in Human Blood Serum Reveals Unique Antibacterial Targets and Compounds against Klebsiella Pneumoniae. Cell Rep 2020, 32 (3), 107927. doi.org / 10.1016 / j.celrep.2020. 107927.Example 2 - Bac Titer Gio AssayAssay protocol:

[0233] 1. Prepare 400 nl of compound solutions in DMSO in a 384 white-bottom plate8 different concentrations: 20 uM, 10 uM, 5 uM, 2.5 uM, 1.25 uM, 0.625 uM, 0.313 uM, 0.156 uMUse compound printer to prepare dilutions from 10 mM compound stocks in DMSO2. Add 40 ul cells at OD600 = 0.1 in M9 media containing 20% serum or 20% heat- inactivated serum (1% DMSO in the assay)3. Incubate for 3 h at 37°C4. Add 20 ul of BacTiter-Glo reagentShake plate for 5 min to complete the lysis5. Read luminescence in Envision plate reader

[0234] 20% regular / heat-inactivated serum in the assayExample 3

[0235] Specific Aims. The rise of antibiotic-resistant infections threatens modern medicine, underscoring the need for novel therapeutic modalities.1'3The complement system is host’s first line of defense against infection that triggers bacterial cytolysis / opsonization and recruits macrophages for phagocytosis (FIGs. 44A, 62A, and 67A).4’5Applicants propose to build chimeric molecules formed by fusion of binders of bacteria surface (blue ball, FIGs. 44A, 62A, and 67A) and complement protein or its recruiter (green square, FIGs. 44A, 62A, and 67A). Such chimeras will recruit immune system to bacteria at high local concentrations leading to bacterial clearance by a natural mechanism.

[0236] The complement system operates via three pathways.6'8The Classical pathway is activated upon complex formation between Clq and the antibody bound to the pathogen. Themannan-binding lectin (MBL) pathway is activated by the binding of MBL to mannose on bacteria. The Alternate pathway activation involves spontaneous hydrolysis of C3 protein to C3b, which binds to the bacterial surface. Following pathway activation, an enzymatic cascade leads to cytolysis and / or recruitment of macrophages. Through the aims below, Applicants will develop six types of chimeras (four are already validated) that selectively activate these three complement pathways (FIGs. 44B, 62B,C, and 67B). To enable chimera optimization by understanding their mode of action, Applicants will build a MicroMap platform (FIGs. 44D, 62E, and 67D) that allows labeling of proteins in the vicinity of the bacterial surface (vs. bulk phase), enabling capture of the dynamics of complement cascade with high spatiotemporal resolution.Aim 1. Optimization of chimeras that recruit Cl a (via IsG) to bacteria.

[0237] Applicants developed robust assays for complement-directed cytolysis (CDC) and ternary-complex formation (bacteria: chimera: complement pathway effector). Using these assays and known bacterial surface binders (e.g., colistin, murepavidin), Applicants generated and validated three classes of chimeras: Class la that recruits serum’s dinitrophenyl (DNP) antibodies; Class lb that recruits total IgG using an Fc-binder These chimeras exhibited low minimum inhibitory concentration (MIC « 0.6-1.2μM) on Pseudomonas aeruginosa (PsA) and E. coll and > 120-fold selectivity for bacterial killing over human cells; and Class Ic that uses an engineered Fc with high affinity and specificity for Clq.

[0238] Next, Applicants will perform medicinal chemistry optimization of binders and linkers in these chimeras to prioritize those that perform well in both ternary complex and cytolysis assays (FIGs. 44C and 62D). To select chimeras with diverse mechanisms, Applicants will apply a MicroMap platform (FIGs. 44D and 62E) to precisely map the complement cascade.Aim 2. Development of chimeras that directly recruit Clq, C3 or MBL to bacteria.

[0239] Using a DNA-encoded library (DEL) screening, Applicants have identified C3 and Clq binders and developed chimeras that exhibit MIC of 150-300 nM. Next, Applicants will discover ligands for MBL (for lectin pathway activation) and perform additional medicinal chemistry optimization of chimeras developed from Clq and C3 binders using the workflow described in Aim 1. By directly recruiting the initiators of the three pathways, these chimeras will enable targeted and selective activation of classical, alternate, and lectin pathway on bacteria.Aim 3. Recruitment of macrophages and in vivo efficacy studies.

[0240] While Aims 1 and 2 focus on cytolysis, the complement system can induce phagocytosis, further amplifying the immune response.9’10Following the workflow in Aims 1 and 2, Applicants will test the chimeras synthesized above in phagocytosis and chemotaxis assays and understand their mechanism using the MicroMap platform. Applicants will test chimeras optimized in Aims 1-3 in for bacterial-to-human cell selectivity (FIGs. 44E and 62G) and activity in antibiotic-resistant strains. Finally, Applicants will test the most optimum chimera for in vivo efficacy in lung infection model using E. coli (FIGs. 44F and 62G).

[0241] By allowing precise control of the complement pathways, Applicants envision these chimeras and MicroMap platform will accelerate the fundamental understanding of the roles of complement components and / or receptors in the initiation, magnitude, maintenance, and quality of immune responses involved in pathogenic infections.A. SignificanceA.1. There is an urgent need to discover novel antimicrobial modalities.

[0242] As the effectiveness of current antibiotics wanes due to the rapid emergence of drugresistant strains, the need for novel antimicrobial modalities has become increasingly urgent. These modalities will not only help prevent the catastrophic consequences of untreatable infections but also safeguard the achievements of modem medicine, including complex surgeries, organ transplantation, and cancer chemotherapy that rely on effective antimicrobials.A.2. Proximity-inducing chimeras are providing novel therapeutic modalities.

[0243] Classical therapeutic modalities operate by inhibiting the target protein’s function (e.g., enzyme inhibition) via “occupancy-driven pharmacology.” Here, the therapeutic effect requires the inhibitor to remain bound to the target protein (Fig. 45A). Molecular chimeras, which are formed by connecting binders of two target proteins (Fig. 45B), are emergent modalities that operate via “event-driven pharmacology.”11'13Here, the therapeutic effect arises from the event arising from the proximity between the two proteins induced by the chimera. For example, Proteolysis-targeting chimeras (PROTACs) induce proximity between ubiquitin ligase and target protein, leading to the latter’s ubiquitination and degradation.14 15The event-driven nature allows one chimera molecule to act on multiple molecules of the target protein (i.e., exhibit turnover),lowering the dose required for the therapeutic effect. Furthermore, the binder of the target protein need not be inhibitory or of ultra-high-affinity, which lowers turnover.

[0244] Applicants will develop a new class of chimeras that will recruit the host’ s complement system to the bacterial surface and the data suggests that the “event-driven pharmacology” may have distinct advantages. First, these chimeras exhibit turnover and require low-affinity binders of target proteins that can be discovered easily. Conveniently, several low-affinity, clinical-grade binders for bacterial surface already exist.16’17By leveraging the turnover feature and requirement to flow-affinity binders, Applicants successfully attained low MIC (150-300 nM) using complement binders with moderate binding affinities (> 5 pM) [see C.2.1.3], Second, the chimeras evade resistance mechanisms encountered by common antibiotics, which often operate by “occupancy-driven mechanisms.” For example, the complement-recruiting chimeras were effective against polymyxin-resistant strains (see C.3.1.1), while “occupancy-driven” polymyxin was not. Chimeras’ extracellular mechanism of action will render them insensitive to efflux pumps. Third, the proposed chimeras are highly modular, allowing swapping one bacterial surface binder with another. For example, Applicants could rapidly generate chimeras that target PsA and E. coli or just PsA by swapping bacterial surface binders (see C.l.1.2).A.3. The complement proteins are ideal targets for the development of chimeras for several reasons:A.3.1. Natural, broad-spectrum, and self-amplifying mechanism of action:

[0245] Developing PROTACs for proteins naturally degraded by the ubiquitin-proteasome pathway is easier than for those that are not.18 19Likewise, chimera-mediated amplification of bacterial clearance by the complement pathway, which naturally neutralizes a wide range of pathogens, should be easier. Furthermore, the complement pathway involves a self-amplifying enzymatic cascade for cytolysis and recruitment of other components of the immune system (e.g., macrophages, neutrophils, B cells).4,5Thus, precise and targeted activation of the pathway can trigger bacterial clearance.A.3.2. High abundance in the body fluids and tissues:

[0246] Complement proteins are present at high micromolar concentrations in serum (Fig. 45C)20and challenges in achieving effective complement inhibition are often attributed to their high concentrations.21Availability of complement proteins is, therefore, not a limiting factor inthe context of complement-recruiting chimeras and engaging complement will likely not perturb its natural functions. The high concentration further reduces the need for high-affinity binders and may also reduce the required dosing of the chimeras as suggested by the preliminary studies.A.3.3. Extracellular mechanism-of-action.

[0247] Extracellular targeting broadens the scope of chemical matter from small molecules to peptides and even proteins.22Restricting chimeras to the extracellular space also circumvents toxicity arising from engaging intracellular proteins of the host, thereby widening the therapeutic window.A.3.4. Globular proteins with available crystal structures.

[0248] Complement proteins are typically large, globular and with available crystal structures.23'27These features improve the chances of finding binders and to leverage the structure- guided ligand design and optimization approaches (see C.2.1.2).A.4. The proposed studies will advance the fundamental understanding of complement biology.

[0249] Studies with the complement-recruiting chimeras will help accelerate the understanding of the roles of complement components and / or receptors in the initiation, magnitude, maintenance, and quality of immune responses involved in pathogenic infections. For example, specific modulation of complement pathways by specific classes of chimeras and deployment of an unbiased MicroMap platform to capture the downstream effects will aid in breaking down the inherent complexity and redundancy in the complement system and tease apart the role of individual complement components.B. InnovationB.l. First-in-class antimicrobial chimeras.

[0250] Approved antibiotics typically target bacterial cell wall, protein, DNA, RNA, mycolic acid, or folic acid synthesis and resistant strains to these mechanisms already exist.28The complement-recruiting chimeras introduce a fundamentally distinct antimicrobial mechanism by amplifying the host’s defense capabilities and exhibiting modularity and turnover.B.2. First-in-class non-inhibitory binders of complement proteins.

[0251] The proposed studies will furnish first-in- class non-inhibitory binders to complement proteins, which can be deployed for several other purposes. For example, these binders can be usedto recruit complement system to cancer cells to induce cytolysis.29Furthermore, these binders can be used to degrade complement proteins using Lysosome-targeting chimeras (LYTACs).30-31Such degradation may be useful for scenarios of excessive complement activation (e.g., inflammatory bowel disease)32-33and this targeted degradation may prove advantageous over occupancy-driven inhibitors, owing to the high concentration of complement proteins.B.3. A DEL screening platforms for rapid discovery of binders.

[0252] The studies will refine the DEL-based ligand discovery platform, which includes a diversity-oriented synthesis (DOS) library, data analysis and visualization app, and a platform to rapidly identify ligand binding pockets. The DOS library includes millions of diverse, natural product-like small molecules that have successfully furnished several anti-infectives.34'39These platforms will allow democratization of ligand discovery efforts.B.4. Development of reagents and methods for monitoring signaling cascade on bacterial surface.

[0253] Mapping signaling cascade on the bacterial surface with high spatiotemporal resolution is non-trivial owing to their small size (1-2 pM). Commonly used proximity-labeling approaches (e g., APEX, TurboID) lack such resolution as they create long-lasting labeling species ( / 1 / 2 = 0.1 ms) that diffuse to large distances from the site being monitored.40’41Applicants will develop a MicroMap-based platform42'45that uses dexter energy transfer to activate diazirine probes with ultrashort half-lives (Z1 / 2 = 1 ns), resulting in =100 nm resolutions. These methods will further the understanding of complement system and provide valuable tools and reagents to those studying bacterial surface phenomenon.B.5. A highly multidisciplinary team.

[0254] The proposed research brings in tools and principles from diverse disciplines, including medicinal and organic chemistry, biochemistry, biophysics, chemoproteomics, microbiology, immunology, and infectious disease. Dr. Amit Choudhary’s (AC) team has expertise in development of chimeras,46'53high-throughput and robust assays (including co-culture assays),?4'60and antimicrobial agents.61 Dr. Arnab Chattel] ee’s (ArC) team has deep expertise in medicinal chemistry, microbiology and immunology. Applicants will be joined by Dr. Aly Shamji (AS, physician-scientist and expert in complement biology), who has guided the trainees to generate the preliminary data described below. Applicants will work with Dr. Steve Carr (Proteomics Platform,Broad Institute, see letter) to develop the MicroMap platform and continue collaborating with Prof. Laura Kiessling (MIT Chemistry, Broad Institute) for testing of chimeras in PsA.C. Approach.Aim 1. Optimization of chimeras that recruit Clq (via JgG) to bacteria.

[0255] Applicants will now optimize the three classes of chimeras that have different complement pathway effectors: Class la (anti-DNP) and Class lb (total IgG). Applicants will prioritize chimeras using three sets of assays that report on: 1) ternary complex (i.e., upstream activity), 2) complement deposition and MicroMap-based pathway mapping (i.e., midstream activity), and 3) bacterial lysis (i.e., downstream activity).C.1.1. Preliminary data.C.1.1.1. Chimera 1 induces ternary complex between anti-DNP and Pseudomonas aeruginosa (PsA).

[0256] Applicants synthesized several Class la chimeras consisting of DNP conjugated to either a Lee A binder or polymyxin B (PMBN) nonapeptide that binds to lipopolysaccharides (LPS) on bacteria without inducing lysis.62’63The PMBN chimeras (1, Fig. 46A) outperformed the Lee A conjugate (data not shown) and was chosen for further characterization. To demonstrate the ternary complex between anti-DNP, 1, and PsA (PA14 strain), Applicants developed a bio-layer interferometry (BLI) assay (Fig. 46B). Briefly, anti-DNP is immobilized on the biosensor tip, and chimera 1 induced capture of

[0257] PsA (i.e., association) and subsequent dissociation in the buffer is monitored. Chimera 1 showed a rapid association and slow dissociation, indicating the formation of a strong ternary complex (Fig. 46C). Such association and dissociation were not observed without either of the three components. Furthermore, Applicants observed competition when half of the chimera (i.e., PBMN nonapeptide or DNP) were used in excess (Fig. 46C). Finally, Applicants confirmed the ternary complex in an orthogonal assay using flow cytometry where anti- DNP is labeled with a fluorophore — 1 resulted in a higher percentage of fluorophore-labeled PsA compared to the controls (Fig. 46D). Working with Prof. Laura Kiessling’s laboratory, Applicants confirmed the formation of a ternary complex between anti-DNP , 1, and various clinical isolates of PsA that represent diverse sources of infections (Fig. 46E,F), suggesting that Class la chimeras can target clinical isolates from diverse infections.C. l.1.2. Chimera 1 induces complement-dependent cytotoxicity (CPC) of PsA

[0258] Applicants evaluated the ability of 1 to induce CDC in a growth inhibition assay using complement-preserved serum (vs. normal serum where complement is typically deactivated). Chimera 1 inhibited PsA growth in a dose-dependent fashion (MIC « 625 nM) only in presence of serum (Fig. 47A). By comparison, growth inhibition of PMBN lacking the DNP (i.e., control compound 3) was considerably lower (Fig. 47B). Furthermore, heat-inactivated serum or complement inhibitor (i.e., C3 inhibitor AMY-101)64-66 lowered the activity of 1 (Fig. 47C). Treatment ofPsA with 1 and serum showed deposition of the complement protein C3b (Fig. 47D), which triggers the formation of membrane attack (MAC) complex and induce bacterial lysis. Importantly, 1 did not induce cytotoxicity in HEK293T cells even at 80 pM, demonstrating >120-fold bacterial selectivity (Fig. 47E).

[0259] To demonstrate the modularity of this approach, Applicants replaced the LPS binder with murepavadin (i.e., chimera 4, Fig. 47F) that binds to the lipopolysaccharide transport protein D (LptD) of PsA.67-70 Like 1, chimera 4 induced ternary complex PsA and inhibited growth with an MIC ~ 2.5 pM (Figs. 47G,H). Like 1, the ternary complex formation was impaired upon adding competitor murepavadin or DNP. While PMBN non-selectively binds to multiple LPS-producing gram-negative bacteria, murepavadin selectively targets P&4.71,67 As such, chimera 4 did not show any CDC towards E.coli (Fig. 471). Together, these studies strongly support the hypothesis that targeted complement activation on bacteria can induce lysis without being toxic to human cells.C.1.1.3. Validation o f Class Ib-c chimeras.

[0260] For Class lb chimera, Applicants envisioned using Fc-III peptide (Fig. 48A)72 73that binds to IgG’s Fc region with high affinity (KD = 185 nM) — this binder may be advantageous over DNP as the total IgG concentration is less prone to intersubject variation. Applicants synthesized the Fc-III- based chimera 5 by linking the Fc-III peptide to PMBN nonapeptide (Fig. 48A). BLI confirmed ternary complex between 5, IgG, and PsA (Fig. 48B), and competitors disrupt ternary complex (Fig. 48B) and chimera 5 showed dose-dependent growth inhibition of PsA in the presence of 5% complement preserved human serum, with a MIC -1.25 pM (Fig. 48C).C. 1.1.4. Development CPC and ternary-complex metrics.

[0261] Identifying Class la-c chimeras with on- mechanism activity requires robust metrics that characterize CDC and ternary-complex formation. For high-throughput and quantitative evaluation of CDC of multiple chimeras, Applicants established a 384-well plate assay (Fig. 49A,B) in which bacteria growing in the log phase (ODeoo ~ 0.4) are diluted to an OD600 of 0.1 and placed in M9 media to assure viability while preventing bacterial growth. Chimeras are added together with human serum, incubated for 3 h and viable cells are detected using a luminescent ATP-Glo assay (BacTiter-Glo) — here BacTiter-Glo measures ATP levels that fuel luciferasedependent conversation of luciferin to oxyluciferin and emits light (Fig. 49A).

[0262] To evaluate the assay’s robustness, Applicants synthesized thirteen Class la and lb chimeras with different linker lengths (i.e., varied number of PEGs) and measured E. coli viability at six different chimera concentrations. Since complement-preserved serum was highly potent at killing E. coli, Applicants used 20% of partially deactivated human serum (Fig. 49C) to differentiate the activity of chimeras with different linkers. Taking inspiration from Basu et al.73who profiled -240 cancer cell lines against 12-point doses of 354 inhibitors, Applicants used the area-under-curve (AUC) metric that allows for comparison of dose-responses of chimeras. Here, an increased cytotoxicity of Class la chimeras was observed with shorter linkers, while Class lb chimeras exhibited an inverted bell-shaped linker length dependency. This linker dependence demonstrates distinct design principles for different chimeras and validates the ability of the assay to rapidly assess CDC. Next, Applicants demonstrated the ternary complex formation of Class la chimeras using BLI (Fig. 49D). Describing the ternary complex formation as chimera-induced association and dissociation rates of bacteria, Applicants can extract k,mik^n metric describing the upstream activity of chimeras (Fig. 49E). Comparison of this upstream metric (ternary complex) with downstream CDC activity can help identify on mechanism chimeras.C.1.2. Experimental planC.1.2.1. Design and synthesis o f chimeras.

[0263] Building upon these studies confirming ternary complex formation and CDC for Class la-c chimeras onE.coli and / or PsA, Applicants will systematically evaluate the contribution of IgG recruiter, linker and bacteria binder, allowing improvement of CDC (FTGs. 50A-D and 64A-D). Previous work with chimeras (e.g., PROTACs) showed that ternary- complex formation,degradation activity and specificity depends on linkers’ length, rigidity, and composition.76’77Furthermore, since the linker has to traverse through the heavily glycosylated extracellular space of the bacterial membrane,78its physiochemical properties are likely important. Thus, Applicants will evaluate different PEG linkers to find the optimal distance for complement recruitment and Applicants will also introduce rigid functional groups into the linkers that can affect the orientation of antibody- and pathogen-binders (FIGs. 50B and 64B). Applicants will incorporate piperazines, pyrrolidines, spirocyclic diamines, and triazoles to rigidify the linkers and alter the linker hydrophobicity by introducing hydrocarbon chains and aromatic spacers. Depending on attachment sites and functional groups, Cu-catalyzed click chemistry, HATU-based amidation, reductive amination, or A-alkylation will be used to conjugate the linkers. These linker optimization strategies mirror those Applicants and others have used for PROTACs to improve activity, specificity, and in vivo pharmacokinetic attributes.77’79(AC, ArC, Yrs 1-2).

[0264] To target endogenous antibodies, several groups, have used a-gal, L-rhamnose, and DNP.80’81Most humans have 2-8% anti-a-gal antibodies, and ~1% of anti-DNP antibodies.82'84’81Rhamnose is preferred for chimeras since it is a monosaccharide as compared to the trisaccharide, a-Gal. Additionally, rhamnose (Rha) is more immunogenic and anti-Rha antibodies are more widely distributed.85-86Because antibody binding to oc-Gal or Rha is relatively weak (micro- millimolar ATd’s), multivalency is necessary to recruit these antibodies.87'89While DNP is a well- established hapten, the low abundance of anti-DNP antibodies in circulation and its strong interactions with membranes and albumins may reduce its therapeutic potential.90'92To further explore Class la chimeras, Applicants will synthesize chimeras containing a-l-rhamnose (aRha), the epitope for the highly abundant anti-Rha antibodies (Fig. 50D). Finally, for Class lb chimeras, the Fc-III peptide offers two linker attachment sites that may alter the surface display orientation (Fig. 50D). (AC, ArC, Yrs 1-2)C.l.2.2. Determination of a ternary complex and CPC metric.

[0265] Applicants will evaluate CDC of the library of Class la-c chimeras in E.coli using the ATP-glo assay while probing for the formation of ternary complexes using BLI in parallel. This will allow Applicants to evaluate both upstream and downstream activities (Fig. 51 A). CDC of Class la-c chimeras will be measured as dose-response in an 8-point, 2-fold dilution series starting from 20 pM using pooled human serum. The assay will be performed in serum (activecomplement) and heat-inactivated serum or C3- inhibited serum (inactive complement) to distinguish complement-dependent and -independent cytotoxicity. Taking inspiration from Basu et al.75, Applicants can describe the effective cytotoxicity difference in active vs. inactive serum as the area under percent viability curves (AUC viability) and thereby characterize CDC in a single metric that is comparable between multiple compounds and chimeras. In parallel, Applicants will assess the formation of ternary complexes by immobilizing antibodies (Class la-b chimeras) and Clq (Class Ic) and measure chimera-induced association / dissociation cfcE.coli. Chimeras showing both CDC and ternary complex formation are on- mechanism, while any toxicity in the absence of ternary complex classifies as an off-target mechanism (Fig. 51 A). The off-target mechanism is possible as some of the hydrophobic linkers (or hydrophobic DNP) can enhance the membrane residence time of PMBN analogs and may cause off-target toxicity and Applicants wish to exclude such chimeras.91’92Chimeras showing a ternary complex without CDC will be included as controls alongside on-mechanism chimeras for deep mechanism-of-action studies described below. (AC, AS, Yrs 2-3)C.1.2.3. Confirmation of CDC mechanism.C.1.2.3.1. Targeted approach:

[0266] Applicants will measure the deposition of complement marker, C3b on the pathogen’s surface using flow cytometry as done above (Fig. 47D).94Applicants have demonstrated that treatment of PsA with chimera 1 in the presence of serum leads to deposition of C3b (Fig. 46D) and anticipate that the degree of C3b deposition will directly correlate with cell lysis for on-target chimeras. Additionally, Applicants will measure levels of sC5b-C9 complex in the serum using a standard ELISA.93Elevated levels of this soluble terminal complex in the serum indicate complement activation. Finally, Applicants will attempt to determine the presence of membraneattack complex (MAC) on the bacterial surface using reported methods.96(AC, AS, Yrs 2-3) C.1.2.3.2. Unbiased approach:

[0267] The preliminary studies indicate that, in some cases, a high degree of ternary complex formation may not correlate with the chimera’s functional ability to induce cell lysis (Fig. 49C-E). Furthermore, Applicants need a metric for selection of chimeras with diverse mechanisms. For example, bacteria often recruit complement inhibitory factors (e.g., Factor H) in the serum to resist CDC,97which can be monitored using proximity-labeling strategies. Therefore, an unbiased andhigh-resolution examination of the bacterial cell surface in the presence / absence of chimeras can help fully understand the mechanism and aid chimera optimization.

[0268] Applicants will employ MicroMap platform42'45that labels proteins near cell surface (vs. those present in the bulk solution) with high spatiotemporal resolution (- 100 nm). In MicroMap an antibody that binds to cell surface marker (e.g., LPS or rhamnose for bacteria) is conjugated to an Iridium photocatalyst (Fig. 5 IB). Upon irradiation with light, a photocaged biotin (bears aryl-azide or diazirine) proximal to the catalyst is activated, resulting in biotinylation of proximal proteins. MicroMap attains high spatiotemporal resolution by two mechanisms. First, the activation of biotin for protein labeling occurs via Dexter transfer from catalyst to photocaged biotin (i.e., not directly by light) and this energy transfer occurs ~10 A radius, allowing highly localized photoactivation and biotinylation. Second, while chemoenzymatic proximity labeling approaches98-40generate species with high half-lives (tm = 0.1 ms), MicroMap employs reactive species with ultrashort half- lives (6 / 2 = 1 ns) that lowers diffusion radius (<10 nm). Thus, this platform has enabled high-resolution mapping in several applications, including profding immunosyanpse proteins, mapping histone reorganization on chromatin, and identifying smallmolecule protein targets.43

[0269] Since MicroMap has not been applied to bacteria that are small 1-2 pm long), Applicants will adapt this platform to understand the molecular basis of CDC efficacy of the chimeras. Briefly, Applicants will append the Iridium photocatalyst to an antibody for the surface marker (e.g., LPS or rhamnose, Fig. 51C). Next, Applicants will treat E. coli with this conjugate in the presence / absence of chimeras and complement-preserved serum. Irradiation will be performed at different time points and the degree of biotinylation will be quantified first by immunoblotting (and subsequently by MS / MS to identify proteins). Applicants will work closely with Dr. Steve Carr (Proteomics Platform, Broad Institute, see letter) whose laboratory working with Dr. Alice Ting has pioneered several proximity-labeling strategies. (AC, Yrs 2-3) C.1.3. Potential pitfalls and proposed solutions.

[0270] Throughout the aim, Applicants describe several alternative strategies (e.g., three chimeras to recruit Clq, > 15 linker strategies, and two orthogonal approaches to understand the mechanism) should any one fail. Applicants have successfully established key assays / metrics, including those for complement deposition, ternary complex formation, and cytotoxicity, andusing these assays Applicants have identified several potent chimeras (MIC ~ 630 nM). Thus, Applicants are positioned well for the success of the proposed studies. Applicants note that several IgG and IgM binders are emerging, which can also be used.85’99’101Aim 3. Crosstalk with macrophages and in vivo efficacy studies.

[0271] Applicants will assess the chimeras synthesized in Aims 1 and 3 for their ability to induce complement pathway activated phagocytosis and chemotaxis. The prioritization strategy will be similar to those used for Aims 1 and 2, but with phagocytosis (in place of cytolysis) as the benchmark. By years 4 and 5, Applicants anticipate to have identified ideal chimeras for in vivo testing. Toward that goal, Applicants first assess the cytotoxicity of the prioritized chimeras in cellular models of hepatotoxicity, nephrotoxicity, and hemolysis. Applicants will also evaluate chimeras in colistin-resistant strains and several other gram -ve / +ve strains, and finally test for in vivo efficacy in a lung model of infection using E. coli.C.3.1. Preliminary data.C.3.1.1. Cytotoxicity assessment in colistin-resistant strains and gram-positive bacteria.

[0272] Complement recruiting chimeras have a fundamentally different mode of action compared to established antibiotics, providing a potential treatment opportunity for emerging multidrug-resistant pathogens. The mobilized colistin resistance-1 (mcr-P) gene confers colistin resistance in clinical isolates of different gram-negative pathogens and threatens the use of lastresort antibiotics.109'111Applicants cloned the mcr-1 gene mXoE.coli and measured the CDC of the complement recruiters 5 and 6 in the ATP-glo assay. While the bactericidal IC50 of polymyxin B (PMB) increased more than 6-fold in mcr-1 vs WT E.coli, chimeras 5 and 6 showed less than a 2- fold loss in IC50 (FIGs. 52A and 66A). The findings suggest that complement-recruiting chimeras can bypass existing antibiotic resistance mechanisms and offer an opportunity to target multidrugresistant clinical strains.

[0273] The preliminary results outlined in Aim 1 and 2 demonstrate that complement recruiting chimeras can induce CDC against gram-negative bacteria susceptible to PMBN or murepavadin. Targeting gram-positive bacteria might pose a bigger challenge due to their thick peptidoglycan layer that resists complement-mediated opsonization112. However, this paradigm assumes a low-micromolar concentration of the complement system in the serum. Chimeric molecules can increase the effective molarity by as much as =1, 000-fold that will likely leading toopsonization.113Indeed, a chimeric fusion consisting of Fc and Factor H domain that binds to the surface of S. pyogenes (but not complement system) was efficacious in attacking and neutralizing several Gram-positive bacteria.114Gram positive bacteria are not tractable with PMBN or murepavadin, but vancomycin binds acyl-D-ala-D-ala sequences of peptidoglycans on the cell wall of gram-positive bacteria and has previously been used for conjugation to chemotractants for neutrophil clearance of S.aureus.113Analogous, Applicants conjugated vancomycin via its carboxy group to the FcIII-peptide to generate a Class lb chimera (Fig. 52B). The chimera showed dosedependent killing of B.subtilis with an MIC50 of ~2.5 pM in the presence of 20% human serum, demonstrating that the chimeras can also direct complement to the surface of gram-positive bacteria and induce CDC.C.3.2. Experimental plan.C.3.2.1. Interaction with macrophages neutrophils.

[0274] While Aims 1 and 2 focus on direct cytolysis by complement activation, the deposition of complement proteins on bacteria activates macrophages. Three major outcomes known for the interaction between complement and immune cells (including macrophages) are phagocytosis, cytokine production, and chemotaxis.9 10First, Applicants will assess the ability of the bestperforming chimeras to recruit macrophages to E. coli by following a reported procedure.116In brief, THP1 cells will be treated with PMA for 48 h, followed by the addition of LPS and IFN-y for another 48 h to differentiate them into macrophages. Macrophages will be incubated with fluorescently labeled E. coli (GFP) at different concentrations of the chimeras in the presence of human serum, and phagocytosis will be assessed by flow cytometry. Since C3b deposition on the surface of bacteria can also trigger macrophage phagocytosis,117Applicants will additionally perform the experiment in C3-inhibited serum to assess dependency on Fey receptor binding to recruited antibodies.

[0275] Mechanistically, phagocytosis through chimeras la-b, synapses can form both as a result of opsonin deposition and via the binding of antibody to FcyRIIIa on macrophages. Mechanistically, phagocytosis through chimeras Ic involves the formation of synapses between complement receptors and opsonin deposited on bacterial cells. Therefore, using the chimeras and MicroMap, Applicants can study the activation of these two pathways independently. Additionally, the binding of anaphylatoxins C3a and C5a to receptors on macrophages leads toactivation and release of pro- inflammatory cytokines IL-1 P and TNFa as well as an increase in ATP release mediated by extracellular signal-regulated kinase 1 / 2 (ERK1 / 2),118which Applicants will monitor using immunoblotting and alphaELISA.119These studies are critical for harnessing the positive effects of complement activation and avoiding deleterious effects caused by dysregulation.

[0276] Leukocyte migration is a key event both in host defense against invading pathogens and in inflammation. Bacteria generate chemoattractants (formylated peptides, fMLPs), primarily by excretion, and subsequently through activation of leukocytes (such as C5a). Chemotaxis of human neutrophils toward chemoattractants C5a and fMLP will be determined using labeled neutrophils and their secreted elastase levels, as previously described120 115. In brief, neutrophils will be incubated with increasing concentrations of the chimera for 30 min at 37°C. The cells will be washed and added to the upper compartment of a Transwell system (3 pm; Costar), which will be placed onto a 24-well plate containing chemo-attractants in the buffer. After incubation at 37°C / 5% CO2, the inlays will be removed and either fluorescence or elastase activity will used a measure of % neutrophils migrated. (AC, ArC, Yrs 2-3) C.3.2.2. Confirmation of mechanism.

[0277] Same as in C.1.2.3 but in a co-culture system with macrophages. (AC, AS, Yrs 2-3) C.3.2.3. Assessment of cytotoxicity across various systems.

[0278] 1) Mammalian cells: Selectivity for bacteria over mammalian cells is a critical determinant for in vivo applications of antibiotics. The lack of selectivity can lead to nephrotoxicity, hepatotoxicity, and hemotoxicity.121'125As outlined in C.l.1.2, Applicants will assess the toxicity of complement-recruiting chimeras in cells of kidney origin (HEK293T) in cell viability assay. Chimeras will be added in different doses and cellular viability will be measured in a 72 h CellTiter-Glo assay. The Class la chimera 1 already showed promising >120-fold selectivity for PsA over HEK293T cells. Applicants will measure toxicity to liver cells (HepG2 cells) to assess potential liver damage. Lastly, Applicants will determine hemotoxicity in hemolysis assays. Red blood cells are freshly isolated from human whole blood, resuspended in PBS, and incubated with chimeras for 30 min. Absorbance at 541 nm of the supernatant will serve as an indication for hemolysis.

[0279] 2) Resistant and other gram-negative and positive strains: Antibiotic resistance is a rising concern in modern medicine,126accounting for more than one million deaths globally in 2019. Opportunistic “ESKAPE” pathogens are on the verge of losing their therapeutic effect due to emerging multidrug resistances, highlighting the dire need for classes of antibiotics. Like E.coli and PsA, K.pneumoniae anA A.baumannii contain LPS and are colistin- susceptible. Applicants will therefore assess the effect of the PMBN-derived chimeras in those pathogens using both the ATP-glo and ternary complex formation assays as described in Aims 1 and 2. Plasmid-derived resistance towards colistin via the mcr-1 gene also spreads rapidly among gram-negative bacteria.127To gain an understanding of how susceptible the chimeras are towards existing antibiotic resistances, Applicants will compare CDC and ternary complex formation of WT and mcr-1 expressing E.coli .

[0280] Unlike gram-negative bacteria, S.aureus and E. faecium are colistin-insensitive but susceptible to vancomycin- chimeras. To test whether Applicants can induce a ternary complex and CDC, Applicants will conjugate haptens to the 2,3,6- trideoxy-3-methyl-3-amino hexose or the C-terminal carboxylic acid on vancomycin via diverse chemical linkers (e.g., see C.1.2.1} and evaluate the resulting chimeras in B.subtilis. (ArC, AC, Yrs 3-4)C.3.2.4. Optimize in vitro and in vivo pharmacokinetic properties of optimum chimera.

[0281] In parallel, Applicants will measure critical physicochemical (e.g., solubility, permeability) and pharmacokinetic (e.g., microsomal stability, plasma binding) properties for downstream development of chimeras for intratracheal delivery. Ideal characteristics include solubility > 50 pM in PBS buffer; plasma stability, with > 75% parent molecule remaining after 1-hour incubation with the mouse or human plasma; and liver microsome stability, such that > 50% parent molecule remains after 1 -hour incubation with the mouse or human liver microsomes. For in vivo optimization, Applicants will assess the in vivo pharmacokinetics of the chimeras in mice, by administering a single dose intratrachealy128’129followed by monitoring the plasma and lung levels of the compound by LC / MS over 24 hours. This experiment will inform the optimal dose to be used later. These quantitative measurements will inform medicinal chemistry efforts to optimize further the activity, selectivity, stability, and toxicity of the chimeras. If these in vitro properties are not ideal, Applicants will leverage bioisosteric replacement strategies to alter thePK properties without dramatically altering the pharmacophore structure (e.g., replacement of aryl ring with a cubane, H with F, or OH with NH2). (ArC, AC, Yrs 4-5) C.3.2.5. Evaluate in vivo efficacy in mouse.

[0282] Applicants will evaluate either complement recruiting chimeras in post- infection treatment model128 129as reported previously. Briefly, 6-8 wk CD1 mice will be infected with E. coli (1 x 103c.f.u) via intratracheal delivery followed by administration of chimeras at 6h or 24 h with vehicle as control. Chimeras will be injected at 2 doses of each optimized chimera (dose to be determined based on PK assay results). A vehicle-treated group will be included as a control (e.g., n = 7 per test group), as well as a group with a meropenem control (~120mg / kg). Mouse weight will be measured every 12 hours, and animal survival will be used to evaluate overall response. All surviving animals will be sacrificed 96 hours post-infection. Spleen, liver, lung, kidney, brain, and bone marrow will be harvested, homogenized, and plated on CASO agar in serial dilutions. The number of CFU will be obtained after overnight incubation at 37°C and will serve as a measure of bacterial burden for each treatment group. The NOAEL (No observable adverse event level) is based on animal observation, weight loss, clinical pathology and histopathological analysis of tissues to determine target organs and safe drug level. Applicants have budgeted for repeat experiments in the event that the doses tested are unable to distinguish between the relative performance of the candidates tested (either due to lack of efficacy or overwhelming efficacy). Repeat experiment, for example, would use 3x and lOx lower or higher dose, depending on results of the first experiment. Mouse of both sexes will be used in the studies.(ArC, AC, Yrs 4-5)C.3.3. Potential pitfalls and proposed solutions.

[0283] Although Applicants will using intratracheal delivery, metabolic stability and clearance can be an issue for chimeras. Many of the conjugates consist of peptides or glycoconjugates, which are prone to enzymatic degradation but are clinical grade and such degradation will be lower for the direct delivery to lung (vs. orally). Applicants anticipate that the development of the platform for synthesizing chimeras to rapidly screen for improved constructs. Additionally, potential peptide-instability can be solved by substituting vulnerable amino acids with unnatural derivatives. SAR data is available for all peptides used in the platform and can be readily implemented to optimize the therapeutic potential.I). Timeline and Outlook.

[0284] All proposed aims are synergistic and independent (e.g., using different chimeras and optimizing for different biological outcome, including cytolysis, phagocytosis, or chemotaxis) and will be pursued simultaneously. The timeline and team members involved with each sub aim are identified in bold. Molecules that allow precise dose- and temporally-controlled recruitment or degradation of the complement components at the target site (e.g., pathogen) will be immensely valuable as basic research tools and may even furnish novel therapeutic modalities. Various assays, reagents, and platforms (e.g., MicroMap, DELs) developed herein will be valuable to the research community at large. Thus, Applicants expect that the proposed studies will produce reagents and tools will be of high interest to the community.References for Example 3(1) The antibiotic resistance crisis: part 1 : causes and threats. Ventola, C. L. P t 2015, 40, 277- 83.PMC4378521(2) Antibiotic resistance: A current epilogue. Dodds, D. R. Biochem Pharmacol 2017, 134, 139- 146(3) Antibiotic resistance in the environment. Larsson, D. G. J.; Flach, C.-F. Nature Reviews Microbiology’ 2022, 20, 257-269(4) Complement System Part II: Role in Immunity. Merle, N. S.; Noe, R.; Halbwachs-Mecarelli, L.; Fremeaux-Bacchi, V.; Roumenina, L. T. Front Immunol 2015, 6, 257.PMC4443744(5) Complement System Part I - Molecular Mechanisms of Activation and Regulation. Merle, N.S.; Church, S. E.; Fremeaux-Bacchi, V.; Roumenina, L. T. Front Immunol 2015, 6, 262.PMC4451739(6) Complement and its role in innate and adaptive immune responses. Dunkelberger, J. R.; Song, W.-C. Cell Research 2010, 20, 34-50(7) The complement system. Sarma, J. V.; Ward, P. A. Cell Tissue Res 2011, 343, 227- 35.PMC3097465(8) The complement system: history, pathways, cascade and inhibitors. Nesargikar, P. N.; Spiller, B.; Chavez, R. Eur J Microbiol Immunol (Bp) 2012, 2, 103-11.PMC3956958(9) The structural basis for anaphylatoxin and chemotactic functions of C3a, C4a, and C5a. Hugli,T. E. Crit Rev Immunol 1981, 1, 321-66(10) Complement-dependent clearance of apoptotic cells by human macrophages. Mevorach, D.; Mascarenhas, J. O.; Gershov, D.; Elkon, K. B. J Exp Med 1998, 188, 2313-20.PMC2212421(11) Induced protein degradation: an emerging drug discovery paradigm. Lai, A. C.; Crews, C. M. Nat Rev Drug Discov 2011, 16, 101-114.PMC5684876(12) Chemically induced proximity in biology and medicine. Stanton, B. Z.; Chory, E. J.; Crabtree, G. R. Science 2018, 359.PMC6417506(13) Unifying principles of bifunctional, proximity-inducing small molecules. Gerry, C. J.; Schreiber, S. L. Nat Chem Biol 2020, 16, 369-378.PMC7312755(14) Proteolysis targeting chimeras (PROTACs) come of age: entering the third decade of targeted protein degradation. Bond, M. J.; Crews, C. M. RSC Chem Biol 2021, 2, 725-742.PMC8190915(15) PROTAC targeted protein degraders: the past is prologue. Bekes, M.; Langley, D. R.; Crews, C. M. Nature Reviews Drug Discovery 2022, 21, 181-200(16) Molecular mechanisms of membrane targeting antibiotics. Epand, R. M.; Walker, C.; Epand, R. F.; Magarvey, N. A. Biochim Biophys Acta 2016, 1858, 980-7(17) Small Molecule Sensors Targeting the Bacterial Cell Wall. Parker, M. F. L.; Flavell, R. R.; Luu, J. M.; Rosenberg, O. S.; Ohliger, M. A.; Wilson, D. M. ACS Infect Dis 2020, 6, 1587- 1598.PMC7808209(18) Exploring Targeted Degradation Strategy for Oncogenic KRAS(G12C). Zeng, M.; Xiong, Y Safaee, N.; Nowak, R. P.; Donovan, K. A.; Yuan, C. J.; Nabet, B.; Gero, T. W.; Feru, F.; Li, L.; Gondi, S.; Ombelets, L. J.; Quan, C.; Janne, P. A.; Kostic, M.; Scott, D. A.; Westover, K. D.; Fischer, E. S.; Gray, N. S. Cell Chem Biol 2020, 27, 19-3 l.e6(19) Mapping the Degradable Kinome Provides a Resource for Expedited Degrader Development. Donovan, K. A.; Ferguson, F. M.; Bushman, J. W .; Eleuteri, N. A.; Bhunia, D.; Ryu, S.; Tan, L.; Shi, K.; Yue, H.; Liu, X.; Dobrovolsky, D.; Jiang, B.; Wang, J.; Hao, M.; You, I.; Teng, M.; Liang, Y.; Hatcher, J.; Li, Z.; Manz, T. D.; Groendyke, B.; Hu, W.; Nam, Y.; Sengupta, S.; Cho, H.; Shin, I.; Agius, M. P.; Ghobrial, I. M.; Ma, M. W.; Che, J.; Buhrlage, S. J.; Sim, T.; Gray, N. S.; Fischer, E. S. Cell 2020, 183, 1714- 1731.el0.PMC10294644(20) The complement system. Kerr, M. A. Biochemical Education 1981, 9, 82-88(21) Expanding horizons in complement drug discovery: challenges and emerging strategies. Harris, C. L. Semin Immunopathol 2018, 40, 125-140. PMC5794834(22) How Big Is Too Big for Cell Permeability? Matsson, P.; Kihlberg, J. Journal of Medicinal Chemistry 2017, 60, 1662-1664(23) The crystal structure of the globular head of complement protein Clq provides a basis for its versatile recognition properties. Gaboriaud, C.; Juanhuix, J.; Gruez, A.; Lacroix, M.; Damault, C.; Pignol, D.; Verger, D.; Fontecilla-Camps, J. C.; Arlaud, G. J. J Biol Chem 2003, 278, 46974-82(24) Structures of complement component C3 provide insights into the function and evolution of immunity. Janssen, B. J.; Huizinga, E. G.; Raaijmakers, H. C.; Roos, A.; Daha, M. R.; Nilsson- Ekdahl, K.; Nilsson, B.; Gros, P. Nature 2005, 437, 505-11(25) Crystal structure and functional characterization of the complement regulator mannosebinding lectin (MBL) / ficolin-associated protein-1 (MAP-1). Skjoedt, M. O.; Roversi, P.; Hummelshoj, T.; Palarasah, Y.; Rosbjerg, A.; Johnson, S.; Lea, S. M.; Garred, P. J Biol Chem 2012, 287, 32913-21. PMC3463328(26) Complement is activated by IgG hexamers assembled at the cell surface. Diebolder, C. A.; Beurskens, F. J.; de Jong, R. N.; Koning, R. I.; Strumane, K.; Lindorfer, M. A.; Voorhorst, M.; Ugurlar, D.; Rosati, S.; Heck, A. J.; van de Winkel, J. G.; Wilson, I. A.; Koster, A. J.; Taylor, R. P.; Saphire, E. O.; Burton, D. R.; Schuurman, J.; Gros, P.; Parren, P. W. Science 2014, 343, 1260- 3.PMC4250092(27) Structural basis for therapeutic inhibition of complement C5. Jore, M. M.; Johnson, S.; Sheppard, D.; Barber, N. M.; Li, Y. I.; Nunn, M. A.; Elmlund, H.; Lea, S. M. Nat Struct Mol Biol 2016, 23, 378- 86.PMC5771465(28) Antibiotics: past, present and future. Hutchings, M. I.; Truman, A. W Wilkinson, B. Current Opinion in Microbiology 2019, 51, 72-80(29) NK-cell activation and antibody-dependent cellular cytotoxicity induced by rituximab-coated target cells is inhibited by the C3b component of complement. Wang, S. Y.; Racila, E.; Taylor, R. P.; Weiner, G. J. B / oot / 2008, 111, 1456-63 PMC2214766(30) Lysosome-targeting chimaeras for degradation of extracellular proteins. Banik, S. M.; Pedram, K.; Wisnovsky, S.; Ahn, G.; Riley, N. M.; Bertozzi, C. R. Nature 2020, 584, 291-297(31) Bifunctional small molecules that mediate the degradation of extracellular proteins. Caianiello, D. F.; Zhang, M.; Ray, J. D.; Howell, R. A.; Swartzel, J. C.; Branham, E. M. J.; Chirkin,E.; Sabbasani, V. R.; Gong, A. Z.; McDonald, D. M.; Muthusamy, V.; Spiegel, D. A. Nature Chemical Biology 2021, 77, 947- 953(32) The complement system in inflammatory bowel disease. Jain, U.; Otley, A. R.; Van Limbergen, J.; Stadnyk, A. W. Inflamm Bowel Dis 2014, 20, 1628-37(33) Phase Ila clinical trial of complement C3 inhibitor AMY-101 in adults with periodontal inflammation. Hasturk, H.; Hajishengallis, G.; Lambris, J. D.; Mastellos, D. C.; Yancopoulou, D. J Clin Invest 2021, 737.PMC8631591(34) Target-oriented and diversity-oriented organic synthesis in drug discovery. Schreiber, S. L. Science 2000, 287, 1964-9(35) Application of natural product-inspired diversity-oriented synthesis to drug discovery. Marcaurelle, L. A.; Johannes, C. W. Prog Drug Res 2008, 66, 187, 189-216(36) Grand challenge commentary: Accessing new chemical space for 'undruggable' targets. Dandapani, S.; Marcaurelle, L. A. Nat Chem Biol 2010, 6, 861-3(37) Hits, leads and drugs against malaria through diversity-oriented synthesis. Dandapani, S.; Comer, E.; Duvall, J. R.; Munoz, B. Future Med Chem 2012, 4, 2279-94(38) Diversity-Oriented Synthesis-Facilitated. Comer, E.; Beaudoin, J. A.; Kato, N.; Fitzgerald, M. E.; Heidebrecht, R. W.; Lee, M. P.; Masi, D.; Mercier, M.; Mulrooney, C.; Muncipinto, G.; Rowley, A.; Crespo-Llado, K.; Serrano, A. E.; Lukens, A. K.; Wiegand, R. C.; Wirth, D. F.; Palmer, M. A.; Foley, M. A.; Munoz, B.; Scherer, C. A.; Duvall, J. R.; Schreiber, S. L. J Med Chem 2014, 57, 8496-502(39) Diversity-oriented synthesis yields novel multistage antimalarial inhibitors. Kato, N.; Comer,E.; Sakata- Kato, T.; Sharma, A.; Shanna, M.; Maetani, M.; Bastien, J.; Brancucci, N. M.; Bittker, J. A.; Corey, V.; Clarke, D.; Derbyshire, E. R.; Dornan, G.; Duffy, S.; Eckley, S.; Itoe, M. A.; Koolen, K. M. J.; Lewis, T. A.; Lui, P. S.; Lukens, A. K.; Lund, E.; March, S.; Meibalan, E.; Meier, B. C.; McPhail, J.; Mitasev, B.; Moss, E. L.; Sayes, M.; VanGessel, Y.; Wawer, M. J.; Yoshinaga, T.; Zeeman, A.-M.; Avery, V. M.; Bhatia, S. N.; Burke, J. E.; Catteruccia, F.; Clardy, J. C.; Clemons, P. A.; Dechering, K. J.; Duvall, J. R.; Foley, M. A.; Gusovsky, F.; Kocken, C. H. M.; Marti, M.; Morningstar, M. L.; Munoz, B.; Neafsey, D. E.; Sharma, A.; Winzeler, E. A.; Wirth, D.F.; Scherer, C. A.; Schreiber, S. L. Nature 2016(40) Directed evolution of APEX2 for electron microscopy and proximity labeling. Lam, S. S.; Martell, J. D.; Kam er, K. J.; Deerinck, T. J.; Ellisman, M. EL; Mootha, V. K.; Ting, A. Y. Nat Methods 2015, 72, 51- 4.PMC4296904(41) Efficient proximity labeling in living cells and organisms with TurboID. Branon, T. C.; Bosch, J. A.; Sanchez, A. D.; Udeshi, N. D.; Svinkina, T.; Carr, S. A.; Feldman, J. L.; Perrimon, N.; Ting, A. Y. Nature Biotechnology 2018, 36, 880-887(42) Microenvironment mapping via Dexter energy transfer on immune cells. Geri, J. B.; Oakley, J. V.; Reyes-Robles, T.; Wang, T ; McCarver, S. J.; White, C. H.; Rodriguez-Rivera, F. P.; Parker, D. L., Jr.; Hett, E. C.; Fadeyi, O. O.; Oslund, R. C.; MacMillan, D. W. C. Science 2020, 367, 1091- 1097.PMC7336666(43) muMap-Red: Proximity Labeling by Red Light Photocatalysis. Buksh, B. F.; Knutson, S. D.; Oakley, J. V.; Bissonnette, N. B.; Oblinsky, D. G.; Schwoerer, M. P.; Seath, C. P.; Geri, J. B.; Rodriguez-Rivera, F. P.; Parker, D. L.; Scholes, G. D.; Ploss, A.; MacMillan, D. W. C. J Am Chem Soc 2022, 144, 6154- 6162.PMC9843638(44) Small molecule photocatalysis enables drug target identification via energy transfer. Trowbridge, A. D.; Seath, C. P.; Rodriguez-Rivera, F. P.; Li, B. X.; Dul, B. E.; Schwaid, A. G.; Buksh, B. F.; Geri, J. B.; Oakley, J. V.; Fadeyi, O. O.; Oslund, R. C.; Ryu, K. A.; White, C.; Reyes- Robles, T.; Tawa, P.; Parker, D. L., Jr.; MacMillan, D. W. C. Proc Natl Acad Sci USA 2022, 119, e2208077119.PMC9407219(45) Tracking chromatin state changes using nanoscale photo-proximity labelling. Seath, C. P.; Burton, A. J.; Sun, X.; Lee, G.; Kleiner, R. E.; MacMillan, D. W. C.; Muir, T. W. Nature 2023, 616, 574- 580.PMC10408239(46) Phosphorylation-Inducing Chimeric Small Molecules. Siriwardena, S. U.; Munkanatta Godage, D. N. P.; Shoba, V. M.; Lai, S.; Shi, M.; Wu, P.; Chaudhary, S. K.; Schreiber, S. L.; Choudhary, A. J Am Chem Soc 2020, 142, 14052-14057(47) Bifunctional modalities for repurposing protein function. Modell, A. E.; Lai, S.; Nguyen, T. M.; Choudhary, A. Cell Chem Biol 2021, 28, 1081-1089(48) Synthetic Reprogramming of Kinases Expands Cellular Activities of Proteins. Shoba, V. M.; Munkanatta Godage, D. N. P.; Chaudhary, S. K.; Deb, A.; Siriwardena, S. U.; Choudhary, A. Angew Chem Int Ed Engl 2022, 61, e202202770.PMC9527066(49) A molecular glue approach to control the half-life of CRISPR-based technologies. Sreekanth, V.; Jan, M.; Zhao, K. T.; Lim, D.; Davis, J. R.; McConkey, M.; Kovalcik, V.; Barkal, S.; Law, B. K.; Fife, J.; Tian, R.; Vinyard, M. E.; Becerra, B.; Kampmann, M. E.; Sherwood, R. I.; Pinello, L.; Liu, D. R.; Ebert, B. L.; Choudhary, A. bioRxiv 2023.PMC 10028966(50) Bifunctional small molecules that induce nuclear localization and targeted transcriptional regulation. Gibson, W. J.; Sadagopan, A.; Shoba, V. M.; Choudhary, A.; Meyerson, M.; Schreiber, S. L. bioRxiv 2023.PMC 10350064(51) Photoswitchable Molecular Glues Enable Optical Control of Transcription Factor Degradation. Arp, C. J.; Reynders, M.; Sreekanth, V.; Kokkonda, P.; Pagano, M.; Choudhary, A.; Trauner, D. bioRxiv 2023.PMC 10104231(52) Development and Applications of Chimera Platforms for Tyrosine Phosphorylation. Pergu, R.; Shoba, V. M.; Chaudhary, S. K.; Munkanatta Godage, D. N. P.; Deb, A.; Singha, S.; Dhawa, U.; Singh, P.; Anokhina, V.; Singh, S.; Siriwardena, S. U.; Choudhary, A. ACS Cent Sci 2023, 9, 1558- 1566.PMC10450875(53) Proximity-inducing modalities: the past, present, and future. Singh, S.; Tian, W.; Severance, Z. C.; Chaudhary, S. K.; Anokhina, V.; Mondal, B.; Pergu, R.; Singh, P.; Dhawa, U.; Singha, S.; Choudhary, A. Chem Soc Rev 2023, 52, 5485-5515(54) Multidimensional chemical control of CRISPR-Cas9. Maji, B.; Moore, C. L.; Zetsche, B.; Volz, S. E.; Zhang, F.; Shoulders, M. D.; Choudhary, A. Nat Chem Biol 2017, 13, 9- 11.PMC5531067(55) A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9. Maji, B.; Gangopadhyay, S. A.; Lee, M.; Shi, M.; Wu, P.; Heler, R.; Mok, B.; Lim, D.; Siriwardena, S. U.; Paul, B.; Dancik, V.; Vetere, A.; Mesleh, M. F.; Marraffmi, L. A.; Liu, D. R.; Clemons, P. A.; Wagner, B. K.; Choudhary, A. Ce / / 2019, 177, 1067-1079.el9.PMC7182439(56) A universal method for sensitive and cell-free detection of CRISPR-associated nucleases. Cox, K. J.; Subramanian, H. K. K.; Samaniego, C. C.; Franco, E.; Choudhary, A. Chem Sci 2019, 10, 2653- 2662.PMC6419926(57) A 3D culture platform enables development of zinc-binding prodrugs for targeted proliferation of 0 cells. Yang, K.; Lee, M.; Jones, P. A.; Liu, S. S.; Zhou, A.; Xu, J.; Sreekanth,V.; Wu, J. L. Y.; Vo, L.; Lee, E. A.; Pop, R.; Lee, Y.; Wagner, B. K.; Melton, D. A.; Choudhary, A.; Karp, J. M. Sci Adv 2020, 6.PMC7673808(58) Native Zinc Catalyzes Selective and Traceless Release of Small Molecules in P-Cells. Lee, M.; Maji, B.; Manna, D.; Kahraman, S.; Elgamal, R. M.; Small, J.; Kokkonda, P.; Vetere, A.; Goldberg, J. M.; Lippard, S. J.; Kulkarni, R. N.; Wagner, B. K.; Choudhary, A. J Am Chem Soc 2020, 742, 6477- 6482.PMC7146867(59) Harnessing reaction-based probes to preferentially target pancreatic P-cells and P-like cells. Kahraman, S.; Manna, D.; Dirice, E.; Maji, B.; Small, J.; Wagner, B. K.; Choudhary, A.; Kulkarni, R. N. Life Sci Alliance 2021, 4.PMC7898467(60) A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules. Lim, D.; Zhou, Q.; Cox, K. J.; Law, B. K.; Lee, M.; Kokkonda, P.; Sreekanth, V.; Pergu, R.; Chaudhary, S. K.; Gangopadhyay, S. A.; Maji, B.; Lai, S.; Amako, Y.; Thompson, D. B.; Subramanian, H. K. K.; Mesleh, M. F.; Dancik, V.; Clemons, P. A.; Wagner, B. K.; Woo, C. M.; Church, G. M.; Choudhary, A. Nat Cell Biol 2022, 24, 1766-1775.PMC9891305(61) Rational Design of Silicon-Based Zinc Ionophores. Yamada, K.; Deb, A.; Shoba, V. M.; Lim, D.; Maji, B.; Modell, A. E.; Choudhary, A. Angew Chem Int Ed Engl 2022, 61, e202201698.PMC9527067(62) Susceptibility of gram-negative bacteria to polymyxin B nonapeptide. Viljanen, P.; Vaara, M. Antimicrob Agents Chemother 1984, 25, 701-5. PMC 185626(63) Binding of polymyxin B nonapeptide to gram-negative bacteria. Vaara, M.; Viljanen, P. Antimicrob Agents Chemother 1985, 27, 548-54.PMC 180093(64) New analogs of the clinical complement inhibitor compstatin with subnanomolar affinity and enhanced pharmacokinetic properties. Qu, H.; Ricklin, D.; Bai, H.; Chen, H.; Reis, E. S.; Maciejewski, M.; Tzekou, A.; DeAngelis, R. A.; Resuello, R. R.; Lupu, F.; Barlow, P. N.; Lambris, J. D. Immunobiology 2013, 218, 496-505. PMC3518557(65) Compstatin: a C3-targeted complement inhibitor reaching its prime for bedside intervention. Mastellos, D. C.; Yancopoulou, D.; Kokkinos, P.; Huber-Lang, M.; Hajishengallis, G.; Biglamia, A. R.; Lupu, F.; Nilsson, B.; Risitano, A. M.; Ricklin, D.; Lambris, J. D. Eur J Clin Invest 2015, 45, 423-40.PMC4380746(66) Inhibition of pre-existing natural periodontitis in non-human primates by a locally administered peptide inhibitor of complement C3. Maekawa, T.; Briones, R. A.; Resuello, R. R.; Tuplano, J. V.; Hajishengallis, E.; Kajikawa, T.; Koutsogiannaki, S.; Garcia, C. A.; Ricklin, D.; Lambris, J. D.; Hajishengallis, G. J Clin Periodontol 2016, 43, 238-49.PMC4803614(67) Peptidomimetic antibiotics target outer-membrane biogenesis in Pseudomonas aeruginosa. Srinivas, N.; letter, P.; Ueberbacher, B. J.; Werneburg, M.; Zerbe, K.; Steinmann, J.; Van der Meijden, B.; Bernardini, F.; Lederer, A.; Dias, R. L.; Misson, P. E.; Henze, H.; Zumbrunn, J.; Gombert, F. O.; Obrecht, D.; Hunziker, P.; Schauer, S.; Ziegler, U.; Kach, A.; Eberl, L.; Riedel, K.; DeMarco, S. J.; Robinson, J. A. Science 2010, 327, 1010-3(68) Pharmacokinetics and Safety of Intravenous Murepavadin Infusion in Healthy Adult Subjects Administered Single and Multiple Ascending Doses. Wach, A.; Dembowsky, K.; Dale, G. E. Antimicrob Agents Chemother 2018, 62.PMC5913942(69) Murepavadin: a new antibiotic class in the pipeline. Martin-Loeches, I.; Dale, G. E.; Torres, A. Expert Rev Anti Infect Ther 2018, 16, 259-268(70) A Peptidomimetic Antibiotic Interacts with the Periplasmic Domain of LptD from Pseudomonas aeruginosa. Andolina, G.; Bencze, L.-C.; Zerbe, K.; Muller, M.; Steinmann, J.; Kocherla, H.; Mondal, M.; Sobek, J.; Moehle, K.; Malojcic, G.; Wollscheid, B.; Robinson, J. A. ACS Chemical Biology 2018, 13, 666-675(71) Polymyxin B sulfate and colistin: old antibiotics for emerging multiresistant gram-negative bacteria, vans, M. E.; Feola, D. J.; Rapp, R. P. Ann Pharmacother 1999, 33, 960-7(72) Convergent Solutions to Binding at a Protein-Protein Interface. DeLano, W. L.; Ultsch, M. H.; de, A. M.; Wells, J. A. Science 2000, 287, 1279-1283(73) Protein Ligand Design: From Phage Display to Synthetic Protein Epitope Mimetics in Human Antibody Fc-Binding Peptidomimetics. Dias, R. L. A.; Fasan, R.; Moehle, K.; Renard, A.; Obrecht, D.; Robinson, J. A. Journal of the American Chemical Society 2006, 128, 2726-2732(74) IgG Fc domains that bind Clq but not effector Fcgamma receptors delineate the importance of complement-mediated effector functions. Lee, C. H.; Romain, G.; Yan, W .; Watanabe, M.; Charab, W.; Todorova, B.; Lee, J.; Triplett, K.; Donkor, M.; Lungu, O. I.; Lux, A.; Marshall, N.; Lindorfer, M. A.; Goff, O. R.; Balbino, B.; Kang, T. H.; Tanno, H.; Delidakis, G.; Alford, C.;Taylor, R. P.; Nimmeijahn, F.; Varadarajan, N.; Bruhns, P.; Zhang, Y. J.; Georgiou, G. Nat Immunol 2017, 18, 889-898.PMC6015732(75) An interactive resource to identify cancer genetic and lineage dependencies targeted by small molecules. Basu, A.; Bodycombe, N. E.; Cheah, J. H.; Price, E. V.; Liu, K.; Schaefer, G. I.; Ebright, R. Y.; Stewart, M. L.; Ito, D.; Wang, S.; Bracha, A. L.; Liefeld, T.; Wawer, M.; Gilbert,J. C.; Wilson, A. J.; Stransky, N.; Kryukov, G. V.; Dancik, V.; Barretina, J.; Garraway, L. A.; Hon, C. S.; Munoz, B.; Bittker, J. A.; Stockwell, B. R.; Khabele, D.; Stern, A. M.; Clemons, P. A.; Shamji, A. F.; Schreiber, S. L. Ce / Z 2013, 154, 1151-1161.PMC3954635(76) Discovery of ARD-69 as a Highly Potent Proteolysis Targeting Chimera (PROTAC) Degrader of Androgen Receptor (AR) for the Treatment of Prostate Cancer. Han, X.; Wang, C.; Qin, C.; Xiang, W.; Fernandez- Sal as, E.; Yang, C.-Y.; Wang, M.; Zhao, L.; Xu, T.; Chinnaswamy,K.; Delproposto, J.; Stuckey, J.; Wang, S. Journal of Medicinal Chemistry 2019, 62, 941-964(77) Current strategies for the design of PROTAC linkers: a critical review. Troup, R. I.; Fallan, C.; Baud, M. G. J. Explor Target Antitumor Ther 2020, 1, 273-312. PMC9400730(78) Synthetic Immunotherapeutics against Gram-negative Pathogens. Feigman, M. S.; Kim, S.; Pidgeon, S. E.; Yu, Y.; Ongwae, G. M.; Patel, D. S.; Regen, S.; Im, W .; Pires, M. M. Cell Chemical Bio!ogy 201Z, 25, 1185-1194.e5(79) Linkers as Game-changers in PROTAC Technology: Emphasizing General Trends in PROTAC Pharmacokinetics for their Rational Design. Cecchini, C.; Tardy, S.; Scapozza, L. CHIMIA 2022, 76, 341(80) Antibody -recruiting molecules: an emerging paradigm for engaging immune function in treating human disease. McEnaney, P. J.; Parker, C. G.; Zhang, A. X.; Spiegel, D. A. ACS Chem Biol 2012, 7, 1139-51. PMC3401898(81) Chemically synthesized molecules with the targeting and effector functions of antibodies. McEnaney, P. J.; Fitzgerald, K. J.; Zhang, A. X.; Douglass, E. F., Jr.; Shan, W.; Balog, A.; Kolesnikova, M. D.; Spiegel, D. A. J Am Chem Soc 2014, 136, 18034-43.PMC4291750(82) Natural antibodies specific to the 2,4-dinitrophenyl group. Farah, F. S. Immunology 1973, 25, 217- 26.PMC1422858(83) Natural DNP-binding immunoglobulins and antibody multispecificity. Ortega, E.; Kostovetzky, M.; Larralde, C. Mol Immunol 1984, 21, 883-8(84) Evolutionary relationship between the natural anti-Gal antibody and the Gal alpha 1 — 3Gal epitope in primates. Galili, U.; Clark, M. R.; Shohet, S. B.; Buehler, J.; Macher, B. A. Proc Natl Acad Set USA 1987, 84, 1369-73.PMC304431(85) Exploring binding and effector functions of natural human antibodies using synthetic immunomodulators. Jakobsche, C. E.; Parker, C. G.; Tao, R. N.; Kolesnikova, M. D.; Douglass, E. F., Jr.; Spiegel, D. A. ACS Chem Biol 2013, 8, 2404-11.PMC3830660(86) Rhamnose glycoconjugates for the recruitment of endogenous anti-carbohydrate antibodies to tumor cells. Sheridan, R. T.; Hudon, J.; Hank, J. A.; Sondel, P. M.; Kiessling, L. L. Chembiochem 2014, 15, 1393-8.PMC4205123(87) Bifunctional ligands that target cells displaying the alpha v beta3 integrin. Owen, R. M.; Carlson, C. B.; Xu, J.; Mowery, P.; Fasella, E.; Kiessling, L. L. Chembiochem 2007, 8, 68-82(88) Selective tumor cell targeting using low-affinity, multivalent interactions. Carlson, C. B.; Mowery, P.; Owen, R. M.; Dykhuizen, E. C.; Kiessling, L. L. ACS Chem Biol 2007, 2, 119-27(89) Multifunctional Glycoconjugates for Recruiting Natural Antibodies against Cancer Cells. Liet, B.; Laigre,E.; Goyard, D.; Todaro, B.; Tiertant, C.; Boturyn, D.; Berthet, N.; Renaudet, O. Chemistry 2019, 25, 15508-15515.PMC6916168(90) Purification of anti-dinitrophenyl antibodies and their fractionation according to affinity. Farah, F. S.; Awdeh, Z. L. Journal of Immunological Methods 1972, 1, 353-361(91) Impact of hapten presentation on antibody binding at lipid membrane interfaces. Jung, H.; Yang, T.; Lasagna, M. D.; Shi, J.; Reinhart, G. D.; Cremer, P. S. Biophys J 2008, 94, 3094- 103.PMC2275692(92) A Remote Arene-Binding Site on Prostate Specific Membrane Antigen Revealed by Antibody-Recruiting Small Molecules. Zhang, A. X.; Murelli, R. P.; Barinka, C.; Michel, J.; Cocleaza, A.; Jorgensen, W. L.; Lubkowski, J.; Spiegel, D. A. Journal of the American Chemical Society 2010, 132, 12711-12716(93) Sortase-Mediated Protein Ligation: A New Method for Protein Engineering. Mao, H.; Hart, S. A.; Schink, A.; Pollok, B. A. Journal of the American Chemical Society 2004, 126, 2670-2671(94) Therapeutic Targeting of the Complement System: From Rare Diseases to Pandemics. Garred, P.; Tenner, A. J.; Mollnes, T. E. Pharmacol Rev 2021, 73, 792-827.PMC7956994(95) Increased complement activation is a distinctive feature of severe SARS-CoV-2 infection. Ma, L.; Sahu, S. K.; Cano, M.; Kuppuswamy, V.; Bajwa, J.; McPhatter, J.; Pine, A.; Meizlish, M.; Goshua, G.; Chang, C. H.; Zhang, H.; Price, C.; Bahel, P.; Rinder, H.; Lei, T.; Day, A.; Reynolds, D.; Wu, X.; Schriefer, R.; Rauseo, A. M.; Goss, C. W.; O'Halloran, J. A.; Presti, R. M.; Kim, A. H.; Gelman, A. E.; Cruz, C. D.; Lee, A. I.; Mudd, P.; Chun, H. J.; Atkinson, J. P ; Kulkarni, H. S. bioRxiv 2021.PMC7924264(96) Soluble MAC is primarily released from MAC-resistant bacteria that potently convert complement component C5. Doorduijn, D. J.; Lukassen, M. V.; van 't Wout, M. F. L.; Franc, V.; Ruyken, M.; Bardoel, B. W Heck, A. J. R.; Rooijakkers, S. H. M. Elife 2022, 77.PMC9402229(97) Complement-resistance mechanisms of bacteria. Rautemaa, R.; Meri, S. Microbes and Infection 1999, 7, 785-794(98) Selective Proteomic Proximity Labeling Assay Using Tyramide (SPPLAT): A Quantitative Method for the Proteomic Analysis of Localized Membrane-Bound Protein Clusters. Rees, J. S.; Li, X. W.; Perrett, S.; Lilley, K. S.; Jackson, A. P. Curr Protoc Protein Sci 2015, 80, 19 27 1-19 27 18(99) Recognition of antigen-specific B-cell receptors from chronic lymphocytic leukemia patients by synthetic antigen surrogates. Sarkar, M.; Liu, Y.; Morimoto, J.; Peng, H.; Aquino, C.; Rader, C.; Chiorazzi, N.; Kodadek, T. Chem Biol 2014, 27, 1670-9.PMC4272629(100) Chemical Tools To Monitor and Manipulate Adaptive Immune Responses. Doran, T. M.; Sarkar, M.; Kodadek, T. J Am Chem Soc 2016, 138, 6076-94.PMC5332222(101) High-throughput Identification of DNA-Encoded IgG Ligands that Distinguish Active and Latent Mycobacterium tuberculosis Infections. Mendes, K. R.; Malone, M. L.; Ndungu, J. M.; Suponitsky- Kroyter, L; Cavett, V. J.; McEnaney, P. J.; MacConnell, A. B.; Doran, T. M.; Ronacher, K.; Stanley, K.; Utset, O.; Walzl, G.; Paegel, B. M.; Kodadek, T. ACS Chem Biol 2017, 72, 234-243.PMC5250564(102) DNA Barcoding a Complete Matrix of Stereoisomeric Small Molecules. Gerry, C. J.; Wawer, M. J.; Clemons, P. A.; Schreiber, S. L. J Am Chem Soc 2019, 141, 10225- 10235.PMC6681660(103) Diversity-oriented synthesis encoded by deoxyoligonucleotides. Hudson, L.; Mason, J. W.; Westphal, M. V.; Richter, M. J.; Thielman, J. R.; Hua, B. K.; Gerry, C. J.; Xia, G.; Osswald, H. L.; Knapp, J. M. Nature Communications 2023, 14, 4930(104) Machine learning on DNA-encoded library count data using an uncertainty-aware probabilistic loss function. Lim, K. S.; Reidenbach, A. G.; Hua, B. K.; Mason, J. W.; Gerry, C. J.; Clemons, P. A.; Coley, C. W. Journal of Chemical Information and Modeling 2022, 62, 2316- 2331(105) The PDBbind database: Collection of binding affinities for protein- ligand complexes with known three- dimensional structures. Wang, R.; Fang, X.; Lu, Y.; Wang, S. Journal of medicinal chemistry 2004, 47, 2977-2980(106) A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules. Lim, D.; Zhou, Q.; Cox, K. J.; Law, B. K.; Lee, M.; Kokkonda, P.; Sreekanth, V.; Pergu, R.; Chaudhary, S. K.; Gangopadhyay, S. A. Nature Cell Biology 2022, 24, 1766-1775(107) Glide: A New Approach for Rapid, Accurate Docking and Scoring. 2. Enrichment Factors in Database Screening. Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W. T.; Banks, J. L. Journal of Medicinal Chemistry 2004, 47, 1750-1759(108) Extra Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein-Ligand Complexes. Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. Journal of Medicinal Chemistry 2006, 49, 6177- 6196(109) An overview of colistin resistance, mobilized colistin resistance genes dissemination, global responses, and the alternatives to colistin: A review. Gharaibeh, M. H.; Shatnawi, S. Q. Vet World 2019, 12, 1735- 1746.PMC6925059(110) Epidemiology of mobile colistin resistance genes mcr-1 to mcr-9. Ling, Z.; Yin, W .; Shen, Z.; Wang, Y.; Shen, J.; Walsh, T. R. Journal of Antimicrobial Chemotherapy 2020, 75, 3087-3095(111) Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane. Sabnis, A.; Hagart, K. L. H.; Klbckner, A.; Becce, M.; Evans, L. E.; Furniss, R. C. D.; Mavridou, D. A. L; Murphy, R.; Stevens, M. M.; Davies, J. C.; Larrouy-Maumus, G. J.; Clarke, T. B.; Edwards, A. M. eLife 2021, 10, e65836(1 12) The key role of peptidoglycan in the opsonization of Staphylococcus aureus. Peterson, P. K.; Wilkinson, B. J.; Kim, Y.; Schmeling, D.; Douglas, S. D.; Quie, P. G.; Verhoef, J . J Clin Invest 1978, 67, 597- 609.PMC372572(113) Covalent Immune Proximity-Induction Strategy Using SuFEx-Engineered Bifunctional Viral Peptides. McCann, H. M.; Lake, B. P. M.; Hoffman, K. S.; Davola, M. E.; Mossman, K. L.; Rullo, A. F. ACS Chem Biol 2022, 17, 1269-1281(114) Factor H-IgG Chimeric Proteins as a Therapeutic Approach against the Gram-Positive Bacterial Pathogen Streptococcus pyogenes. Blom, A. M.; Magda, M.; Kohl, L.; Shaughnessy, J.; Lambris, J. D.; Ram, S.; Ermert, D. J Immunol 2017, 199, 3828-3839. PMC5698128(115) Antibiotic-chemoattractants enhance neutrophil clearance of Staphylococcus aureus. Payne, J. A. E.; Tailhades, J.; Ellett, F.; Kostoulias, X.; Fulcher, A. J.; Fu, T.; Leung, R.; Louch, S.; Tran, A.; Weber, S. A.; Schittenhelm, R. B.; Lieschke, G. J.; Qin, C. H.; Irima, D.; Peleg, A. Y.; Cryle, M. J. Nat Commun 2021, 72, 6157.PMC8546149(116) Protocol for detecting macrophage-mediated cancer cell phagocytosis in vitro and in vivo. Xu, C.; Wu, H.; Liu, Y.; Li, F.; Manne, R. K.; Lin, H.-K. STAR Protocols 2023, 4, 101940(117) Coupling of 02 integrins to actin by a mechanosensitive molecular clutch drives complement receptor-mediated phagocytosis. Jaumouille, V.; Cartagena-Rivera, A. X.; Waterman, C. M. Nature Cell Biology 2019, 27, 1357-1369(118) Complement, clq, and clq-related molecules regulate macrophage polarization. Bohlson, S. S.; O'Conner, S. D.; Hulsebus, H. J.; Ho, M. M.; Fraser, D. A. Front Immunol 2014, 5, 402.PMC4139736(119) p38MAPK, ERK and PI3K signaling pathways are involved in C5a-primed neutrophils for ANCA-mediated activation. Hao, J.; Meng, L. Q.; Xu, P. C.; Chen, M.; Zhao, M. H. PLoS One 2012, 7, e38317.PMC3365028(120) Chemotaxis inhibitory protein of Staphylococcus aureus, abacterial antiinflammatory agent, de Haas, C. J.; Veldkamp, K. E.; Peschel, A.; Weerkamp, F.; Van Wamel, W. J.; Heezius, E. C.; Poppelier, M. J.; Van Kessel, K. P.; van Strijp, J. A. J Exp Med 2004, 799, 687-95.PMC2213298(121) Hepatotoxicity of antibiotics. Hautekeete, M. L. Acta Gastroenterol Belg 1995, 58, 290-6(122) Drug-induced immune hemolytic anemia. Garratty, G. Hematology Am Soc Hematol Educ Program 2009, 73-9(123) Hepatic safety of antibiotics used in primary care. Andrade, R. J.; Tulkens, P. M. J Antimicrob Chemother 2011, 66, 1431-46.PMC3112029(124) Nephrotoxicity of Antimicrobials and Antibiotics. Morales- Alvarez, M. C. Adv Chronic Kidney Dis 2020, 27, 31-37(125) Drug-Induced Immune Hemolytic Anemia due to Amoxicillin-Clavulanate: A Case Report and Review. Chan Gomez, J.; Saleem, T.; Snyder, S.; Joseph, M.; Kanderi, T. Cureus 2020, 12, e8666.PMC7370667(126) Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Murray, C. J. L.; Ikuta, K. S.; Sharara, F.; Swetschinski, L.; Robles Aguilar, G.; Gray, A.; Han, C.; Bisignano, C.; Rao, P.; Wool, E.; Johnson, S. C.; Browne, A. J.; Chipeta, M. G.; Fell, F.; Hackett, S.; Haines-Woodhouse, G.; Kashef Hamadani, B. H.; Kumaran, E. A. P.; McManigal, B.; Achalapong, S.; Agarwal, R.; Akech, S.; Albertson, S.; Amuasi, J.; Andrews, J.; Aravkin, A.; Ashley, E.; Babin, F.-X.; Bailey, F.; Baker, S.; Basnyat, B.; Bekker, A.; Bender, R.; Berkley, J. A.; Bethou, A.; Bielicki, J.; Boonkasidecha, S.; Bukosia, J.; Carvalheiro, C.; Castaneda-Oijuela,C.; Chansamouth, V.; Chaurasia, S.; Chiurchiu, S.; Chowdhury, F.; Clotaire Donatien, R.; Cook, A. J.; Cooper, B ; Cressey, T. R.; Criollo-Mora, E.; Cunningham, M.; Darboe, S.; Day, N. P. J.; De Luca, M.; Dokova, K.; Dramowski, A.; Dunachie, S. J.; Duong Bich, T.; Eckmanns, T.; Eibach,D.; Emami, A.; Feasey, N.; Fisher-Pearson, N.; Forrest, K.; Garcia, C.; Garrett, D.; Gastmeier, P.; Giref, A. Z.; Greer, R. C.; Gupta, V.; Haller, S.; Haselbeck, A.; Hay, S. I.; Holm, M.; Hopkins, S.; Hsia, Y.; Iregbu, K. C.; Jacobs, J.; Jarovsky, D.; Javanmardi, F.; Jenney, A. W. J.; Khorana, M.; Khusuwan, S.; Kissoon, N.; Kobeissi, E.; Kostyanev, T.; Krapp, F.; Krumkamp, R.; Kumar, A.; Kyu, H. H.; Lim, C.; Lim, K.; Limmathurotsakul, D.; Loftus, M. J.; Lunn, M.; Ma, J.; Manoharan, A.; Marks, F.; May, J.; Mayxay, M.; Mturi, N. The Lancet 2022, 399, 629-655(127) The global distribution and spread of the mobilized colistin resistance gene mcr-1. Wang, R.; van Dorp, L.; Shaw, L. P.; Bradley, P.; Wang, Q.; Wang, X.; Jin, L.; Zhang, Q.; Liu, Y.; Rieux, A.; Dorai- Schneiders, T.; Weinert, L. A.; Iqbal, Z.; Didelot, X.; Wang, H.; Balloux, F. Nature Communications 2018, 9, 1179(128) Porous Silicon Nanoparticle Delivery of Tandem Peptide Anti-Infectives for the Treatment of Pseudomonas aeruginosa Lung Infections. Kwon, E. J.; Skalak, M.; Bertucci, A.; Braun, G.; Ricci, F.; Ruoslahti, E.; Sailor, M. J.; Bhatia, S. N. Adv Mater 2017, 29.PMC5765747(129) Engineering synthetic breath biomarkers for respiratory disease. Chan, L. W .; Anahtar, M. N.; Ong, T. H.; Hern, K. E.; Kunz, R. R.; Bhatia, S. N. Nat Nanotechnol 2020, 75, 792- 800.PMC8173716.Example 4 - Complement-recruiting chimeras for immunotherapies against microbial pathogens

[0285] The excessive and inappropriate use of antimicrobials has led to a dramatic surge in the number of multidrug-resistant bacteria. There is an urgent need for novel antibiotics. The complement system is part of the innate immune system and a natural defense mechanism against bacterial infections: 1. Classical pathway: Activated through antibody -mediated deposition of Clq on the bacterial surface; 2. Alternative pathway: Activated through direct deposition of C3 on the bacterial surface; 3. Mannan-binding lectin (MBL) pathway: Activated through MBL binding to lectin on the bacterial surface. Applicants developed chimeric molecules that recruit complement proteins to the surface of bacteria to locally activate the immune system. The approach holds the potential to fight against the emerging threat of multi drug-resistant bacteria.

[0286] Characteristic features of complement-recruiting chimeras: Exhibits catalytic turnover and can act on multiple targets; Typically requires a lower therapeutical dose; No need for high- affinity and inhibitory binders; Acts on extracellular targets (no need for cell permeability; not affected by efflux pumps); Highly modular: flexible assembly, rapid response to emerging threats; Orthogonal to existing antibiotics; Potentially generalizable to other disease areas.

[0287] Applicants developed chimeras that can activate either the classical or alternative complement pathway on the bacterial surface to locally induce an immune response.

[0288] The DEL platform allows to rapidly select binders for complement proteins.

[0289] Future directions: 1) Expand the platform to other gram negative and gram positive pathogens; 2) Demonstrate in vivo capability in a rodent infection model (P. aeruginosa), 3) Use DEL platform to identify chimeras for the MBL complement pathway.References for Example 41) Dunkelberger, J.R. and Song WC. “Complement and its role in innate and adaptive immune responses.” WT MCR1 5 10 15 20 25 Cell Research. 20:34-50 (2010).2) Hudson, L., Mason, J.W., Westphal, M.V. et al. “Diversity-oriented synthesis encoded by deoxyoligonucleotides.” Chimera retains CDC against colistin-resistant Chimera inhibits growth of PA 14 at Nat Commun.14, 4930 (2023).Example 5 - Develop lEDs that enhance the immunogenicity of bacterial and viral proteins

[0290] Multiple reports3’4and Applicants data show that MHC peptides covalently linked to small molecules exhibit tighter binding to MHC and enhance immunogenicity. The MHC peptides have anchoring residues (for peptide: MHC interaction) restricting the sequence space.11However, the appendage of organic moiety to peptides can enhance their binding to MHC- 1 via hydrophobic interactions or other mechanisms. Furthermore, the covalent modification of the target can alter peptide processing by the immunoproteasome. Using SARS-CoV-2 proteins12,13and those secreted by Mycobacterium tuberculosis into the macrophage (e.g., PtpA, SapM) and working with a proteomics platform,14 15 16Applicant’s will develop universal linkers that enhance the residence time of peptides on MHC (and higher immunogenicity), enabling MHC display of any peptide of interest.

[0291] Haptenizing Chimeras (HaCs) enable the display of non-canonical MHC peptides. Per contemporary algorithms that predict the sequence of MHC-1 displayed peptides, the cysteine- bearing peptides for most targets above are not predicted to be displayed. Typically, the MHC -I displayed peptides have anchoring residues to enhance the interaction between the peptide and MHC-1, which restricts the sequence space.32However, Applicant hypothesizes that the covalent addition of organic fragments to peptides enhances their binding to MHC-1 via potentially hydrophobic interactions. Furthermore, the covalent modification of the oncogene can alter the nature of peptides processed by the immunoproteasome. Thus, non-canonical peptides can be displayed by chemically modifying the target protein, enabling the generation of neo-antigens for targets not previously predicted to be displayed.

[0292] In example embodiments, the HaC has the formula according to:o OMeReferences for Example 5(3) A covalent inhibitor of K-Ras(G12C) induces MHC class I presentation of haptenated peptide neoepitopes targetable by immunotherapy. Zhang, Z.; Rohweder, P. J.; Ongpipattanakul, C.; Basu,K.; Bohn, M. F.; Dugan, E. J.; Steri, V.; Hann, B.; Shokat, K. M.; Craik, C. S. Cancer Cell 2022, 40, 1060-1069 e7(4) Creating MHC-Restricted Neoantigens with Covalent Inhibitors That Can Be Targeted by Immune Therapy. Hattori, T.; Maso, L.; Araki, K. Y.; Koide, A.; Hayman, J.; Akkapeddi, P.; Bang, I.; Neel, B. G.; Koide, S. Cancer Discov 2023, 13, 132-145.PMC9827112(11) A pattern search method for putative anchor residues in T cell epitopes. Hobohm, U.; Meyerhans, A. Eur J Immunol 1993, 23, 1271-6(12) Profiling SARS-CoV-2 HLA-I peptidome reveals T cell epitopes from out-of-frame ORFs. Weingarten- Gabbay, S.; Klaeger, S.; Sarkizova, S.; Pearlman, L. R.; Chen, D. Y.; Gallagher, K. M. E.; Bauer, M. R.; Taylor, H. B.; Dunn, W. A.; Tarr, C.; Sidney, J.; Rachimi, S.; Conway, H.L.; Katsis, K.; Wang, Y.; Leistritz- Edwards, D.; Durkin, M. R.; Tomkins-Tinch, C. H.; Finkel, Y.; Nachshon, A.; Gentili, M.; Rivera, K. D.; Carulli, I. P.; Chea, V. A.; Chandrashekar, A.; Bozkus, C. C.; Carrington, M.; Bhardwaj, N.; Barouch, D. H.; Sette, A.; Maus, M. V.; Rice, C.M.; Clauser, K. R.; Keskin, D. B.; Pregibon, D. C.; Hacohen, N.; Carr, S. A.; Abelin, J. G.; Saeed, M.; Sabeti, P. C. Cell 2021, 184, 3962-3980.el7.PMC8173604(13) The HLA-II immunopeptidome of SARS-CoV-2. Weingarten-Gabbay, S.; Chen, D. Y.; Sarkizova, S.; Taylor, H. B.; Gentili, M.; Hernandez, G. M.; Pearlman, L. R.; Bauer, M. R.; Rice,C. M.; Clauser, K. R ; Hacohen, N.; Carr, S. A.; Abelin, J. G.; Saeed, M.; Sabeti, P. C. Cell Rep 2023, 43, 113596(14) Mycobacterium tuberculosis suppresses innate immunity by coopting the host ubiquitin system. Wang, J.; Li, B. X.; Ge, P. P.; Li, J.; Wang, Q.; Gao, G. F.; Qiu, X. B.; Liu, C. H. Nat Immunol 2015, 16, 237-45(15) Mycobacterium tuberculosis Mce3E suppresses host innate immune responses by targeting ERK1 / 2 signaling. Li, J.; Chai, Q. Y.; Zhang, Y Li, B. X.; Wang, J.; Qiu, X. B.; Liu, C. H. J Immunol 2015, 194, 3756-67(16) Mycobacterium tuberculosis-secreted phosphatases: from pathogenesis to targets for TB drug development. Wong, D.; Chao, J. D.; Av-Gay, Y. Trends Microbiol 2013, 21, 100-9(32) A pattern search method for putative anchor residues in T cell epitopes. Hobohm, U.; Meyerhans, A. Eur J Immunol 1993, 23, 1271-6Example 6

[0293] Applicants developed a Class 1c chimera consisting of Fc-PMBN-based protein- peptide conjugate using Fc that is engineered to be Clq selective (Fig. 63D).74Applicants confirmed using BLI that the engineered Fc binds better to Clq than the wt Fc (Fig 63E). Next, Applicants prepared Fc-PMBN conjugate using sortase chemistry and demonstrated dosedependent E. coli growth inhibition (Fig. 63F).

[0294] For Class Ic chimeras that directly recruit the complement protein Clq, Applicants will vary the number of targeting ligands (PMBN) per Fc molecule using Sortase-mediated ligation (Fig. 64E).93 Here as well, Applicants will study the effect of linker length and rigidity on Clq recruitment. If needed, Applicants will also explore recently reported concatenation strategy where multiple Fc are linked using bi orthogonal click chemistry. Since antibodies have excellent pharmacological properties, Applicants expect enhancement in systemic efficacy with these Fc- PMBN conjugates. Moreover, the large size of the Fc protein relative to the PMBN allows room for optimization of cationic and hydrophobic contents and attenuates toxicity observed for PMBN analogs. (AC, Yrs 1-2).Aim 2. Development of chimeras that directly recruit Cig to bacteria.

[0295] For targeted and selective activation of classical pathways, Applicants will build chimeras from direct small molecule binders for Clq. The workflow for prioritization of chimeras will mirror those in Aim 1 .C.2.1. Preliminary data.C.2.1.1. DNA-encoded library screening (DELs).

[0296] DEL screening is an ultra-high throughput screening method that enables the simultaneous screening of millions of compounds in a single Eppendorf tube using only low microgram quantities of a protein. Each compound in the DEL library is appended to a DNA sequence that encodes for the compound’s identity. For screening, the protein is immobilized on a bead, incubated with DEL and unbound small molecules are washed away. Subsequent PCR amplification and sequencing provide identities of bound compounds. The DELs approach incorporates several features that are critical for success:

[0297] 1) Natural-product-like library with structural and stereochemical analogs: The DELs utilizes diversity-oriented synthesis (DOS)102that are rich in molecules with high rigidity and a high fraction of sp3- hybridized carbons. The natural- product- like DOS libraries have been particularly successful at furnishing potent and selective binders of microbial proteins.34'39Furthermore, each compound is built from three building blocks (i.e., “cycle’): a central scaffold and two side chain building blocks with stereoisomers.102This cycle approach allows deep stereochemistry (and structure)-activity relationship (SAR) information in DEL screening, enabling rapid identification of bona fide hits from false positives.103

[0298] 2) Innovative computational method to denoise screening data: The computational team (led by Drs. Paul Clemons and Connor Coley) has developed methods to reduce the noise inherent in DEL screening, including those for calculation of enrichment ratios (defined as enrichment of compound sequence in protein-loaded beads vs. control beads) with a range of confidence.104

[0299] 3) User-friendly app: The computational team has also built a user-friendly app that chemists can use to explore SAR via different data visualization approaches. For example, a chemist can use this app to generate 3D box plots with each building block / cycle shown on a separate axis. Each point in this plot represents a compound in the library, with the size of eachpoint proportional to its enrichment value in the screen. Failed screens or noisy binding data results in a 3D plot with diffuse, randomly scattered points that do not converge on any set of building block(s). In contrast, successful screens will result in patterns of building blocks converging on one or more axes, indicating enrichment of classes of compounds containing similar building blocks. The C3 screen displayed good enrichment of certain classes of building blocks, indicated by the formation of SAR ‘lines’ in the 3D box plots.

[0300] 4) Hits analogs readily available: Broad Institute’s in-house compound collection includes several analogs of compounds in DEL, dramatically speeding up hit optimization and lowering the chemistry workload. As Applicants complete the testing of remaining analogs, Applicants will now identify patterns from the SPR data to identify key pharmacophore following an approach Applicants have taken previously.53’60C.2.1.4. DNA-encoded library screening (DELs) on Chib.

[0301] Using the aforementioned DEL screening pipeline, Applicants also performed DEL screening on Clqb (Fig. 7A). The Clqb screen converged on azetidine and azetidine- RCM libraries (Fig. 7B). Representative compounds from these two classes with high enrichment ratio and target counts in the DEL screening were synthesized and validated for binding to Clqb using SPR. Next, Applicants identified 500 structurally similar compounds and completed testing 104 analogs in SPR assay (Fig. 7C). This effort led to the identification of BRD5001 and BRD7021 with Kd of 9 pM and 5.1 pM, respectively (Fig. 7D,E). Since C3 binders with similar affinity exhibited nanomolar MIC, Applicants will now generate chimeras and test them in cytolysis assay. C.2.2.2. Ligand optimization of Cl a.

[0302] The preliminary data suggests that binders with 0.5-1 pM potency will furnish chimeras with low nM MIC. Highly enriched libraries have been identified for Clq and following the above workflow(Fig. 7G).

[0303] For Clq, Applicants have obtained binders of ~ 5 pM affinity and Applicants will further optimize this affinity by testing the remaining 396 analogs. Applicants will also explore the activity of azetidine library members by SPR. Compounds will be tested at 10 and 20 pM concentrations and those with the highest SPR RU (Fig. 7G) will then be further tested in full dose to determine their binding affinity constants. Applicants expect this exhaustive SPR screening will generate robust stereochemistry / structure-activity data to identify the pharmacophore.

[0304] To identify the binding pockets, Applicants will follow the lysine-warhead approach as described in C.2.1.2. Briefly, the lysine warhead will be conjugated to the hit compound and dosedependent labeling will be assessed by clicking fluorophore and analyzing the mixture using SDS PAGE. MS / MS experiments will also be used to confirm the dose-dependent labeling. Upon identification of bona fide lysine, Applicants will use Schrodinger to identify the binding pocket and rationalize the binding potencies. Key information from previous studies such as stereocenters, ring conformations, functional group orientations, and side chain substituents, will be carefully altered to attain the target affinities in 0.5—1 pM range. Applicants will also use the lysine warhead- conjugated ligands for specificity studies by determining if such ligands crosslink other serum proteins (Fig. 7G) or mammalian cell surface proteins. Applicants will leverage SAR to reduce any off-targets that may arise. (AC, AS, Yrs 2-3)C.2.3.3. Generation of chimeras and determination of a ternary complex and CPC metric.

[0305] Same as C.1.2.2. The optimized high-affinity Clq ligands will be conjugated to PMBN to build chimeras, which will tested in assays described above. (AC, AS, Yrs 2-3)C.2.3.4. Validation of mechanism-of-action.

[0306] Same as C.1.2.3. (AC, AS, Yrs 2-3)C.2.3. Potential pitfalls and proposed solutions.

[0307] Here as well Applicants have outlined multiple strategies and Applicants anticipate recruitment of Clq will also induce a comparable antimicrobial response. Recruitment via three distinct pathways reduces the risk of microbial evasion from a specific pathway. Independent recruitment of the pathways may also modulate the degree of immune response and circumvent potential immune overreaction. Applicants anticipate the efficacy of Clq recruitment will likely be enhanced by higher affinity binders for these proteins. As such, Applicants will use DEL screening data, SPR, chemoproteomics, and docking studies to develop chimeras with nanomolar affinity for recruiting these proteins.Example 7Aim 2. Development of chimeras that directly recruit C3 or MBL to bacteria.

[0308] For targeted and selective activation of alternate and lectin pathways, Applicants will build chimeras from direct small molecule binders for C3 and MBL, respectively. The workflow for prioritization of chimeras will mirror those in Aim 1.C.2.1. Preliminary data.C.2.1.1. DNA-encoded library screening (DELs) on C3.

[0309] DEL screening is an ultra-high throughput screening method that enables the simultaneous screening of millions of compounds in a single Eppendorf tube using only low microgram quantities of a protein. Each compound in the DEL library is appended to a DNA sequence that encodes for the compound’s identity. For screening, the protein is immobilized on a bead, incubated with DEL and unbound small molecules are washed away. Subsequent PCR amplification and sequencing provide identities of bound compounds. The DELs approach incorporates several features that are critical for success:

[0310] 1) Natural-product-like library with structural and stereochemical analogs: The DELs utilizes diversity-oriented synthesis (DOS)102that are rich in molecules with high rigidity and a high fraction of sp3- hybridized carbons. The natural- product- like DOS libraries have been particularly successful at furnishing potent and selective binders of microbial proteins.34'39Furthermore, each compound is built from three building blocks (i.e., “cycle’): a central scaffold and two side chain building blocks with stereoisomers (Fig. 3A, B).102This cycle approach allows deep stereochemistry (and structure)-activity relationship (SAR) information in DEL screening, enabling rapid identification of bona fide hits from false positives.103

[0311] 2) Innovative computational method to denoise screening data: The computational team (led by Drs. Paul Clemons and Connor Coley) has developed methods to reduce the noise inherent in DEL screening, including those for calculation of enrichment ratios (defined as enrichment of compound sequence in protein-loaded beads vs. control beads) with a range of confidence (Fig. 3B).104

[0312] 3) User-friendly app: The computational team has also built a user-friendly app that chemists can use to explore SAR via different data visualization approaches. For example, a chemist can use this app to generate 3D box plots with each building block / cycle shown on a separate axis. Each point in this plot represents a compound in the library, with the size of each point proportional to its enrichment value in the screen (Fig. 3A). Failed screens or noisy binding data results in a 3D plot with diffuse, randomly scattered points that do not converge on any set of building block(s). In contrast, successful screens will result in patterns of building blocks converging on one or more axes, indicating enrichment of classes of compounds containing similarbuilding blocks (Fig. 3 A). The C3 screen displayed good enrichment of certain classes of building blocks, indicated by the formation of SAR Tines’ in the 3D box plots (Fig. 3A).

[0313] 4) Hits analogs readily available: Broad Institute’s in-house compound collection includes several analogs of compounds in DEL, dramatically speeding up hit optimization and lowering the chemistry workload. For example, the C3 screen converged on the Sultam library (Fig. 3B), which has 6677 analogs readily available. To confirm Sultam library as true hit, Applicants selected one of the analogs and validated for binding to C3 using BLI (Fig. 3C). Here, the biotinylated compound was immobilized on streptavidin biosensors and the binding affinities >20 pM were observed. Leveraging the in-house collection, Applicants assembled 831 structurally similar analogs of the Sultam hit and have completed testing of 204 analogs using a Surface Plasmon Resonance (SPR) assay (Fig. 3D). This approach allowed rapid identification of several binders (Fig. 3E-H) with deep structure and stereochemical activity relations ship. Currently, the best binder exhibit an affinity of 1.6 pM — a > 12-fold increase in potency form initial hit (Fig. 3F, G). Since the hits possess three stereocenters, Applicants tested binding of all the stereoisomes and observed a clear trend of (17?, 27?, 37?) outperforming others in the SPR assay (Fig. 3G). The stereochemical dependence on binding points to the specific nature of molecular interactions between the ligand and C3 (Fig. 3G). As Applicants complete the testing of remaining analogs, Applicants will now identify patterns from the SPR data to identify key pharmacophore following an approach Applicants have taken previously.53’60C.2.1.2. Binding-pocket identification.

[0314] An examination of structures in Protein Data Bank (PDB) with bound small molecules suggests that nearly 97 % of proteins have a lysine residue near the bound small molecule (Fig. 1OA).105Applicants had previously used a diazirine-based photo-crosslinking method to identify the binding pocket of a small molecule. However, this method is prone to false positives, requiring specialized instruments and software (and takes ~4-6 months to identify binding pocket).106

[0315] Applicants have developed a miniature lysine-reactive warhead, which Applicants appended to the DEL hit to identify lysine proximal to the binding pocket (Fig. 4B). Here, the warhead transfers an alkyne handle on the lysine of C3 protein to which a fluorophore can be appended using click chemistry (Fig. 4B). Applicants observed a dose-dependent labeling of C3 (Fig. 4C) with most of the labeling occurring on the a chain (Fig. 4D) — the a chain has a highermolecular weight than the P chain and the two chains can be separated using reducing conditions. Tryptic digestion of the labeled C3 and subsequent MS / MS led to the identification of lysine-1436 in the C3a chain as target lysine (Fig. 4E, F). Dose-dependent labeling C3 was also observed using mass spectrometry — Applicants observed a dose-dependent increase in the intensity of the total ion current (TIC) of the labeled peptide (Fig. 4E).

[0316] Subsequently, Applicants conducted docking studies using all the stereoisomers of the hit (BRD8499) using the Maestro program from Schrodinger.107’108Notably, the docking conformation of the (1R2R3R) stereoisomer that exhibited good affinity (Ad = 2.92 pM) showed good fitting within the pocket with the sulfonamide group oriented toward the exit site of the pocket (Fig. 4G). This orientation agrees with the DNA attachment site (terminal CH20H) in BRD8499. In contrast, the (1S2R3S) stereoisomer, exhibiting lower affinity (Ad ~ 30 pM) was unable to dock within the binding pocket.C.2.1.3. Chimeras formed from C3 binder induces CDC in PsA with MIC ~ 300 nM.

[0317] Applicants synthesized a chimera from the identified C3 binder (BRD6432, Fig. 3E-F) to PMBN (Fig. 4H) Upon incubation with chimera 6 in the presence of 5% complement preserved serum led growth suppression for up to 24 hours, demonstrating an MIC50 in the 150-300 nM range (Fig. 4H). Applicants note that BRD6432 has a binding affinity of 5 pM and the low nanomolar MIC can be attributed to the turnover / catalytic nature of the chimeras as well as the high concentration of the complement proteins present in the serum. Thus, ultrahigh affinity binders are not necessarily needed to attain low MIC.

[0318] C.2.2. Experimental plan.C.2.2.1. DEL screening on MBL, an initiator of the lectin pathway.

[0319] Applicants have initiated bead binding optimization experiments on his- tagged MBL and found that 5 pM MBL was sufficient for bead saturation (Fig. 5)— optimizing bead saturation is important as undersaturated beads can lead to non- specific binding. DEL screening, analysis, and hit validation will be performed as outlined for C3 and Clq. (AC, AS, Yr 1)C.2.2.2. Ligand optimization of C3 andMBE

[0320] The preliminary data suggests that binders with 0.5-1 pM potency will furnish chimeras with low nM MIC. Highly enriched libraries have been identified for C3 and following the above workflow, Applicants will identify promising libraries for MBL (Fig. 5). For C3, 204compounds out of 831 analogs of Sultam library has been tested in SPR (6677 analogs available) and Applicants will conclude the testing of the remaining analogs.

[0321] Compounds will be tested at 10 and 20 pM concentrations and those with the highest SPRRU (Fig. 5) will then be further tested in full dose to determine their binding affinity constants. Applicants expect this exhaustive SPR screening will generate robust stereochemistry / structure- activity data to identify the pharmacophore.C.2.3.3. Generation of chimeras and determination of a ternary complex and CDC metric.

[0322] Same as C.l.2.2. The optimized high-affinity C3 and MBL ligands will be conjugated to PMBN to build chimeras, which will tested in assays described above. (AC, AS, Yrs 2-3) C.2.3.4. Validation of mechanism-of-action. Same as C.l.2.3. (AC, AS, Yrs 2-3)C.2.3. Potential pitfalls and proposed solutions.

[0323] Here as well Applicants have outlined multiple strategies and already shown that C3 recruitment is sufficient to trigger CDC on PsA, and Applicants anticipate recruitment of MBL will also induce a comparable antimicrobial response. Recruitment via three distinct pathways reduces the risk of microbial evasion from a specific pathway. Independent recruitment of the pathways may also modulate the degree of immune response and circumvent potential immune overreaction. Applicants anticipate the efficacy of MBL and C3 recruitment will likely be enhanced by higher affinity binders for these proteins. As such, Applicants will use DEL screening data, SPR, chemoproteomics, and docking studies to develop chimeras with nanomolar affinity for recruiting these proteins.***

[0324] Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.

Claims

CLAIMSWhat is claimed is:

1. A complement recruiting chimeric (CRC) molecule according to the formula R1-L-R2, wherein Ri is a pathogen binding moiety;R2 is a complement recruiting moiety; andL is a linker, wherein the linker may optionally comprise an electrophilic reactive group.

2. The CRC molecule of claim 1, wherein the complement recruiting moiety is an antibody binding moiety that binds an antibody that recruits one or more components of the complement system.

3. The CRC molecule of claim 3, wherein the compliment recruiting moiety binds to the light chain or the heavy chain of the antibody.

4. The CRC molecule of claim 3, wherein the compliment recruiting moiety binds to the antigen binding site of the light chain.

5. The CRC molecule of any one of claims 2-4, wherein the antibody binding moiety is a small molecule, a polypeptide, or a hapten.

6. The CRC molecule of claim 5, wherein the hapten is 2,4-dinitrophenyl (DNP)," '> :, or an analog thereof, and wherein the hapten is bound by an anti -DNP antibody.

7. The CRC molecule of any one of claims 2-6, wherein the antibody binding moiety comprisesrhamnose, , galactose, , or an analog or derivative thereof.

8. The CRC molecule of any one of claims 2-7, wherein the antibody binding moiety comprises or an analog or derivative thereof.

9. The CRC molecule of any one of claims 2-8, wherein the antibody binding moiety comprises of10. The CRC molecule of any one of the preceding claims, wherein the pathogen is a virus, bacteria, fungi, or protozoa.

11. The CRC molecule of claim 10, wherein the bacteria is a gram-negative bacteria.

12. The CRC molecule of claim 11, wherein the gram-negative bacteria pathogen is Pseudomonas Aeruginosa (PsA).

13. The CRC molecule of any of the preceding claims, wherein the pathogen binding moiety binds a protein, lipopolysaccharide, or a polysaccharide.

14. The CRC molecule of claim 13, wherein the protein, lipopolysaccharide, or polysaccharide is pathogen-specific.

15. The CRC molecule of any of the preceding claims, wherein the pathogen binding moiety is a peptidomimetic antibiotic macrocycle, or a lectin binder.

16. The CRC molecule of any of the preceding claims, wherein the pathogen binding moiety comprises of glucopyranoside, fucopyranoside, or an analog or derivative thereof.

17. The CRC molecule of any of the preceding claims, wherein the pathogen binding moiety comprises of: an analog or derivative thereof.

18. The CRC molecule of any of claims 1-14, wherein the pathogen binding moiety is a colistin-targeting liposaccharide.

19. The CRC molecule of any one of claims 1-14, wherein the pathogen binding moiety comprises:

20. The CRC molecule of any one of claims 1-14, wherein the pathogen binding moiety comprises: / OH or an analog or derivative thereof.

21. The CRC molecule of any one of claims 1-14, wherein the pathogen binding moiety is murepavadin, polymyxin, vancomycin, or an analog or derivative thereof.

22. The CRC molecule of any of the previous claims, wherein L comprises alkane; alkene; alkyne; amine; ether; thiol; sulfone; carbonyl; acyl; ketone; carboxylate ester; amide; enone; anhydride; imide; PEG; or any combination thereof.

23. The CRC molecule of any of the preceding claims wherein L comprises:n; or an analog or derivative thereof, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14; or an analog or derivative thereof.

25. The CRC molecule of any of the preceding claims wherein L comprises:O CN OI ,nand n is 1, 2, 3, 4, or 5;0 0 iii) n and n is between 0 and 6.

26. The CRC molecule of claim 1 wherein L is selected from: '' or27. The CRC molecule of claim 1 or 2 according to the formula: j.( ,U. _ . R'S-* , wherein R1is the pathogen binding moiety and R2is the antibody binding moiety.

28. The CRC molecule of claim 27, wherein R1is galactopyranoside and R2is DNP.

29. The CRC molecule of claim 1 or 2 according to the formula:, wherein R1is the pathogen binding moiety and R2is the antibody binding moiety.

30. The CRC molecule of claim 30, wherein R1is fucopyranoside and R2is DNP.

31. The CRC molecule of claim 1 or 2 according to the formula:wherein the top R1and bottom R1are independently selected pathogen binding moieties and R2is the antibody binding moiety.

32. The CRC molecule of claim 32, wherein the top R1and bottom R1are fucopyranoside and R2is DNP.

33. The CRC molecule of claim 1 or 2 according to the formula:, wherein R1is the pathogen binding moiety and R2is the antibody binding moiety.

34. The CRC molecule of claim 33, wherein R1is polymyxin B and R2is DNP.

35. The CRC molecule of claim 1 or 2 according to the formula: wherein the circle comprises ofthe triangle comprises of any of the hexagon comprises of and optionally comprise an electrophilic reactive group.

36. The CRC molecule of claim 1 or 2 according to the formula: wherein the triangle comprises of the complement recruiting moiety.

37. The CRC molecule of claim 36, wherein the complement recruiting moiety is a FcIII- peptide.

38. The CRC molecule of claim 1 or 2 according to the formula:AD616H2NJM.QH39. The CRC molecule of claim 1 or 2 according to the formula:wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12.

40. The CRC molecule of claim 1 or 2 according to the formula:AD616or41. A method of targeted activation of the complement immune system against pathogens comprising administering to a cell or cell population a molecule of any one of claims 1 to 40, wherein binding of the complement recruiting moiety to an antibody activates the complement immune system against a target pathogen.

42. A method of treating pathogenic infections comprising administering to a cell or cell population a molecule of any one of claims 1 to 40, wherein binding of the complement recruiting moiety to an antibody activates the complement immune system against a target pathogen.

43. The method of claim 41 or 42, wherein the pathogen is a virus, bacteria, fungi, or protozoa.

44. The method of claim 43, wherein the gram-negative bacteria pathogen is Pseudomonas aeruginosa (PsA).

45. A complement recruiting chimeric (CRC) molecule comprising an antibody fragment bound to one or more pathogen binding moieties via one or more linkers, wherein the linker may optionally comprise an electrophilic reactive group.

46. The CRC molecule of claim 45, wherein the antibody fragment is an Fc fragment.

47. The CRC molecule of claim 46, wherein the Fc fragment is an IgG Fc fragment.

48. A CRC molecule according to the formulaR1-L-R2, wherein Ri is a pathogen binding moiety;R2 is a complement recruiting moiety; andL is a linker, wherein the linker may optionally comprise an electrophilic reactive group.

49. The CRC molecule of claim 48, wherein the complement recruiting moiety binds one or more Clq polypeptides.

50. The CRC molecule of claim 49, wherein the complement recruiting moiety binds a Clq A-chain, B-chain, C-chain, or a combination thereof.

51. The CRC molecule of any one of claims 48 to 50, wherein the complement recruiting moiety is a small molecule, a polypeptide, or a hapten.

52. The CRC molecule of any one of claims 45-51, wherein the compliment recruiting moiety comprises of wherein R1 comprises ofR2 comprises of53. The CRC molecule of any one of claims 45-52, wherein the pathogen is a virus, bacteria, fungi, or protozoa.

54. The molecule of claim 53, wherein the bacteria is a gram-negative bacteria.

55. The molecule of claim 54, wherein the gram-negative bacteria pathogen is Pseudomonas aeruginosa (PsA).

56. The CRC molecule of any one of claims 45-55, wherein the pathogen binding moiety binds a protein, lipopolysaccharide, or a polysaccharide.

57. The molecule of claim 56, wherein the protein, lipopolysaccharide, or polysaccharide is pathogen-specific.

58. The CRC molecule of any one of claims 45-57, wherein the pathogen binding moiety is a peptidomimetic antibiotic macrocycle, or a lectin binder.

59. The CRC molecule of any one of claims 45-58, wherein the lectin binder is LecA (PAIL) or LecB (PA-IIL).

60. The CRC molecule of any one of claims 45-59, wherein the pathogen binding moiety comprises of glucopyranoside, fucopyranoside, or an analog or derivative thereof.

61. The CRC molecule of any one of claims 45-60, wherein the pathogen binding moiety comprises of:an analog or derivative thereof.

62. The molecule of any of claims 45-57, wherein the pathogen binding moiety is a colistintargeting liposaccharide.

63. The molecule of any one of claims 45-57, wherein the pathogen binding moiety comprises:

64. The CRC molecule of any one of claims 45-57, wherein the pathogen binding moiety comprises: / OH or an analog or derivative thereof.

65. The CRC molecule of any one of claims 45-57, wherein the pathogen binding moiety is murepavadin, polymyxin, vancomycin, or an analog or derivative thereof.

66. The CRC molecule of any one of claims 45-65, wherein the linker(s) are individually selected from: alkane; alkene; alkyne; amine; ether; thiol; sulfone; carbonyl; acyl; ketone; carboxylate ester; amide; enone; anhydride; imide; PEG, or any combination thereof.

67. The CRC molecule of any one of claims 45-66, wherein linker(s) are selected from:n; or an analog or derivative thereof, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

68. The CRC molecule of any one of claims 45 to 65 claims, wherein the linker(s) are selected from:or derivative thereof.

69. The CRC molecule of any one of claims 45 to 65 claims, wherein the linker(s) are selected from:i) the group consisting of70. The CRC molecule of any one of claims 45 to 65, wherein the linker(s) are selected from: or71. A method of targeted activation of the complement immune system against pathogens comprising administering to a cell or cell population a molecule of any one of claims 45 to 47, wherein the antibody fragment induces recruitment of the complement immune system upon binding of the one or more pathogen moieties to a target pathogen.

72. A method of targeted activation of the complement immune system against pathogens comprising administering to a cell or cell population a molecule of any one of claims 48 to 52, wherein the complement recruiting moiety induces recruitment of the complement immune system upon binding of the pathogen binding moiety to a target pathogen.

73. The method of claim 71 or 72, wherein the pathogen is a virus, bacteria, fungi, or protozoa.

74. The method of claim 73, wherein the bacteria is a gram-negative bacteria.

75. The method of claim 74, wherein the gram-negative bacteria pathogen is Pseudomonas aeruginosa (PsA).

76. A method of treating pathogenic infections comprising administering to a cell or cell population a molecule of any one of claims 45 to 47, wherein the antibody fragment induces recruitment of the complement immune system upon binding of the one or more pathogen moi eties to a target pathogen.

77. A method of treating pathogenic infections comprising administering to a cell or cell population a molecule of any one of claims 48 to 52, wherein the complement recruiting moiety induces recruitment of the complement immune system upon binding of the pathogen binding moiety to a target pathogen.

78. The method of claim 76 or 77, wherein the pathogen is a virus, bacteria, fungi, or protozoa.

79. The method of claim 78, wherein the gram-negative bacteria pathogen is Pseudomonas aeruginosa (PsA).

80. A complement recruiting chimeric (CRC) molecule according to the formula R1-L-R2, wherein Ri is a pathogen binding moiety;R2 is a complement recruiting moiety; andL is a linker, wherein the linker may optionally comprise an electrophilic reactive group.

81. The CRC molecule of claim 80, wherein the complement recruiting moiety binds to C3 or C5.

82. The CRC molecule of any one of claims 80-81, wherein the complement recruiting moiety is a small molecule, a polypeptide, or a hapten.

83. The CRC molecule of any one of claims 80-82, wherein the complement recruiting moiety comprises of: wherein R2comprises of84. The CRC molecule of claim 80, wherein the complement recruiting moiety isor an analog or derivative thereof.

85. The CRC molecule of any one of claims 80-84, wherein the pathogen binding moiety binds a virus, bacteria, fungi, or protozoa.

86. The molecule of claim 10, wherein the bacteria is a gram-negative bacteria.

87. The molecule of claim 11, wherein the gram-negative bacteria pathogen is Pseudomonas Aeruginosa (PsA).

88. The CRC molecule of any one of claims 80-87, wherein the pathogen binding moiety binds a protein, lipopolysaccharide, or a polysaccharide.

89. The molecule of claim 13, wherein the protein, lipopolysaccharide, or polysaccharide is pathogen-specific.

90. The CRC molecule of any one of claims 80-89, wherein the pathogen binding moiety is a peptidomimetic antibiotic macrocycle, or a lectin binder.

91. The CRC molecule of any one of claims 80-90, wherein the lectin binder is LecA (PAIL) or LecB (PA-IIL).

92. The CRC molecule of any one of claims 80-91, wherein the pathogen binding moiety comprises of glucopyranoside, fucopyranoside, or an analog or derivative thereof.

93. T The CRC molecule of any one of claims 80-92, wherein the pathogen binding moiety comprises:or an analog or derivative thereof.

94. The CRC molecule of any one of claims 80-93, wherein the pathogen binding moiety is a colistin-targeting liposaccharide.

95. The CRC molecule of any one of claims 80-88, wherein the pathogen binding moiety96. The CRC molecule of any one of claims 80-88, wherein the pathogen binding moiety comprises: / OH or an analog or derivative thereof.

97. The CRC molecule of any one of claims 80-88, wherein the pathogen binding moiety is murepavadin, polymyxin, vancomycin, or an analog or derivative thereof.

98. The CRC molecule of any one of claims 80-97, wherein the linker is selected from: alkane; alkene; alkyne; amine; ether; thiol; sulfone; carbonyl; acyl; ketone; carboxylate ester; amide; enone; anhydride; imide; PEG, or any combination thereof.

99. The CRC molecule of any one of claims 80-98, wherein the linker is selected from:n; or an analog or derivative thereof, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14; or an analog or derivative thereof.

101. The CRC molecule of any one of claims 80-100, wherein the linker is selected from:O i) the group consisting ofHN NH iii) and n is between 0 and 6.

102. The CRC molecule of claim 80 wherein L is selected from: or103. The CRC molecule of claim 80 according to the formula: wherein 2, 5, and 6 are independently selected from R or 5;R1comprises:, Br, or an analog or derivative thereof; andR2comprises: an analog or derivative thereof.

104. The CRC molecule of claim 80, according to the formula:wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and positions 2, 5, and 6 are independently selected from R and 5.

105. A method of targeted activation of the complement immune system against pathogens comprising administering to a cell or cell population a molecule of any one of claims 80 to 104, wherein binding of C3 or C5 activates the complement system upon binding of the pathogen binding moiety to a target pathogen.

106. A method of treating pathogenic infections comprising administering to a cell or cell population a molecule of any one of claims 80 to 104, wherein binding of C3 or C5 activates the complement system upon binding of the pathogen binding moiety to a target pathogen.

107. The method of claim 105 or 106, wherein the pathogen is a virus, bacteria, fungi, or protozoa.

108. The method of claim 107, wherein the gram -negative bacteria pathogen is Pseudomonas aeruginosa (PsA).

Images