Small-molecule inhibitors of c1s protease and classical complement pathway

Abstract

A small-molecule C1 s inhibitor compound. A competitive inhibitor of C1 s, the compound having the structure of formula A1 comprising 3 rings designated as A, B, and C, and an amidine group attached to the A-ring: pharmaceutically-acceptable salts thereof, prodrugs thereof, or analogs thereof comprising an effective substitution at the A, B, and / or C ring or amidine group. Methods of inhibiting C1 s activation and treating or preventing complement-mediated diseases or conditions.

Description

[0001] SMALL-MOLECULE INHIBITORS OF CIS PROTEASE AND CLASSICAL COMPLEMENT PATHWAY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 612,044, filed December 19, 2023, and Serial No. 63 / 593,780, filed October 27, 2023, each entitled SMALL-MOLECULE INHIBITORS OF CIS PROTEASE AND CLASSICAL COMPLEMENT PATHWAY, and each incorporated by reference in its entirety herein.

[0004] BACKGROUND OF THE INVENTION

[0005] Field of the Invention

[0006] The present invention relates to inhibitors of complement activation, and in particular, small-molecule Cis inhibitors, compositions thereof, treatment methods using the same, and methods of making the same.

[0007] Description of Related Art

[0008] The complement system (CS) plays a critical role in innate host defense and tissue homeostasis. However, the CS is also associated with various diseases, such as autoimmune, inflammatory, and neurodegenerative diseases. When it becomes dysregulated or altered, CS activation can be prolonged or misdirected toward healthy cells within the host. Inhibitors of the CS could provide novel therapeutic interventions against such complement-mediated conditions.

[0009] The CS is activated via three different initiation pathways: the classical pathway (CP), lectin pathway (LCP), or alternative pathway (AP). The early events in all pathways consist of a sequence of cleavage reactions in which the larger cleavage product binds covalently to a surface (e.g., of a pathogen) or other nearby biomaterial and contributes to the activation of the next component. The CP is a potent mechanism for initiating complement activity and is a driver of pathology in many complement-mediated diseases. The CP is initiated via activation of complement component Cl, which consists of Clq, Clr, and Cis proteins, in which pattern recognition molecule Clq is bound to a tetrameric assembly of proteases Clr and Cis. Upon binding of Cl to an immune complex or other activator, the Cis component, a serine protease, cleaves complement components C4 and C2 to initiate activation of the complement pathway. Thus, enzymatically active Cis provides the catalytic basis for cleavage of the downstream CP components, C4 and C2, and is therefore an attractive target for therapeutic intervention in CP- driven diseases.

[0010] Inhibitors of Cis activity have been described in the literature. However, many such inhibitors that are both known and publicized are macromolecules or biologies (e.g., proteins). These include: (i) Cl -inhibitor (commonly abbreviated Cl -INH in the literature), which is the body’s physiological regulator of Cis activity, (ii) Sutimlimab, an inhibitory monoclonal antibody developed initially by True North Therapeutics (now Bioverativ, part of Sanofi), and (iii) Gigastasin, an inhibitor found in the saliva of the Giant Amazon Leach, Haementeria ghilianii. Some small molecule inhibitors have also been described. Yet, these molecules are either promiscuous inhibitors of many serine proteases (e.g., Futhan, also known as ‘nefamostat mesylate’; chemical name 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate) or they have not been thoroughly evaluated in both in vitro assays of Cis enzyme activity or functional assessments of complement activity. These gaps have presented an opportunity for discovery and improvement of the current state of the art. Further, although an anti-Cls monoclonal antibody has been FDA-approved, identifying chemically-defined, small-molecule Cis inhibitors remains a priority.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention is broadly concerned with a family of small molecule compounds that are effective as selective competitive inhibitors of Cis, the compound comprising formula Al comprising 3 rings designated as A, B, and C, and an amidine group attached to the A Ring: and the analogs thereof comprising any effective substitution at the A, B, and / or C Ring, or amidine group. For example, analogs are contemplated herein where the C Ring is a substituted phenyl with a halogen or alkyl substitution. As used herein, the term “analog” refers to chemical analogs of Al and specifically a variant compound of Al having a chemical structure similar to that of Al, but differing from it in at least one aspect, while retaining the Cis binding and inhibitory activity. In particular, the analogs are variants derived from the scaffold of Al, preferably where the A, B, and / or C Ring or amidine group has been modified, while still retaining Cis binding and inhibitory activity (although such activity may be higher or lower than the original Al scaffold). In one or more embodiments, the compounds dose-dependently inhibit CP activation by heparin-induced immune complexes, CP-driven lysis of antibody-sensitized erythrocytes, CP activation in a pathway-specific ELISA, and cleavage of C2 by Cis. The Examples exemplify a series of small molecule compounds, including Compound Al and its analogs.

[0013] The present disclosure provides small molecule compounds that bind to and inhibit the activity of complement protein Cis. The present disclosure also provides compositions comprising such small molecule compounds and methods of using such small molecule compounds in therapeutic interventions.

[0014] Biochemical experiments demonstrated that Compound Al binds directly to C 1 s with a Kd of ~9.8 pM and competitively inhibits its enzymatic activity with a Ki of ~5.8 pM. A 1.8 A resolution crystal structure revealed the physical basis for Cis inhibition by Al and provided information on the structure-activity relationship of the Al scaffold, which was supported by evaluating a panel of Al analogs. Together, our work identifies Al as a new class of smallmolecule Cis inhibitor and lays the foundation for development of increasingly potent and selective Al analogs for both research and therapeutic purposes.

[0015] The present disclosure provides therapeutic compositions comprising small molecule inhibitors of Cis according to any of the embodiments disclosed herein and a pharmaceutically acceptable carrier. Some embodiments include a sterile container comprising such a composition. In some embodiments, the container is selected from the group consisting of a bottle and a syringe.

[0016] The present disclosure provides methods of treating a subject having or at risk of a complement-mediated disease, disorder, or condition. The methods generally comprise administering to the subject a small molecule inhibitor of Ci s of any of the embodiments disclosed herein or a therapeutic composition thereof.

[0017] A kit is also provided herein. The kit comprises: a small molecule inhibitor of Cis according to the various embodiments described herein; and instructions for administering the compound to a subject in need thereof.

[0018] Methods of preventing or inhibiting Cis activation in a subject are also disclosed. The methods generally comprise administering to the subject a small molecule inhibitor of Cis of any of the embodiments disclosed herein or a therapeutic composition thereof.

[0019] The disclosure is also concerned with the use of a compound according to the various embodiments described herein to prepare a therapeutic or prophylactic medicament for preventing or inhibiting Cis activation in a subject in need thereof or treating a subject having or at risk of a complement-mediated disease, disorder, or condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0021] FIG. 1. Screening Workflow for the Identification of Novel Small-Molecule Cis Inhibitors. AtomNet, a deep learning neural network developed by Atomwise, was used to screen a molecular library of several million compounds for small molecules that bind near the Cis active site. The crystal structure of C1S-2SP was used as the basis for the screen (PDB: 1ELV). Top scoring compounds were filtered to select for favorable physicochemical properties, including solubility, and a selection of 94 distinct compounds were obtained commercially for testing. Each of these 94 compounds was tested for direct binding to Cis by SPR, inhibition of Cis in purified enzyme assays, and inhibition of the CP in serum-based assays. Based on its performance across all three assay formats, compound Al (i.e., 6-(4-phenylpiperazin-l-yl)pyridine-3- carboximidamide, or alternatively known as 6-(4-phenylpiperazin-l-yl)nicotinimidamide) was selected for further characterization. The dose-dependent binding and inhibitory properties of Al were evaluated in five independent assays of complement function and a 1.8 A crystal structure of a Cis fragment inhibited by Al was obtained. SAR was then developed by synthesis and characterization of a limited series of Al analogs.

[0022] FIG. 2. Evaluation of Cis Binding and Inhibition by Putative Hit Compounds. (A) The pool of 94 candidate hit molecules was screened initially for solubility in HBS-T + 5% (v / v) DMSO. Those that exhibited signs of precipitation were excluded (compounds E3-E12, grey open circles). All other compounds were assayed at 100 pM for direct binding to Cl s-2SP that had been previously immobilized on an SPR sensor chip. Six compounds exhibited binding responses greater than the mean value + S.D., as judged by the background- and solvent-corrected resonance units (RU) immediately prior to injection stop (Al, purple; D4, teal; DIO, pink; F2 grey; Hl, brown; H9, cyan). (B) The ability of Cls-2SP to cleave the substrate ZGR was measured in the presence of 100 pM of each compound. The initial reaction velocities from individual trials were plotted and compared to the mean. Four molecules diminished Cis activity to a rate of less than one standard deviation below the mean (Al, purple; C7, blue; E3, blue-green; and E6, orange). (C) The activity of the CP was measured in triplicate by ELISA in the presence of 100 pM compound. Microtiter plates were coated with human IgM, washed, and incubated at 37 °C with a 2% (v / v) dilution of pooled, complement preserved human serum in a Ca2+and Mg2+-supplemented buffer that contained 100 pM of each compound. Following a series of washes, CP activity in the presence of each compound was quantified by detection with an anti-C4b antibody coupled to colorimetric development with an HRP -conjugated secondary antibody. Mean values are presented for each compound. Only two molecules diminished CP activity to less than two standard deviations below the mean (Al, purple and B5, light brown). Since Al was the lone compound that met each of these screening criteria, it was carried forth for further analyses. The outcome of screening assays shown in panels A-C are data representative of two replicates.

[0023] FIG. 3. Compound Al is a Dose-Dependent Inhibitor of the Classical Pathway. Al was tested for the ability to dose-dependently inhibit CP activity in a series of functional assays. (A) ACD-anticoagulated whole blood was incubated with either a dilution series of Al ranging from 1,000 - 10 pM, BmA-C (positive control), BmB-C (negative control), or buffer alone, and combined with samples of an anti-PF4 / heparin monoclonal antibody and PF4 / heparin antigen to form immune complexes. Each reaction was incubated at 37 °C for 30 min, and the extent of CP activity was assessed by isolating the plasma fraction and processing its contents for anti-C5a ELISA. The outcomes of triplicated measurements are shown with the error bars representing the standard deviation. Statistical significance was assessed by unpaired t-test of the relevant experimental groups versus buffer alone: *, p < 0.05, ***, p < 0.005. (B) A fixed dilution of complement preserved AS was mixed with a dilution series of Al ranging from 100 - 3.1 pM. Following addition of the serum mixture to antibody-sensitized sheep erythrocytes, each reaction was incubated at 37 °C for 1 h, and the extent CP -mediated hemolysis was measured spectrophotometrically. The outcomes of triplicate measurements are shown as the mean (filled circles) with error bars representing the standard deviation. The change in hemolysis (%) versus Al concentration was fit to a dose-response curve (solid line). The apparent IC50 for Al in this assay is 16 pM. (C) A fixed dilution of complement preserved AS was mixed with a two-fold dilution series of Al ranging from 100 - 3.1 pM. Following addition of the serum mixture to IgM- coated microtiter wells, each reaction was incubated at 37 °C for 1 h, and the activity of the CP was measured by ELISA using levels of C4b deposition as a readout. The outcome of triplicate measurements is shown as the mean (filled circles) with error bars representing the standard deviation. The change in complement activity versus concentration was fit to a dose-response curve (solid line). The apparent IC50 for Al in this assay is 22 pM. (D-E) The ability of Al to inhibit Cis cleavage of C2 was measured using an electrophoresis-based assay that monitored the appearance of the cleavage product C2b. A two-fold dilution series of Al ranging from 100 - 1.56 pM was prepared and added to reactions containing 6.25 nM Ci s and 670 nM C2. Following incubation at 37 °C for 1 h, the reaction contents were separated by SDS-PAGE. The results of densitometry analysis on triplicate measurements are shown in panel D, while a representative gel is shown in panel E. The experiments shown in panels A-E were performed a minimum of three times.

[0024] FIG. 4. Compound Al is a Competitive Inhibitor of Cis. The influence of increasing concentrations of Al on the activity of Cis was examined by steady-state kinetics. Note that symbols identifying the plots corresponding to various concentrations of Al are inset, with values in micromolar. (A) Apparent turnover number of Cis purified from human serum versus substrate concentration in the presence of five different concentrations of Al. Data were collected in triplicate. Fitting these data to a competitive inhibition model yields a Ki of 5.8 pM. (B) Analogous experiment to that in Panel A, except for using recombinant Cls-2SP expressed and purified from E. coli. Fitting these data to a competitive inhibition model yields a Ki of 8.5 pM. (C) Analogous experiment to that in Panel A, except for using recombinant mouse C 1 s-2SP expressed and purified from E. coli. Fitting these data to a competitive inhibition model yields a Ki of 24.6 pM. (D) Analogous experiment to that in Panel A, except for using Clr purified from human serum. Fitting these data to a competitive inhibition model yields a Ki of 842 pM. The experiments shown in panels A-D were performed a minimum of three times.

[0025] FIG. 5. Crystal Structure of Human eCls-2SP Inhibited by Al at 1.8 A Resolution. Preformed crystals of eCls-2SP were incubated with saturating concentrations of Al prior to crystal harvest. Following collection of X-ray diffraction data to 1.8 A limiting resolution, the structure was solved by molecular replacement and refined to an Rfreevalue of 19.6%. (A) Representation of the eCl s-2SP active site following initial structure solution and refinement, but prior to modeling Al . The 2Fo-Fc electron density map contoured at 1 ,3G is shown as a blue cage, while the Fo-Fc electron density map contoured at 3.0c is shown as a green cage. eCls-2SP is drawn in ball-and-stick convention with its carbon atoms colored orange. Residues corresponding to the catalytic triad appear with their carbon atoms colored silver. (B) Representation of the final model for eCls-2SP inhibited by Al. eCls-2SP is shown as an orange molecular surface, with the exception that atoms from catalytic triad residues are colored silver (carbon), red (oxygen), and blue (nitrogen). The Al molecule is drawn in ball-and-stick convention with its carbon atoms in yellow. The 2Fo-Fc electron density map corresponding to Al and contoured at 1.3o is shown as a blue cage. Positions of key residues are denoted by text. (C) Schematic representation of the Al- binding site of eCls-2SP in the final model. The Al molecule and its corresponding electron density map are drawn identically to panel B, while key residues from eCls-2SP are drawn in ball- and-stick convention. Catalytic triad residues appear with their carbon atoms colored silver. Likely hydrogen bonds between the enzyme and Al (green), or with the catalytic triad (blue) are drawn as dashed lines.

[0026] FIG. 6. Molecular Dynamics Analysis of eCls-2SP Inhibited by Al. The final crystallographic model of eCl-2SP inhibited by Al was used as a starting point for three independent molecular dynamics simulations lasting 100 ns each. A still image from a movie taken of the progress of a representative simulation is shown in FIG. 7. (A) RMSD of the eCls-2SP Ca atoms across the duration of the simulation. A legend is inset. (B) RMSD of the Al heavy atoms across the duration of the simulation. A legend is inset.

[0027] FIG. 7. Molecular Dynamics Analysis of eCls-2SP Inhibited by Al. Still image from a movie taken of the progress of a representative molecular dynamics simulation of eCls-2SP inhibited by Al. The final coordinates from PDB entry 8TYP were used as the starting point of the simulation as described in Materials and Methods. The color scheme is identical to that used in Fig. 5.

[0028] FIG. 8. Binding Studies of Al with Cis and Selectivity Information Versus Other Serine Proteases Found in the Bloodstream. In panels A-B, SPR was used to study binding of an Al dilution series (100 - 1.25 pM) to immobilized forms of Cls-2SP. (A) Binding of Al to immobilized Cls-2SP expressed and purified from E. coli. Triplicate data were fit to a single site binding isotherm (inset), which revealed an apparent Ka of 9.8±1.1 pM. (B) Binding of Al to immobilized, activated eCls-2SP expressed and purified from HEK293(t) cells. Triplicate data were fit to a single site binding isotherm (inset), which revealed an apparent Ka of 3.8±0.2 pM. (C) The ability of 100 pM Al to inhibit the LP was assessed in triplicate using mannan as a specific activator of the LP and 2% (v / v) Clq-depleted serum followed by detection of C4b by ELISA. Note that the data shown here for the CP are replotted from the 100 pM wells from the doseresponse assay shown in Fig. 3C and are redrawn here for the sake of comparison. In panels D-F, the influence of increasing concentrations of Al on the activities of various proteases was examined by steady-state kinetics. Insets show either symbols identifying the curves corresponding to various concentrations of Al (values of micromolar), or apparent turnover number at various concentrations of Al. (D) Apparent turnover number of thrombin versus substrate concentration in the presence of five different concentrations of Al . Data were collected in triplicate. Fitting these data to a competitive inhibition model yields a Ki of 51.2 pM. (E) Reaction progress curves for Factor Xa versus substrate concentration in the presence of five different concentrations of Al. (F) Reaction progress curves for Neutrophil Elastase versus substrate concentration in the presence of five different concentrations of Al.

[0029] DETAILED DESCRIPTION

[0030] The present invention is concerned with a series of new small molecule inhibitors of Cis shown to possess dose-dependent modulation of Cis activity. In particular, 6-(4-phenylpiperazin- l-yl)pyridine-3-carboximidamide (designated as compound Al) was first identified as a candidate Cis binding agent through in silico analysis and synthesized for further study. In subsequent testing, Al was shown to inhibit Cis dose-dependently with low micromolar potency in functional assays and is a selective, competitive inhibitor of Cis. Al is comprised of 3 rings (A, B and C) having the following structure, with an amidine group attached to the A-ring:

[0031] In one or more embodiments, compound Al is provided as an HC1 (3X) salt. Analogs of Al are synthesized or envisioned by considering alternatives for each of the three rings, both in terms of substitution on the 6-membered rings as shown as well as alternatives to the 6-membered rings. Analogs of Al may also include scaffolds having substitutions or alternatives for the amidine group. Based on our preliminary data and the X-ray structure, ideal groups are those able to donate an H-bond. Compound Al and its analogs are competitive inhibitors of Cis activity with very high selectivity and exhibit direct binding to the SI pocket of Cis.

[0032] Al and its analogs contemplated herein are advantageous over other Cis inhibitors because they: (i) display favorable solubility properties, (ii) exhibit selective inhibition of Cis when compared to other closely related serine proteases, (iii) potently block the activities of Cis and the Classical Complement Pathways, and (iv) are small molecules compliant with Lipinski’s “Rule of Five.” These features are described below:

[0033] First, Al and several analogs are soluble to concentrations of at least 10 mM in distilled, deionized water. Advantageously, this means that preparation of solutions comprising Al and its analogs does not require any organic co-solvents or other toxic chemicals, such as dimethyl sulfoxide.

[0034] Second, Al exhibits selective inhibition of Cis when compared to related proteases found in the human bloodstream. Whereas Al inhibits Cis with a Ki value of 8.5 pM, it inhibits Clr (its closest homolog in humans) at least 100-fold less potently. Although Al inhibits thrombin with a Ki value of 51 pM, it does not inhibit Factor Xa. Al also does not inhibit the neutrophil serine proteases, cathepsin-G or neutrophil elastase. Therefore, Al represents an improvement over other small-molecule serine protease inhibitors, such as Futhan (alternatively known as nafamostat mesylate), which are promiscuous in their activities.

[0035] Third, the inhibitory concentrations of Al in established assays for Cis enzyme activity and activity of the Classical Complement Pathway lie within the low micromolar range. Specifically, Al (a) acts as a competitive inhibitor of human Cis with a Ki value of 8.5 pM in an enzyme activity assay, (b) inhibits activity of the Classical Complement Pathway in human serum with an IC50 value of 22 pM, as judged by a C4b-specific ELISA assay, and (c) inhibits activity of the Classical Complement Pathway in human serum with an IC50 value of 16 pM, as judged by an erythrocyte hemolysis assay. The strong correlation between these inhibitory parameters in both purified and complex systems indicates that Al retains its inhibitory activity against Cis in biologically-relevant settings.

[0036] In contrast to macromolecules that inhibit Cis, Al has a low molecular weight. At 390 Da (molecular weight provided for the 3-HC1 salt), it is less than 500 Da. Al also has a cLogP value of 1.67. Al also has a total of 3 rotatable bonds, as well as a total of 3 Hydrogen Bond Donors and a total of 5 Hydrogen Bond Acceptors. Finally, Al has a total Polar Surface Area of 69.24 A2.

[0037] Moreover, the analogs synthesized to date have Cls-inhibitory properties comparable to those of the parental compound Al.

[0038] At a minimum, Al and the analogs have the potential to be used as research tools where they would find utility as high-affinity, specific Ci s inhibitors. Al and the analogs will be useful as therapeutic inhibitors of the Classical Complement Pathway. This is clearly the most impactful future use of Al and the analogs.

[0039] Starting from Ring C, the present disclosure includes analogs of the parent compound Al, as follows: where Ring C is selected from the group consisting of substituted phenyls: where the squiggle line indicates attachment to the rest of the structure, and where each R is independently selected from the group consisting of: H, branched or unbranched alkyls (e.g., Ci- C4alkyls), halogens (e.g., F, Cl), ORa, -CN, -CF3, -CO2Ra, -C(O)NHRa, -C(O)NRxRy, NH2, NHRX, NRxRy, -CH2ORa, -CH2NHRa, -SO2Ra, S(O)NHRX, each Rais selected from H, branched or unbranched alkyls (e.g., C1-C4 alkyls), halogens (e.g., F, Cl), and each Rx= Ryis selected from alkyls (e.g., C1-C2 alkyls), cycloalkyls (e.g., C3-C6 alkyls), aryls, and further each Rx= Rycould be connected to form a ring such as piperidine or morpholine.

[0040] In one or more embodiments, Ring C is a substituted phenyl ring having a halogen substitution. In one or more embodiments, Ring C is a substituted phenyl ring having a metahalogen substitution. In one or more embodiments, Ring C is a substituted phenyl ring having an alkyl substitution. In one or more embodiments, Ring C is a substituted phenyl ring having a metaalkyl substitution.

[0041] In one or more embodiments, Ring C is not a moiety selected from the group consisting of benzopyridines, phalazines, 1-aminophalazine, and isoquinolines.

[0042] In one or more embodiments, Ring C is selected from the group consisting of heterocyclic rings (e.g., pyridine, pyrimidine, 1,4- and 1,2-pyrazine) potentially substituted with R groups defined as above: where n is 0-2, each R is independently defined as above; each “B” in the structures above designates the location of attachment to the adjacent B Ring; each X is independently selected from CH2, O, NH, or S; each Y is independently selected from CH or N, and further where X or Y is a carbon in the depicted ring, it can be further substituted with an R group as defined above. In one or more embodiments, each “B” in the above structures designates that the B “ring” could instead be replaced by any non-ring (non-cyclic) linker between A and C, such as a simple heteroatom such as oxygen, sulfur, nitrogen, etc. attached to a carbon(s). Alternatively, the non- cyclic linker could be selected from ureas or amides. It will be appreciated that the B component (whether cyclic or not) could be connected to variable positions in adjacent Rings C and A.

[0043] In the three-ring structure of parental compound Al, the central ring (piperazine) designated as the B Ring is the linker group between the A Ring and C Ring.

[0044] As such, in one or more embodiments, the linker could be a ring or it could be a non-cyclic linker, such as a simple alkyl or heteroalkyl group to connect the adjacent A Ring and C Ring. For example, such non-cyclic linkages can be envisioned as: where n is 1, 2, or 3; “X” denotes the B Rring and is NH, O, or S; “A” and “C” designate the respective A Ring and C Ring adjacent the B linker (which is shown as a heteroatom X in the structures above). Likewise, other non-cyclic B linkages can be envisioned as:

[0045] In one or more embodiments, alternative cyclic or ring systems for use in the B linker position include:

[0046] Likewise, aromatic ring options for use in the B linker position include: where R can be H, or branched or unbranched alkyls (e.g., C1-C4 alkyls), and where the “(N)” indicates that the nitrogen, for example, could be at any open position around the ring, including pyrimidines or pyrazines, and there are more than one N substitution pattern that could be achieved. In one or more embodiments, it is contemplated that the analog structure does not include a linker, that is, the B Ring is not present, nor is there any non-cyclic B linker. For example, in some embodiments, substituents used in the C Ring fill in the space without a separate B Ring at all. Examples include where the C Ring is substituted by a bicyclic ring or various ring fusions of AZB and B / C. So, a separate B Ring or non-cyclic B linker might not be needed. Examples of potential C / B Ring fusions include: 2,;, In the three-ring structure of parental compound Al, the pyridine ring, designated as the A Ring, could be modified as using the same aromatic or heteroaromatic ring options described above for the C Ring. The proviso is that the alternative structure needs to facilitate the amidine (Z group) or related substituent. Thus, for example, options for the A Ring position can be envisioned as: where the “(N)” indicates that the pyridine nitrogen, for example could be at any open position around the ring, including pyrimidines or pyrazines, and there are more than one N substitution pattern that could be achieved, each X is CH2, NH, O, S, and each Y is CH or N, and further where X or Y is a carbon in the depicted ring, it can be further substituted with an R group as defined above.

[0047] It is also contemplated that the A Ring could be substituted by a bicyclic heterocycle, where the Z group itself is accounted for via the heterocyclic moiety of the ring: Finally, the Z group of parental compound Al can be modified using any suitable H bond donor group to interact with the Asp626 group in the SI site. See e.g., Fig. 5C. Among the residues important in this interaction are Asp-626, Ser-627, Pro-657, and Tyr-662

[0048] Examples of potential bio-isosteres that could be used include -CH2NH2, -CO2H, - C(O)NH2, -C(O)NHRX, -S(O)2NH2, -S(O)2NHRX, -SO2H, -PO3H, -B(OH)2, as well as

[0049]

[0050] In one or more embodiments, analogs of Al modified according to the above principles can include any of the following:

[0051] SLUPP-2356 SLUPP-2565 ci

[0052] Particularly preferred analogs are selected from the group consisting of

[0053] Embodiments described herein include Cis inhibitor compounds having dose-dependent and selective inhibition activity against Cis as well as in the classical pathway. The compounds directly bind at or near the active site of Cis, such as in the SI site responsible for substrate specificity adjacent to the Asp-His-Ser catalytic triad, in a competitive manner to inhibit Cis activity and activation of the classical pathway, thereby intervening in a wide variety of complement-mediated diseases, disorders, and conditions. It will be appreciated that pharmaceutically-acceptable salts of any of the compounds described here as well as their prodrug forms are also contemplated herein.

[0054] The term “pharmaceutically-acceptable salt” refers to an acid or base salt of a compound of the disclosure, which salt possesses the desired inhibitor activity and is neither biologically nor otherwise undesirable. Hydrochloride, mesylate, hydrobromide, acetate, and fumarate salts can be used. The present disclosure may also include prodrugs (ester, amide, carbamate, carbonate, ether, imine, phosphate, etc. derivatives) of the disclosed compounds.

[0055] Prophylactic and / or therapeutic compositions comprising inhibitors of Cis are also disclosed. Compositions that comprise combinations of one or more of the foregoing compounds are also contemplated. The compositions comprise a small molecule inhibitor compound described herein dispersed in a pharmaceutically-acceptable carrier. The term carrier is used herein to refer to diluents, excipients, vehicles, and the like, in which the small molecule inhibitor may be dispersed for administration. Suitable carriers will be pharmaceutically acceptable. As used herein, the term “pharmaceutically acceptable” means not biologically or otherwise undesirable, in that it can be administered to a subject without excessive toxicity, irritation, or allergic response, and does not cause unacceptable biological effects or interact in a deleterious manner with any of the other components of the composition in which it is contained. A pharmaceutically-acceptable carrier would be selected to minimize any degradation of the compound or other agents and to minimize any adverse side effects in the subject. Pharmaceutically-acceptable ingredients include those acceptable for veterinary use as well as human pharmaceutical use, and will depend on the route of administration. For example, compositions suitable for administration via injection are typically solutions in sterile isotonic aqueous buffer. Similarly, compositions suitable for oral administration can be oral solutions, including for example various flavoring agents / taste modifiers, antioxidants, preservatives, pH adjusters, surfactants and lubricants, and the like. Exemplary carriers include aqueous solutions such as normal (n.) saline (-0.9% NaCl), phosphate buffered saline (PBS), sterile water / distilled autoclaved water (DAW), various oil-in-water or water-in-oil emulsions, as well as dimethyl sulfoxide (DMSO), glycerol, propylene glycol, polyethylene glycol, sucrose, sorbitol, ethanol, chloride salts, buffers, acids, or other acceptable vehicles, and the like. In one or more embodiments, the compositions can be formulated as tablets or capsules, including vehicles such as microcrystalline cellulose, sodium carboxymethyl cellulose, hydroxyl propyl methyl cellulose (HPMC), xanthan gum, acacia, tragacanth, alginates, guar gum, colloidal silicon dioxide, and the like.

[0056] The composition can comprise a therapeutically effective amount of the compound or analog thereof dispersed in the carrier. As used herein, a “therapeutically effective” amount refers to the amount that will elicit the biological or medical response of a tissue, system, or subject that is being sought by a researcher or clinician, and in particular elicit some desired therapeutic or prophylactic effect as against activation of the complement system, such as by binding and / or inhibiting Cis, inhibiting of complement activation, and / or inhibiting the complement system activation via the classical pathway. One of skill in the art recognizes that an amount may be considered therapeutically “effective” even if the complement-mediated condition is not totally eradicated or prevented, but it or its symptoms and / or effects are improved or alleviated partially in the subject. In some embodiments, the composition will comprise from about 5% to about 95% by weight of the small molecule inhibitor compound described herein, and preferably from about 30% to about 90% by weight of the small molecule inhibitor compound, based upon the total weight of the composition taken as 100% by weight. In some embodiments, combinations of more than one type of the described small molecule inhibitor compounds can be included in the composition, in which case the total levels of all such compounds will preferably fall within the ranges described above.

[0057] Other ingredients may be included in the composition, such as other active agents, preservatives, buffering agents, salts, other pharmaceutically-acceptable ingredients.

[0058] Compounds and compositions according to the embodiments disclosed herein are useful in inhibiting protease activity. Compounds and compositions according to the embodiments disclosed herein are useful in inhibiting Cis such as by binding and blocking Cis. More specifically, the compositions according to the embodiments disclosed herein are useful in treating complemented-mediated diseases, disorders, or conditions in a subject. The terms “therapeutic” or “treat,” as used herein, refer to processes that are intended to produce a beneficial change in an existing condition (e.g., complemented-mediated effects) of a subject, such as by reducing the severity of the clinical symptoms and / or effects of the condition, and / or reducing the duration of the symptoms / effects. References to “treatments” can also refer to “prophylactic” uses that are intended to inhibit or reduce the effects of a future complement-mediated condition that the subject may be at risk of, but which the subject is not currently experiencing symptoms. In some cases, the composition may prevent the development of observable clinical symptoms and / or effects of the condition. In other cases, the composition may only partially prevent and / or lessen the extent of the clinical symptoms and / or effects of the condition, such as by reducing the severity of the symptoms and / or effects of the condition, and / or reduce the duration of the symptoms / effects, or increase the rate of recovery from the condition. In either case, the compounds are still considered to “treat” the target condition, disorder, or disease.

[0059] In use, a therapeutically-effective amount of the inhibitor compound (Al or analog thereof) is administered to a subject. In some embodiments, a composition comprising a therapeutically- effective amount of an inhibitor compound is administered to a subject.

[0060] In some embodiments, the subject is afflicted with or suffering from a complement- mediated condition before the compounds are administered, wherein methods described herein are useful for treating the condition and / or ameliorating the effects of the condition. In other embodiments, the subject is at risk of, but currently free of a given condition before administering the compound, wherein the methods described herein are useful for preventing the occurrence or incidence of the condition and / or preventing the effects of the condition, as described above.

[0061] The disclosed embodiments are suitable for various routes of administration, depending upon the particular carrier and other ingredients used. For example, the prophylactic and / or therapeutic compounds or compositions can be injected intramuscularly, subcutaneously, intradermally, or intravenously. They can also be administered via mucosa such as intranasally, orally, ocularly, sublingually, buccally, or through intranasal, inhalation, rectal, or vaginal routes of administration. The compounds or compositions can also be administered through the skin via a transdermal patch, or topical administration of sprays, ointments, gels, or lotions. The compositions can be formulated as liquids, solids, powders, patches, sprays, tablets, gelcaps, creams, gels, ointments, pessaries, depots, and the like.

[0062] In some embodiments, the compound or compositions can be provided in unit dosage form in a suitable container. The term “unit dosage form” refers to a physically discrete unit suitable as a unitary dosage for human or animal use. Each unit dosage form may contain a predetermined amount of a compound disclosed herein (and / or other active agents) in the carrier calculated to produce a desired effect. In other embodiments, the compound can be provided separate from the carrier (e.g., in its own vial, ampule, sachet, or other suitable container) for on-site mixing before administration to a subject. A kit comprising the inhibitor compound(s) is also disclosed herein. The kit further comprises instructions for administering the compound to a subject. The compound(s) can be provided as part of a dosage unit, already dispersed in a pharmaceutically- acceptable carrier, or it can be provided separately from the carrier. The kit can further comprise instructions for preparing the compounds for administration to a subject, including for example, instructions for dispersing the compounds in a suitable carrier.

[0063] It will be appreciated that therapeutic and prophylactic methods described herein are applicable to humans as well as any suitable animal, including, without limitation, dogs, cats, and other pets or captive animals (e.g., zoo animals, research subjects), as well as, rodents, primates, horses, cattle, pigs, etc. The methods can be also applied for clinical research and / or study.

[0064] Additional advantages of the various embodiments of the invention will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present invention encompasses a variety of combinations and / or integrations of the specific embodiments described herein.

[0065] As used herein, the phrase "and / or," when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and / or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination. In the foregoing structures, R groups may be independently defined as indicated in the associated description for the depicted structures.

[0066] The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting "greater than about 10" (with no upper bounds) and a claim reciting "less than about 100" (with no lower bounds).

[0067] EXAMPLES

[0068] The following examples set forth methods in accordance with the invention. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention. Except where noted, precursor, intermediate, and final compounds described in the synthesis reactions below are independently numbered in each Example.

[0069] EXAMPLE 1

[0070] Pyridine-3-carboximidamide Does Not Inhibit Cis

[0071] Studies were carried out which demonstrated that the pyridine-3-carboximidamide (e.g., A Ring and amidine group attached to the A Ring, above) itself only inhibits Cis very weakly. The Ki value of this compound is ~9.3 mM, which is nearly 1,000-fold weaker than that of compound, Al.

[0072] EXAMPLE 2

[0073] In this work, Al was identified as candidate Cis binder through an in silico analysis.

[0074] Al inhibits Cis dose-dependently with low micromolar potency in functional assays. And the structure of Cis inhibited by Al suggests strategies for next generation Al analogs.

[0075] MATERIALS AND METHODS

[0076] Proteins - Samples of endogenously expressed C Ir and C 1 s that were purified from human serum were purchased from Complement Technologies. Samples of activated thrombin and Factor Xa that were purified from serum were obtained from Enzyme Research Laboratories. Samples of neutrophil elastase that was purified from human sputum were obtained from Elastin Products Corp. Samples of cathepsin-G that was purified from human neutrophils were obtained from Innovative Research.

[0077] Plasmids for prokaryotic expression of the 2SP region from human Cis, murine Cis, and human Clr were obtained from GenScript, Inc. Gene fragments encoding the CCP2 and SP regions of various proteases were synthesized and subcloned in the expression vector pT7HMT. An identical procedure was used to prepare each protease, with the exception that the corresponding expression vector was used. Briefly, the expression plasmids were transformed into E. coli strain BL21(DE3) and transformants were cultured for recombinant protein expression according to the general published methods. The induced cells were harvested by centrifugation and extracted with denaturant; following this, the extract was clarified by high-speed centrifugation prior to purification of the recombinant protein by IMAC performed under denaturing conditions. After elution, the recombinant protein was reduced with 1 mM TCEP for 30 min at room temperature prior to dilution into 10 volumes of 50 mM Tris-HCl (pH 8.3), 500 mM L-arginine, 5 mM EDTA,

[0078] 3 mM reduced glutathione, and 1 mM oxidized glutathione; following an overnight incubation at

[0079] 4 °C, the sample was buffer-exchanged into PB S (pH 7.4) and concentrated thereafter by tangential flow filtration. Soluble, monomeric protein was isolated by gel filtration chromatography on a Superdex S200 26 / 60 column that had been previously equilibrated in HBS (pH 7.4). Column fractions containing the target protein (as judged by SDS-PAGE) were pooled and concentrated. When necessary, the target protein was digested overnight with recombinant TEV protease at a 1 : 100 molar ratio to remove the fusion tag; upon verification of cleavage (as judged by SDS- PAGE), the target protein was separated from the free fusion tag, TEV protease, and other contaminants by IMAC where the unbound fraction was collected. The purified protein was stored temporarily at 4 °C until needed, or in 50% (v / v) glycerol at -20 °C for long-term storage.

[0080] A plasmid for expression of the C 1 s-2SP region of human C 1 s in HEK293(t) cells was also obtained from GenScript, Inc. A gene fragment encoding the native Cis signal peptide, followed by the CCP2 and SP domains of Cis, and a C-terminal octahistidine tag was synthesized and subcloned into pcDNA3. 1+. Following preparation of a large quantity of plasmid, HEK293(t) cells were transiently transfected according to the published procedures. Once approximately 1 L of conditioned culture medium was collected, the sample was concentrated by tangential flow filtration to a volume of -200 mL, exchanged into native binding buffer (20 mM tris (pH 8.0), 500 mM NaCl, 20 mM imidazole), and applied to a 5 mL HisTrap HP column (Cytiva). Following extensive washing with native binding buffer, the bound proteins were eluted with a linear gradient to 500 mM imidazole. Column fractions containing eCls-2SP (as judged by SDS-PAGE) were determined to be of sufficient purity without further chromatographic procedures, and were pooled, concentrated, and stored at 4 °C until further use.

[0081] Both Clr-2SP and Cls-2SP expressed and purified from A. coli displayed catalytic activity without further modifications, as determined by their ability to cleave the model substrate ZGR (MP Biomedicals) in chromogenic assays (please see Protease Activity and Inhibition Assays)' . For Clr-2SP, this feature was expected since Clr is capable of self-activation. For Cls-2SP, this feature was surprising because Cis normally requires activation by Clr to exhibit catalytic activity. Although the reason for activation of C 1 s-2SP is not known, it is possible that either self-activation during refolding or prolonged incubation with a contaminating protease during the days-long purification and refolding procedure could be responsible. Nevertheless, the comparable catalytic properties between Cis purified from human serum and Cls-2SP strongly suggest that this recombinant protein replicates essential features of the native molecule. In the case of eCls-2SP, the recombinant protein did not exhibit catalytic activity following purification. Therefore, purified eCls-2SP was activated by incubation with a 1 :200 molar ratio of Clr enzyme for 24 h at 37 °C. Following verification of cleavage (as judged by SDS-PAGE), the activated Cls-2SP was stored at 4 °C until further use. Surface Plasmon Resonance - Direct binding of small molecules to C 1 s-2SP was assessed by surface plasmon resonance using a T200 Biacore instrument (Cytiva) and general published methods. Cls-2SP (50 mg / mL in 10 mM acetate buffer (pH 4.0)) was used to modify flow cells 2, 3, and 4 of an HC1500M sensor chip (Xantec Bioanalytics, GmbH) that had been previously activated with EDC andNHS according to published procedures. A reference surface was prepared on flow cell 1 by EDC / NHS activation, followed by immediate quenching with ethanolamine. All experiments were performed in HBST-DMSO running buffer (HBS with 0.005% (v / v) Tween-20 and 5% (v / v) DMSO) using a flow rate of 30 pL / min. Solvent correction curves were obtained for DMSO concentrations ranging from 4.0% to 5.9% (v / v). Correction curves were obtained after every 10 compounds for initial screening, while only a single correction curve series was collected at the beginning of each dose-response cohort, as described below.

[0082] An initial screen for binding to Cls-2SP was performed at a single concentration of each compound (i.e., 100 pM) using C1S-2SP surface densities of 37,488, 39,074, and 38,653 RU on flow cells 2, 3, and 4, respectively. Compounds that gave visual evidence of precipitation in the HBST-DMSO running buffer were excluded from further consideration. However, those compounds that exhibited a binding response greater than 25% of the theoretical maximum signal expected for saturation of the average Cls-2SP surface used (i.e., 38,405 RU) with the average molecular mass of the compounds in the library (i.e., 435.9 Da) were carried forward. Assuming equimolar binding of these compounds to Cls-2SP (i.e., 36,966 Da), this threshold value was 113 RU (i.e., [(38,405*(435.9 / 36,966))*0.25] = 113.2 RU).

[0083] Dose-dependent binding to immobilized C1S-2SP was investigated for the remaining compounds in cohorts of 2-3 at a time. Solutions of each compound were prepared at 1.56, 3.13, 6.25, 12.5, 50, 75, and 100 pM in HBST-DMSO and injected in order of increasing concentration over the sensor chip used for initial screening in reference subtraction mode. The association phase was 30 sec, while the dissociation phase was 30 sec. Dose-dependent binding to immobilized eCls-2SP that had been enzymatically activated by Clr was also investigated for compound Al. In this case, triplicate injections over a surface with 7,116 RU of eCls-2SP were performed using the same concentration series (absent the 75 pM sample) with an association phase of 90 sec and a dissociation phase of 150 sec. The resulting sensorgrams were analyzed using Biacore T200 Evaluation Software (Cytiva) using the steady-state affinity model and fit to a single-site binding curve using GraphPad Prism. For determination of apparent Ka values, the fitted number derived from each experimental flow cell was averaged and the standard deviation was calculated.

[0084] Protease Activity and Inhibition Assays - The ability of Al to inhibit the activity of Cis, Clr, and various other bloodstream proteases was assessed at 25 °C through microplate-format chromogenic assays employing substrates appropriate to each protease. In the cases of Cis, Clr, thrombin, and factor Xa,, Z-Gly-Arg-thiobenzyl ester (ZGR; 8412= 13.6 mM'1cm'1) was used as primary substrate, while DTNB was included in the assay mixture as a fixed-concentration indicator. For the initial screening efforts, assays measuring the initial velocity of the Cis reaction (10 nM enzyme) in the presence of Kmconcentrations of ZGR (i.e., -0.16 mM) were followed in 96-well microplate format. The final concentration of each small molecule was 100 pM, while DMSO (5% (v / v)) was included in the reaction buffer to promote small-molecule solubility.

[0085] Cis. The inhibition constant of Al was determined using a classical 4x5 assay. The initial velocities of the Cis reaction (10 nM enzyme) were determined at four ZGR concentrations (~ 0.3

[0086] - 1.8 x Km) using five inhibitor concentrations (0 - -3.8 x Ki) per substrate concentration. The buffer conditions consisted of 50 mM HEPES (pH 7.4), 140 mM NaCl, 0.005% (v / v) Tween-20, 5% (v / v) DMSO, and 0.44 mM DTNB. The data (n = 3) were globally fit to a competitive inhibition model using GraphPad Prism.

[0087] Cls-2SP. The inhibition constant of Al was determined using a classical 4x5 assay. The initial velocities of the Cls-2SP reaction (2.4 nM enzyme) were determined at four ZGR concentrations (~ 0.2 - 1.2 x Km) using five inhibitor concentrations (0 - -2.6 x Ki) per substrate concentration. The buffer conditions consisted of 50 mM HEPES (pH 7.4), 140 mM NaCl, 0.005% (v / v) Tween-20, 5% (v / v) DMSO and 0.44 mM DTNB. The data (n = 3) were globally fit to a competitive inhibition model using GraphPad Prism.

[0088] Mouse Cls-2SP. The inhibition constant of Al was determined using a classical 4x5 assay. The initial velocities of the mouse Cls-2SP reaction (2.5 nM enzyme) were determined at four ZGR concentrations (~ 2.3 - 9.4 x Km) using five inhibitor concentrations (0 - -8.4 x Ki) per substrate concentration. The buffer conditions consisted of 50 mM HEPES (pH 7.4), 140 mM NaCl, 0.005% (v / v) Tween-20, 5% (v / v) DMSO and 0.44 mM DTNB. The data (n = 3) were globally fit to a competitive inhibition model using GraphPad Prism.

[0089] Clr. The inhibition constant of Al was determined using a classical 4x5 assay. The initial velocities of the Clr reaction (10 nM enzyme) were determined at four ZGR concentrations (~ 0.1

[0090] - 0.7 x Km) using five inhibitor concentrations (0 - -0.8 x Ki) per substrate concentration. The buffer conditions consisted of 50 mM HEPES (pH 7.4), 140 mM NaCl, 0.005% (v / v) Tween-20, 10% (v / v) DMSO and 0.44 mM DTNB. The data (n = 3) were globally fit to a competitive inhibition model using GraphPad Prism. Thrombin. The inhibition constant for Al was determined using a classical 4x5 assay. The initial velocities of the thrombin reaction (2.7 nM enzyme) were determined at four ZGR concentrations (~ Km- 9 x Km) using five inhibitor concentrations (0 — 3.4 x Ki) per substrate concentration. The buffer conditions consisted of 50 mMHEPES (pH 7.4), 140 mMNaCl, 0.005% (v / v) Tween-20, 5% (v / v) DMSO with 0.59 mM DTNB as an indicator. The data (n = 3) were fit globally to a competitive inhibition model using GraphPad Prism.

[0091] Factor Xa. Enzyme activity (3.5 nM enzyme) was determined at inhibitor concentrations that ranged from 0 to 226 pM. The concentration of ZGR was fixed at 42 pM. Buffer conditions consisted of 50 mM HEPES (pH 7.4), 140 mM NaCl, 0.025% (v / v) Tween-20, 5% (v / v) DMSO with 0.59 mM DTNB as an indicator.

[0092] Neutrophil Elastase. Enzyme activity (6.9 nM enzyme) was determined at inhibitor concentrations that ranged from 0 to 29.4 pM. The concentration of the substrate (methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanalide; 8400 = 12.3 mM'1cm'1) was fixed at 52 pM. Buffer conditions consisted of 50 mM HEPES (pH 7.4), 140 mM NaCl, and 0.05% (v / v) Tween- 20.

[0093] Cathepsin G. Enzyme activity (9.5 nM enzyme) was determined at inhibitor concentrations that ranged from 0 to 29.4 pM. The concentration of the substrate (succinyl-Ala- Ala-Pro-Phe-p-nitroanalide; 8400 = 12.3 mM'1cm'1) was fixed at 0.92 mM. Buffer conditions consisted of 50 mM HEPES (pH 7.4), 140 mM NaCl, 0.025% (v / v) Tween-20, and 20.7% (v / v) DMSO.

[0094] ELISAs for Complement Activity - The ability of Al to inhibit the CP was assessed through a pathway-specific ELISA using published methods. A dilution series of Al (100 - 3.1 pM) was incubated with 2% (v / v) AS (Innovative Research) as a source of complement components in HBS++ buffer (10 mM HEPES (pH 7.3), 140 mM NaCl, 0.15 mM CaCl2, 0.5 mM MgCh) plus 0.1% (w / v) gelatin. Reactions were added to the wells of ELISA plates that had been previously coated with human IgM (MP Biomedicals). Following a 1 h incubation at 37 °C, the wells were washed with TBS-T and probed with a mouse monoclonal antibody against C4b (Quidel). The wells were then washed and processed for detection using an HRP-conjugated anti-mouse IgG secondary according to standard procedures. Data were normalized to both an uninhibited reaction as a positive control and a serum-free reaction as a negative control. Triplicate data (n = 3) were fitted by non-linear regression to a four-parameter dose-response model using GraphPad Prism. The ability of Al to inhibit the LP was assessed at a single 100 pM concentration using an identical protocol as for the CP with the following exceptions: IgM was replaced with 20 pg ml'1mannan from Saccharomyces cerevisiae (Sigma) and reactions were incubated with 2% (v / v) Clq-depleted serum (Complement Technologies).

[0095] Activation of the Classical Pathway by Heparin-Induced Immune Complexes - The inhibitory effects of Al on CP activation were examined using published methods. Freshly drawn whole blood was obtained from a healthy donor following written consent using an institutional review board-approved protocol (Duke IRB#: Pro00010740); human subjects were enrolled in accordance with the Declaration of Helsinki. Acid-ci trate-dextrose (ACD) anti coagulated whole blood was prepared by mixing 1.4 mL of ACD solution (0.75% (w / v) citric acid, 2.20% (w / v) sodium citrate, 2.45% (w / v) dextrose) with 8.6 mL of whole blood. Samples were then preincubated either with buffer, a dilution series of Al (1000 - 10 pM), 100 pg / mL Borrelia miyamotoi FbpA-C (BmA-C; positive control (28)) or with 100 pg / mL B. miyamotoi FbpB-C (BmB-C; negative control) for 30 min at room temperature prior to addition of a monoclonal anti- PF4 / heparin antibody (KKO, 50 pg / mL) and antigen (platelet factor 4, 25 pg / mL and heparin 1 U / mL) to form immune complexes in situ. Following a 30 min incubation at 37 °C, the whole blood mixture was separated by centrifugation to allow plasma collection. After separation, plasma was incubated with 10 mM EDTA to quench further complement activation, and immunoassays were used to detect C5a by a sandwich ELISA. Anti-human C5a / C5a(desArg) antibody (clone C17 / 5; Biolegend) was used for capture, while biotinylated anti-human C5a / C5a(desArg) / C5 Antibody (clone G25 / 2; Biolegend) was used for detection according to standard procedures.

[0096] Lysis of Erythrocytes by the Classical Pathway - The ability of Al to inhibit the CP was assessed via a hemolytic assay using published methods. A dilution series of Al (100 - 3.1 pM) was incubated with antibody-opsonized sheep red blood cells (Complement Technologies) that had been freshly washed and 2% (v / v) AS (Innovative Research) as a source of complement components in HBS++ buffer plus 0.1% (w / v) gelatin. Following a 1 h incubation at 37 °C, the reaction was terminated by low-speed centrifugation. The absorbance of the supernatant was read at 412 nm using a multi-mode plate reader (Perkin Elmer). All data were normalized to an uninhibited control representing 100% lysis under the same reaction conditions. By contrast, background hemolysis from a negative control reaction consisting of buffer and red blood cells alone was subtracted from each reading. Triplicate data (n = 3) were fitted by non-linear regression to a four-parameter dose-response model using GraphPad Prism.

[0097] Cleavage of C2 by Cis - The ability of Al to inhibit cleavage of C2 by activated Ci s was investigated using published methods. A dilution series of Al and Cis (6.25 nM enzyme) was prepared in HBS++ buffer prior to adding 1.25 pL of C2 (0.5 pg / pL). The reaction was allowed to proceed at 37 °C for 1 hr prior to the addition of SDS-PAGE sample buffer. The reaction contents were separated by SDS-PAGE and visualized by Coomassie staining. Following staining, gels were imaged using a ChemiDocTM XRS+ (Bio-Rad) and processed for quantitation. Bands corresponding to C2b were corrected for total C2, including both cleavage products, and normalized to 100% cleavage relative to a control reaction consisting of Cis and C2 alone. Triplicate data (n = 3) were fitted by non-linear regression to a four-parameter dose-response model using GraphPad Prism.

[0098] X-ray Crystallography - The structure of eCls-2SP inhibited by Al was obtained by soaking preformed crystals of eCls-2SP with saturating concentrations of Al. First, crystals of eCls-2SP were obtained by vapor diffusion of hanging drops prepared by mixing 1 pL of enzymatically-activated eCls-2SP (5 pg / pL protein in 10 mM HEPES (pH 7.5), 50 mM NaCl) with 1 pL of precipitant solution (0.1 M Tris-HCl (pH 8.1), 0.2 M sodium acetate, 24% (w / v) PEG- 4000) and incubating over 500 pL of precipitant solution at 20 °C. Plate-shaped crystals appeared in 2-3 days and grew to their final size in -10-14 days. Droplets containing several single crystals were prepared for further use by adding 0.5 pL of an Al stock solution (10 mM in ddH2O) and incubating for 4-5 days prior harvest. Individual crystals were cryopreserved by a brief incubation in a buffer consisting of precipitant solution adjusted to 35% (w / v) PEG-4000 supplemented with 0.5 mM Al, followed by flash cooling in liquid N2.

[0099] Monochromatic X-ray diffraction data extending to 1.8 A limiting resolution were collected using beamline 5.0.1 of the Advanced Light Source at Lawrence Berkeley National Laboratory. Individual reflections were indexed, integrated, and scaled using HKL-2000. The structure was solved by molecular replacement using PHASER as implemented in the PHENIX software suite and the structure of Cls-2SP (PDB entry 1ELV) as a search model. Following placement of the search model, an initial round of bulk solvent scaling, positional and B-factor refinement was performed using PHENIX.REFINE to calculate electron density maps. Thereafter, the Al inhibitor was placed into the Fo-Fc map using PHENIX.LIGANDFIT. Iterative rounds of manual rebuilding using COOT and refinement using PHENIX.REFINE were used to generate the final model. The final model consists of residues 358-434, 438-495, 500-598, 609-683 of human Cis (UniProtKB Accession Number P09871; uniprot.org) separated by chain breaks due to either weak electron density or cleavage by Clr during the activation step, an oligosaccharide chain linked to Asn-406, a single copy of Al, and 335 ordered solvent molecules. Residual positive signal in the Fo-Fc electron density map was noticed near the sidechain of His-507; although this is consistent with phosphorylation of His-507, this modification was not modeled due to lack of supporting biochemical evidence or direct influence on Al binding to eCls-2SP. Additional information on X-ray diffraction data collection statistics and properties of the final model can be found in Table I. The final model and structure factors have been deposited in the RCSB Protein Data Bank with accession code 8TYP.

[0100] Molecular Dynamics Simulations - The crystal structure of eCls-2SP in complex with Al was used to perform molecular dynamics simulations. An all atom CHARMM molecular force field was used to parameterize the protein molecule. Topologies and parameters for Al were obtained from the CGENFF webserver after adding explicit hydrogens to the compound via OpenBabel 2.1 and converting the crystal structure coordinates of Al to the mol2 format. The system was then solvated in a cubic box of dimensions of 101.1 A, followed by the addition of 32,225 TIP3P waters with a cutoff of 10 A set as the boundary of the periodic box. The solvated complex system was next charge neutralized by adding 11 Na+ions in exchange for an equal number of TIP3P waters resulting in a total of 101,459 atoms for the system. Bond lengths were constrained by the LINCS algorithm followed by the application of Particle Mesh Ewald electrostatics. Heavy atom positional restraints were applied to compound Al via the “genrestr” program of Gromacs v2021.3. The system was then subjected to an energy minimization protocol by the steepest descent method until the forces on the system converged to a minimum of -1000 steps.

[0101] An isochoric-isothermal NVT ensemble (constant number of particles, volume, and temperature) was utilized to equilibrate the system to a final temperature of 300K for 100 ps using a Berendsen thermostat. Further equilibration of the system to maintain a constant pressure of 1 bar was achieved in an NPT ensemble (constant no. of particles, pressure, and temperature) following the Parinello-Rahman protocol for 100 ps. All production MD runs for the system were performed in this NPT ensemble in triplicate for a period of 100 ns with coordinates being saved at 10 ps intervals. Programs “gmx tijconv”, “gmx rms”, and “gmx distance” were used to convert trajectories to different file formats (z.e., PDB), to calculate RMSDs for different group(s) of atoms, and to calculate pairwise atomic distances. Average RMSD values were calculated after each simulation equilibrated for 1 ns. Further analyses were performed using scripts written in Fortran 90, Python and C-shell.

[0102] Small-Molecule Compounds - Compound Al and the other compounds in the screening library were obtained commercially from Enamine and were used for all experimental validations described here. Al and its analogs were also prepared in-house using the following general synthetic methods. Unless otherwise noted, all materials were obtained from commercial vendors and used as is without further purification. All reactions were monitored by using thin-layer chromatography (TLC) on silica gel coated glass plates or by high-pressure liquid chromatography (HPLC: Ascentis express peptide ES C-18 column, OD 3 cm X 4.6 cm; 6 min separation time, flow rate 1 mL / min; mobile phase gradient = 95 / 5 —> 5 / 95 CH3CN-H2O). Reverse-phase chromatography was performed on a Teledyne-Isco instrument using a Gold 50g C18 reversephase column with a mobile phase gradient of 100-0% — > 0-100% acetonitrile-EbO (0.1% TFA).!H NMR spectra were recorded on a Bruker Avance II nuclear magnetic resonance spectrometer (400 MHz) with chemical shifts reported in 5 ppm relative to DMSO-de (s = singlet, d = doublet, t = triplet, m = multiplet, dd = doublet of doublets, etc.).13C NMR and19F NMR spectra were obtained on the same instrument at 100 MHz and 376 MHz, respectively. Final compound purities were determined by HPLC using the conditions described above.

[0103] RESULTS

[0104] Structure-Based in silica Screening for Small-Molecule Inhibitors of Cis

[0105] The general workflow for our study was as follows (Fig. 1). We began by utilizing a machine learning approach for in silico drug discovery developed by AtomWise. The deep neural network AtomNet® was employed to predict small -molecule compounds that bind to the active site of Cis. A total of 94 compounds were selected for procurement and carried forward for bench validation in single concentration assays (i.e., 100 pM compound) using three orthogonal approaches that included: i) Cls-binding assays using surface plasmon resonance (SPR), ii) Cis enzyme inhibition assays, and iii) serum-based CP activation ELISAs. From an initial 94 compounds, a single compound met our hit criteria in all three assays: 6-(4-phenylpiperazin-l- yl)pyridine-3-carboximidamide, which we refer to hereafter as compound “Al”. We proceeded with quantitative characterization of Al activity in five independent dose-response assays. Finally, we developed a structure-activity relationship (SAR) for Al by solving a 1.8 A crystal structure of Cis inhibited by Al, followed by subsequent synthesis and evaluation of Al analogs.

[0106] Virtual Screening Identifies Putative Cls-Binding Small Molecule Inhibitors

[0107] The SI site of Cis is critical for substrate recognition (i.e., C2 and C4). This functional importance arises due to SI -residue interactions with a specific substrate residue - conventionally called Pl - that lies immediately N-terminal to the scissile peptide bond. Therefore, we hypothesized that any small molecules that bind to the SI site of Cis would inhibit its catalytic activity. To test this idea, the previously solved 1.7 A crystal structure of an activated form of truncated human Cis composed of the complement control protein domain 2 (CCP2) and serine protease (SP) domains (hereafter Cls-2SP) (PDB: 1ELV; ) was used with AtomNet to screen for compounds predicted to bind within the SI site of Cis. Top scoring compounds from the virtual screen were clustered and filtered for favorable predicted physicochemical properties related to favorable aqueous solubility, a lack of toxic functional groups, improved blood-brain barrier penetration, and a predicted lack of pan-assay inhibitor (PAINS) behavior. The top 94 compounds were obtained commercially, dissolved in neat dimethyl sulfoxide (DMSO) to 10 mM final concentration, and added to a 96-well sample block to facilitate further characterization. For the purposes of initial screening, we refer to these compounds hereafter according to their address in the sample block, as rows (A-H) and columns (1-12) (e.g Al, D4, F2, etc ).

[0108] Empirical Validation of In Silico Hit Compounds

[0109] We then sought empirical validation of the hit compounds identified via virtual screening. All 94 compounds were first tested for their ability to directly interact with Cls-2SP at a single high concentration (i.e., 100 pM) using a biosensor surface that had been modified with a recombinant form of human C1S-2SP. A compound was initially considered positive for binding if it met the following criteria: (i) it exhibited clearly discernable association / dissociation phases in the sensorgram, and (ii) its background- and solvent-corrected response (i.e., resonance units (RU)) at the injection stop was greater than one standard deviation above the mean. A total of only six compounds met these stringent criteria, denoted here by their position in the 96-well plate: Al, D4, DIO, F2, Hl, and H9 (Fig. 2A). However, we noticed that approximately one-third of the screening compounds exhibited 25% of the theoretical maximum response for the average compound in this library (MW = 435.9 Da) if 1: 1 stoichiometry was assumed and the immobilization level of C1S-2SP was considered (please see Materials & Methods').

[0110] Therefore, we rescreened each of these thirty molecules, along with six non-binding compounds as negative controls, against replicate Cls-2SP biosensor surfaces to test for dosedependent binding behavior (Data Not Shown). Dose-dependent data were obtained for compounds that displayed binding signals to Cls-2SP greater than 25% of the theoretical maximum response in the single concentration assays shown in Fig. 2A. A two-fold dilution series of each compound from 100 pM (purple) to 1.6 pM (orange) was injected over replicate surfaces consisting of recombinant Cls-2SP expressed and purified from E. coli. Six compounds that gave no initial evidence for binding to C1S-2SP were included as negative controls. Of the 30 other candidates evaluated in this way, multiple compounds exhibited clear evidence of dose-dependent binding to immobilized C 1 s-2SP. However, the same six compounds identified as hits in the single concentration screen again showed the greatest binding responses (highest SPR signals in the dosedependent experiment) across all the concentrations examined.

[0111] In addition to the SPR studies, we also tested whether any of the 94 compounds identified in the virtual screen were capable of inhibiting Cis in two different functional assays. The first assay monitored spectrophotometrically the ability of Cls-2SP to cleave the synthetic substrate, Z-Gly-Arg-Thiobenzyl ester (ZGR), followed by reaction of this product with the thiol indicator, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). Initial reaction velocities for C1S-2SP were compared for trials conducted either in the absence or presence a single, fixed concentration of inhibitor (i.e., 100 pM). We found that Cls-2SP activity was reduced to levels lower than one standard deviation below the mean by four compounds: Al, C7, E3, and E6 (Fig. 2B). In a second functional assay, we measured the ability of each compound to block Cis cleavage of C4 in the context of serum-mediated CP activation. Using IgM-coated microtiter wells as a specific initiator of the CP, we measured C4b deposition in the presence of a fixed concentration of compound (i.e., 100 pM) using an anti-C4b ELISA. Only two compounds reduced C4b deposition to less than two standard deviations below the mean: Al and B5 (Fig. 2C). Of these, compound Al alone resulted in essentially complete inhibition of C4b deposition at the concentration examined.

[0112] Compound Al is a Dose-dependent Inhibitor of the Classical Pathway

[0113] Our initial validation assays identified compound Al as the only molecule in our hit library capable of binding to Cis and inhibiting its enzyme activity in both purified and serological settings. Therefore, we obtained a resupply of the purified compound (-100 mg) from a commercial source and confirmed its identity using 'H,13C, and NMR spectroscopies as well as MALDI-TOF mass spectrometry (Data Not Shown) as a prelude to more detailed analyses. Although each of our original compounds was dissolved in neat DMSO, the Al obtained commercially was provided as a trihydrochloride salt; this feature, along with a favorable calculated partition coefficient (cLogP) of 1.67 suggested that the compound would be water soluble. Indeed, we found that aqueous solutions of this compound could be prepared to concentrations of at least 10 mM. Thus, we performed all subsequent characterization of compound Al in the absence of DMSO. Aside from its solubility, Al had other favorable physicochemical properties including a relatively low molecular weight (281.36 Da), 2 hydrogen bond donors, 3 hydrogen bond acceptors, 3 rotatable bonds, and a total polar surface area of 70 A2. Based upon these features, Al met all Lipinski’s criteria for Rule of Five compliance. To test whether compound Al exhibited dose-dependent and saturable inhibition of Cis, and to define its mechanism of inhibition, we carried out a series of functional assays in systems of decreasing overall complexity. We first examined whether Al could block activation of the CP in whole blood triggered by formation of immune complexes consisting of platelet factor 4 (PF4) / heparin and an anti-PF4 / heparin monoclonal antibody. Using a dilution series of Al that ranged from 1,000 - 10 pM, we found that increasing concentrations of Al resulted in progressively greater inhibition of C5a generation (Fig. 3A). The greatest change in the extent of CP inhibition occurred between Al concentrations of 50 and 250 pM, at which point a statistically significant decrease in C5a generation was observed when compared to buffer control. Although Al resulted in only ~75% inhibition at the highest concentration examined, these experiments showed clear evidence of dose-dependent inhibition of the CP in whole blood.

[0114] Next, we tested Al in two independent serum-based CP assays. The first assay utilized a fixed dilution of human serum incubated with antibody-sensitized sheep erythrocytes to assess hemolysis spectrophotometrically. Using a dilution series of Al that covered 100 - 3.1 pM, we observed saturable, dose-dependent inhibition of CP activity (Fig. 3B). The IC50 value for Al in this experiment was 16 pM, which was consistent with the potency observed in the previous whole blood study. We then repeated the CP-specific ELISA for C4b deposition (described above in the wider screening of all compounds) in the presence of dilution series of Al. Using a concentration range that spanned 100 - 3.1 pM, we likewise observed saturable, dose-dependent inhibition of the CP (Fig. 3C). The IC50 value of Al in this experiment was 22 pM, which again corresponded well to our earlier experiments.

[0115] While the CP ELISA provided insight into cleavage of C4 by Ci s, cleavage of the other Cis substrate, C2, was not readily measured in that assay format. To test whether Al could inhibit proteolysis of C2 by Cis in a dose-dependent manner, we performed a gel electrophoresis-based assay to monitor Cis cleavage of C2 in the presence of a dilution series of Al. Purified Cis enzyme was incubated with C2 substrate and a two-fold dilution series of Al ranging from 100 - 1.56 pM and the products were separated by electrophoresis. We observed dose-dependent inhibition of C2 cleavage by Cis, as judged by densitometry of band intensities corresponding to the substrate, C2, and the cleavage product, C2b (Fig. 3D, E). Following fitting both band series to a dose-response curve, the IC50 values for each was ~85 pM. Collectively, these results demonstrate that compound Al is a dose-dependent inhibitor of CP activity in both serum and whole blood via its ability to block Cis cleavage of C4 and C2.

[0116] Compound Al is a Competitive and Selective Inhibitor of Cis To gain further insight into the mode of Cis inhibition, we determined an apparent inhibition constant (Ki) for Al against both native Cis isolated from human serum and a recombinant Cls-2SP fragment, as well as recombinant mouse Cl s-2SP. In these experiments, we carried out replicate measurements of enzyme activity across a range of substrate and inhibitor concentrations. We determined initial reaction rates for each substrate and inhibitor condition, which allowed derivation of apparent Ki values and other kinetic parameters using non-linear curve fitting. We found that compound Al behaved as a competitive inhibitor of native human Cis, Cls- 2SP, and mouse C1S-2SP with Ki values of 5.8, 8.5, and 24.6 pM, respectively (Fig. 4A-C). We noted that the Ki parameters for Al inhibition of the human Cls-2SP fragment corresponded well to its apparent Ka of 9.8 ± 1.1 pM, as determined by SPR (Fig. 8A, B). This would be expected for a purely competitive inhibitor of enzyme activity. These observations were also consistent with what might be expected for a small molecule that binds within the SI site, which is known to play an important role in substrate binding to the C 1 s enzyme. Interestingly, and in contrast to its effects on Cis, we found that compound Al was a substantially weaker inhibitor of Clr with a Ki of 842 pM (Fig. 4D) This ~145-fold selectivity was surprising since Cis and Clr are closely related to one another and share -40% sequence identity. This result also strongly suggests that the ability of Al to block the CP arises from competitive and selective inhibition of Cis, rather than by broadbased inhibition of other proteases, including Clr.

[0117] Although we found that Al was selective in its inhibition of Cis when compared to Clr, there are numerous other serine proteases found in the bloodstream that may be subject to off- target effects of Al. To gain initial insight into this issue, we examined if Al could block the LP. We incubated Clq-depleted serum with 100 pM Al and monitored LP activation on ELISA plates previously coated with mannan. We found that Al had relatively weak activity against the LP compared to the CP under these conditions (FIG. 8C). Next, we tested whether Al had any effect on the activities of several other non-complement bloodstream serine proteases. Whereas we found that Al was a competitive inhibitor of thrombin (FIG. 8D), its Ki value for thrombin of 51.2 pM was nearly an order of magnitude weaker than that for native Cis. Importantly, Al did not inhibit Factor Xa (FIG. 8E), nor the two most abundant neutrophil serine proteases Neutrophil Elastase (FIG. 8F) or Cathepsin-G (Data Not Shown). These data, in conjunction with those for Clr, demonstrate that Al is a selective serine protease inhibitor. Combined with its ability to inhibit murine Cis, these features support Al as a potential lead compound for further development of C 1 s-targeted complement inhibitors and research tools.

[0118] Insights into the Structure-Activity Relationship for Inhibition of Cis by Al. Al was identified using the structure of unbound Cis as template for virtual screening. However, we sought empirical structural information on Cis inhibited by Al to establish a structure-activity relationship and to lay a foundation for future medicinal chemistry efforts. We succeeded in growing crystals of a recombinant version of human Cls-2SP secreted from HEK293(t) cells (hereafter eCls-2SP) that diffracted to 1.4 A resolution in their apo-form (Data

[0119] Not Shown). Thereafter, we incubated pre-formed crystals of eCls-2SP with saturating concentrations of Al and collected X-ray diffraction data to 1.8 A limiting resolution (Table I). Following structure solution, we observed unambiguous electron density corresponding to Al within the SI site that is adj acent to the C 1 s catalytic site (Fig. 5A). We then modeled the inhibitor at full occupancy and refined the structure to an Rfreeof 19.6% (Fig. 5B, C & Table I). aValues in parentheses are for the hi hest-resolution shell.

[0120] We found that the pyridine-3-carboximidamide group of Al lay deep within the SI substrate binding pocket where it engaged in apparent hydrogen bonds with both main chain- and side chain- derived groups from multiple residues of the protease, including Asp-626, Ser-627, Lys-629, Pro- 657, and Tyr-662. By contrast, the terminal phenyl group of Al occupied a position near several voids and channels that could be targeted during future hit expansion efforts (Fig. 5B, C).

[0121] To gain additional insight into the binding mode of Al, we used the crystal structure of eCls-2SP inhibited by Al to carry out 100 ns molecular dynamics (MD) simulations in triplicate. Root mean square deviations (RMSD) were monitored across the time course of each simulation to assess the stability of eCls-2SP (CaRMSD) and Al (Ligand RMSD) relative to the conformation observed in the crystal structure. The backbone eCls-2SP RMSD (i.e., CaRMSD) equilibrated by ~1 ns and ranged between average values of 2.07 - 2.21 A (SD = 0.23 - 0.38 A) across the three independent simulations (Fig. 6A). By contrast, the Al RMSD values ranged between 3.18 - 4.18 A (SD = 0.69 - 1.18 A) across the three simulations (Fig. 6B). Notably, we found that the terminal phenyl moiety is relatively dynamic in these simulations when compared to the other Al ring structures.

[0122] With empirical structural information in place, we set out to develop an initial SAR model for Al. To do this, we synthesized a limited panel of analogs based upon the Al scaffold. Al and various analogs were synthesized with using a three-step process generally in good yields.

[0123]

[0124] ^Conditions a) XPhos, Pd2(dba)3, NaOtBu, Toluene. 90 ’C, 12h, b) 2.0M AIMe3-Toluene, NH4CL Toluene, c) HCIjc;Dioxane, rt, 48h

[0125] Step 1 : Synthesis of 6-(4-phenylpiperazin-l-yl)nicotinonitrile. XPhos (212 mg, 0.44 mmol) and Pd2(dba)a (230mg, 0.22 mmol) were added to a 4-dram reaction vial and dissolved in toluene (2 mL). The mixture was heated to 60 °C for 15 min under argon. The vial was allowed to cool to room temperature and 1 -phenylpiperazine (180 mg, 1.1 mmol) and 6-bromonicotinonitrile (243 mg, 1.3 mmol) were added to the reaction vessel followed by NaO'Bu (149 mg, 1.55 mmol). The vessel was heated to 90 °C and stirred overnight under Argon. The reaction was then allowed to cool to room temperature and the crude reaction mixture was filtered through a PFFE syringe filter with washing from EtOAc to remove the palladium precipitate. The mixture was then concentrated to dryness and taken back up in a small amount of DMSO. The DMSO mixture was purified by reverse-phase chromatography to give 320 mg of the desired product as a tan solid.1H NMR (400 MHz, DMSO-rL) d 8.51 (d, J= 2.2 Hz, 1H), 8.7.87 (dd, J= 2.3 Hz, 9.1 Hz, 1H), 7.26-7.21 (m, 4), 7.00-6.97 (m, 4H), 6.81 (t, J = 7.2 Hz 1H), 3.81-3.80 (m, 4H), 3.25-3.22 (m, 4H); LCMS: m / z calcd. for Ci6Hi6N4 [M+H]+: 265.2; found [M+H]+: 265.2.

[0126] Step 2: Synthesis of 6-(4-phenylpiperazin-l-yl)nicotinimidamide, A 2-dram vial was equipped with a stir-bar. Ammonium chloride (364 mg, 6.81 mmol, 15 eq.) and 0.5 mL of toluene were added to the vial. The mixture was placed on an ice bath and an argon balloon was applied. To the reaction mixture was added 2 M trimethylaluminum solution in toluene (3.41 mL, 6.81 mmol, 15 eq.) and allowed to stir on ice, warming to room temperature, for three hours. The above nitrile (120 mg, 0.454 mmol, 1.0 eq.) was added to the vial as a solution in 2.0 mL of toluene. The reaction mixture was allowed to stir overnight at 95 °C. The toluene was removed by passing air through the reaction vessel. The resulting residue was dissolved in 15 mL of CH2CI2, diluted with water, and 1 N NaOH was added until the mixture was at a pH of 14. The layers were separated, and the aqueous layer was extracted three times with CH2CI2. The organic layers were combined, dried, and concentrated in vacuo. The resulting residue was dissolved in minimal DMSO and purified by reverse-phase chromatography. This yielded 24.6 mg of desired compound as a TFA salt in the form of a white powder.

[0127] Step 3: 6-(4-phenylpiperazin-l-yl)nicotinimidamide hydrochloride salt. The above TFA salt (25mg, 0.09 mmol) was dissolved in 1.5 mb of dioxane. 37% HC1 (0.073 mb, 0.9 mmol) was added to this mixture. The reaction was stirred at room temperature for 2 days. The solvent was removed by evaporation by passing air over the top of the vessel. The resulting residue was slurried in diethyl ether and filtered through a fritted funnel. The white solid was rinsed with additional ether and dried to give 18.1 mg of the desired product.!H NMR (400 MHz, DMSO-t / g) d 9.20 (s, 2H), 8.91 (s, 2H), 8.68 (d, J= 2.6 Hz, 1H), 8.04 (dd, J= 2.6 Hz, 9.2 Hz, 1H), 7.38-7.30 (m, 4H),

[0128] 7.10 (d, , / = 9.2 Hz, 1H), 7.06-7.02 (m, 1H), 4.1-4.0 (m, 4H), 3.5-3.4 (m, 4H);13C NMR (100 MHz, DMSO-t / d) d 163.1, 160.0, 148.7, 137.2, 129.4, 117.7, 111.4, 106.2, 62.8, 49.9, 43.3. LCMS: m / z calcd. for Ci6Hi9N5[M+H]+: 282.3; found [M+H]+: 282.2.

[0129] Using this scheme, we prepared the parent molecule along with seven different analogs that represented simple substitutions at positions within the phenyl ring (Table II & Table III).

[0130] Table. IL Characterization of Analogs

[0131] Inhibitor Km (pM) SE fcaf(s-1) SE Ki (pM) SE

[0132] SLUPP-2398 83.2 8.6 17.5 0.7 50.8 3.6

[0133] SLUPP-2397 69.4 9.4 16.7 0.8 19.5 1.8

[0134] SLUPP-2396 69.8 10.7 15.5 0.9 149.2 16.9

[0135] SLUPP-2395 84.8 5.4 14.9 0.4 22.6 1.0

[0136] SLUPP-2394 113.4 18.9 18.0 1.6 52.2 5.2

[0137] SLUPP-2393 91.2 11.2 17.7 1.0 32.6 2.6

[0138] SLUPP-2392 68.5 11.1 15.7 1.0 76.0 9.5

[0139] SLUPP-2383 / j

[0140] SLUPP-2380ft

[0141] 6-(4-phenylpiperazin-l- 75.5 9.7 15.2 0.8 21.5 2.1 yl)pyridine-3- carboximidamide (Al)

[0142] “Assay conditions: 50 mM HEPES (pH: 7.4), 140 mM NaCl, 0.05 % Tween-20, 10 % DMSOhNO inhibition at 440 pM

[0143] SLUPP-2380: SLUPP-2383:

[0144] The effects of various substitutions within the terminal phenyl ring of the Al molecule (top panel) were evaluated in a Cis enzyme activity assay. The results were used to generate information on the structure-activity relationship of the Al scaffold in conjunction with the crystallographic information presented earlier (please see Fig. 5). Ki determinations are reported as the mean of three independent replicates.

[0145] Table III. Structure-Activity Relationships of Al and Panel of Al Analogs.

[0146] Following chemical characterization of each analog, we determined Ki values against human C1S-2SP in the same enzyme activity assay used previously. Although none of these analogs had improved potency when compared to the parental Al compound, several had comparable potency that allowed us to discern some SAR information. For example, substitutions at the para position of the C Ring (i.e., -R3) were deleterious as replacement of the hydrogen with even small groups like fluorine or chlorine resulted in noteworthy loss of activity (i.e. > ~10-fold). By contrast, substitution at the ortho position of the C Ring (i.e., -Rl) was better tolerated, since replacement of the hydrogen with chlorine resulted in only a ~5-fold loss in activity when compared to Al. Most importantly, we found that substitutions at the meta position of the C Ring (i.e., -R2) appeared to be preferred. Replacement of the hydrogen with fluorine (Ki = 19.5 pM), chlorine (Ki = 32.6 pM), or methyl (Ki = 22.6 pM) yielded compounds of comparable potency to Al itself. We noted that each of these observations was consistent with our crystal structure of Al bound to eCls-2SP, which showed obvious voids projecting to either side of phenyl ring (Fig. 5B). Although the panel of analogs we prepared was limited in both its extent and chemical diversity, we believe this existing SAR information alongside the co-crystal structure suggests a productive trajectory for the development of improved compounds based on the existing Al scaffold.

[0147] ABBREVIATIONS

[0148] Al; 6-(4-phenylpiperazin-l-yl)pyridine-3-carboximidamide

[0149] ACD; acid citrate dextrose

[0150] Al; artificial intelligence

[0151] AP; alternative pathway of complement

[0152] AS; active human serum

[0153] BSA; bovine serum albumin

[0154] CAD; cold agglutinin disease

[0155] CCP; complement control protein module

[0156] CP; classical pathway of complement

[0157] Cl; complement component Cl

[0158] Clr: complement component Clr

[0159] Clr-2SP; recombinant Clr fragment containing the second CCP domain and the SP domain Cis: complement component Cis

[0160] Cls-2SP: recombinant Cis fragment containing the second CCP domain and the SP domain eCls-2SP: Cls-2SP expressed and secreted from HEK293(t) cells

[0161] C3; complement component C3

[0162] C3b; complement component C3b

[0163] DMSO; dimethyl sulfoxide

[0164] DTNB; 5, 5'-dithio-bis-(2 -nitrobenzoic acid) EDC; 1 -Ethyl -3-(3-dimethylaminopropyl)carbodiimide

[0165] FB; complement factor B

[0166] FD; complement factor D

[0167] FH; complement factor H

[0168] HPLC; high-performance liquid chromatography

[0169] HBS; HEPES buffered saline

[0170] HBS-T; HEPES buffered saline with tween -20

[0171] HBS++; HEPES buffered saline with calcium and magnesium chloride

[0172] HIS; heat-inactivated human serum

[0173] HIT; heparin-induced thrombocytopenia

[0174] HSA; human serum albumin

[0175] IMAC; immobilized metal ion affinity chromatography

[0176] LP; lectin pathway of complement

[0177] MD; molecular dynamics

[0178] NHS; N-hydroxy-succinimide

[0179] NMR; nuclear magnetic resonance spectroscopy

[0180] PBS; phosphate buffered saline

[0181] RU; resonance unit

[0182] SAR; structure-activity relationship

[0183] SCR; short consensus repeat domain

[0184] SPR; surface plasmon resonance

[0185] TBS-T; tris buffered saline with tween-20

[0186] TCC; terminal complement complex

[0187] TCEP; tris(2-carboxyethyl)phosphine

[0188] ZGR; Z-Gly-Arg thiobenzyl ester

Claims

Claims:

1. A small molecule compound that is a competitive inhibitor of Cis, the compound having the structure of formula Al comprising 3 rings designated as A, B, and C, and an amidine group attached to the A-ring:pharmaceutically-acceptable salts thereof, prodrugs thereof, or analogs thereof comprising a substitution at the A, B, and / or C ring or amidine group.

2. The compound of claim 1, wherein the analog is selected from the group consistingGt3. A therapeutic composition for inhibiting Cis in a subject in need thereof, the composition comprising a small molecule inhibitor of Cis according to claim 1 or claim 2 and a pharmaceutically acceptable carrier.

4. A method of treating a subject having or at risk of a complement-mediated disease, disorder, or condition, the method comprising administering to the subject a small molecule inhibitor of C 1 s according to according to claim 1 or claim 2 a or a therapeutic composition thereof.

5. The method of claim 4, wherein said compound is dispersed in a pharmaceutically- acceptable carrier.

6. The method of claim 5, further comprising providing a unit dosage form of said compound dispersed in said pharmaceutically-acceptable carrier prior to said administering.

7. The method of any one of claims 4-6, wherein said subject is suffering from a complement-mediated disease, disorder, or condition prior to said administering.

8. The method of any one of claims 4-6, wherein said subject is free of symptoms of said a complement-mediated disease, disorder, or condition prior to said administering.

9. The method of any one of claims 4-8, wherein said compound is administered intramuscularly, subcutaneously, intradermally, intranasally, intravenously, orally, buccally, ocularly, sublingually, vaginally, rectally, or via a transdermal patch.

10. The method of any one of claims 4-9, wherein said subject is a human.

11. A kit comprising: a small molecule inhibitor of Cis according to claim 1 or claim 2; and instructions for administering said compound to a subject in need thereof.

12. The kit of claim 11, wherein said compound is provided in unit dosage form.

13. The kit of claim 12, wherein said compound is provided in a first container, said kit further comprising a carrier in a second container; and instructions for preparing said inhibitor compound for administration to said subject.

14. A method of preventing or inhibiting Cis activation in a subject in need thereof, said method comprising administering to the subject a small molecule inhibitor of Cis according to claim 1 or claim 2 or a therapeutic composition thereof.

15. Use of a small molecule inhibitor of Cis according to any of the embodiments disclosed herein to prepare a therapeutic or prophylactic medicament for preventing or inhibiting Cis activation in a subject in need thereof or treating a subject having or at risk of a complement- mediated disease, disorder, or condition.

16. A therapeutic or prophylactic medicament for preventing or inhibiting Cis activation in a subject in need thereof comprising a small molecule inhibitor of Cis according to claim 1 or claim 2.

17. The therapeutic or prophylactic medicament of claim 16, further comprising a pharmaceutically-acceptable carrier, wherein said compound is dispersed in saidpharmaceutically-acceptable carrier.

18. A therapeutic composition for inhibiting Cis in a subject in need thereof, the composition comprising a small molecule inhibitor of Cis according to any of the embodiments disclosed herein and a pharmaceutically acceptable carrier.

19. A method of treating a subject having or at risk of a complement-mediated disease, disorder, or condition, the method comprising administering to the subject a small molecule inhibitor of C 1 s according to any of the embodiments disclosed herein or a therapeutic composition thereof.

20. A kit comprising: a small molecule inhibitor of Cis according to any of the embodiments disclosed herein; and instructions for administering said compound to a subject in need thereof.

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