Screening method for Acinetobacter baumannii SpoT enzyme modulators

Abstract

The present invention relates to a screening method for identifying compounds that modulate the activity of the Acinetobacter baumannii SpoT enzyme, particularly compounds that can partially or completely inhibit the hydrolase activity of the enzyme. The screening method relies on evaluating the fit of candidate compounds to the three-dimensional structure of the A. baumannii SpoT protein and / or the A. baumannii SpoT-ppGpp complex, which is represented by a well-defined set of atomic coordinates. The screening method can further rely on evaluating the interaction of the candidate compounds with one or more amino acid residues in a region on the surface of the SpoT protein.

Description

[Technical Field]

[0001] The present invention relates to the elucidation of the crystal structure of the Acinetobacter baumannii SpoT enzyme and screening methods for identifying Acinetobacter baumannii SpoT enzymes that bind to the catalytic site of the crystal structure. The present invention is of particular interest to the field of molecular biology, and more specifically to the development of drugs against antibiotic-resistant Acinetobacter baumannii. [Background technology]

[0002] The overuse and misuse of antibiotics, combined with slow progress in the development of new antimicrobial drugs, has led to the emergence of pathogenic antibiotic-resistant bacteria. The incidence of these bacteria (also known as "superbugs") is increasing at an alarming rate, and bacterial infections have once again emerged as a prominent threat to human health (Ventola, The antibiotic resistance crisis, Pharmacy and therapeutics, 2015). Over the past few years, multiple health organizations have repeatedly warned about these pathogenic antibiotic-resistant (multidrug) bacteria and the threat they pose to human health (Michael et al., Frontiers in public health, 2013). Six of the most highly pathogenic and antibiotic-resistant bacterial pathogens, which can evade or escape commonly used antibiotics due to increasing multidrug resistance, have recently been designated by the acronym "ESKAPE" (group): Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.

[0003] One mechanism these bacteria use to survive in the presence of antibiotics is through the phenomenon of bacterial resistance and persistence. While a large portion of the bacterial population rapidly proliferates within an infected host organism, a smaller proportion of this population actively suppresses growth. Because the majority of all clinically used antibiotics target rapidly dividing bacteria, the small population of bacteria in a persister state is unaffected by these agents and can revert to a normal, non-persistent state after antibiotic treatment. An essential mediator for achieving the typical phenotype of resistant cells (also known in the art as the stringent response) is the alarmone guanosine polyphosphate (guanosine 3',5'-bisdiphosphate and guanosine 5'-triphosphate-3'-diphosphate) (abbreviated as (p)ppGpp). The level of (p)ppGpp is tightly controlled by the coordinated antagonism of RelA / SpoT homologous (RSH) enzymes, which can transfer the pyrophosphate group of ATP to the 3' position of GDP (or GTP) or remove the 3' pyrophosphate moiety from (p)ppGpp (Geiger et al., Infection and immunity, 2010).

[0004] The RelA-SpoT pair is the product of a gene duplication of the ancestral ribosome-associated bifunctional RSH Rel, a pair whose taxonomic distribution is restricted to the Betaproteobacteria and Gammaproteobacteria (Atkinson et al., PLoS One, 2011; Mittenhuber et al., J Mol Microbiol Biotechnol, 2001).

[0005] Subfunctionalization (the division of function between two paralogs through gene duplication) appears to have occurred at least twice in gammaproteobacteria. First, relatively soon after the duplication that gave rise to RelA and SpoT, RelA lost its alarmone hydrolysis ability and evolved into a monofunctional synthetase-only RSH. Second, during the evolution of protobacteria in the Moraxellaceae lineage, SpoT likely lost its synthetase function, as indicated by the lack of sequence conservation at sites essential for nucleotide pyrophosphorylation (Atkinson et al., PLoS One, 2011). This resulted in further specialization into a monofunctional (p)ppGpp hydrolase, SpoT[Hs] (capital "H" indicates hydrolase capacity, lowercase "s" indicates "synthetase incompetent"), in contrast to the bifunctional HD and SYNTH-competent SpoT[HS] found in other Beta- and Gamma-proteobacteria.

[0006] In particular, recent studies in A. baumannii have demonstrated the loss of (p)ppGpp in ΔrelA strains (i.e., A. baumannii strains in which the relA gene has been inactivated or deleted) both in the presence and absence of acute amino acid starvation induced by serine hydroxamate (SHX) (Jung et al., J Antimicrob Chemother, 2020; Perez-Varela et al., J Bacteriol, 2020). This observation is consistent with the hypothesis that RelA is indeed the sole source of alarmones in this bacterium. Furthermore, consistent with the important role of (p)ppGpp-mediated signaling in bacterial virulence and antibiotic resistance (Kundra et al., Front Microbiol, 2020), it is likely that ppGpp 0A. baumannii ΔrelA strains show increased susceptibility to multiple antibiotics (Jung et al., J Antimicrob Chemother, 2020; Perez-Varela et al., J Bacteriol, 2020), reduced virulence in the Galleria mellonella wax model, and a lack of switch from a pathogenic opaque to a non-pathogenic translucent colony variant (Perez-Varela et al., J Bacteriol, 2020).

[0007] Rel, RelA, and SpoT all share the same conserved domain composition, revealing a common structure underlying the intramolecular allosteric regulation of long RSH (Atkinson et al., PLoS One, 2011). When recruited to starved ribosomes, both Rel and RelA adopt highly extended, elongated conformations. In these complexes, the regulatory C-terminal domains (CTD: TGS, HEL, ZFD, and RRM domains) are highly structured, whereas the N-terminal catalytic domains (NTD: HD and SYNTH domains) and interdomain linker regions are highly dynamic and remain unresolved in some structures (Arenz et al., Nucleic Acids Res, 2016; Brown et al., Nature 2016; Loveland et al., Elife 2016; Pausch et al., Cell Rep, 2020). Outside of the ribosome, structural understanding of long RSHs relies on the structures of isolated NTDs of several Rel representatives (Pausch et al., Cell Rep, 2020; Hogg et al., Cell, 2004; Tamman et al., Nat Chem Biol, 2020; Mojr et al., ACS Chem Biol, 2021). Although the physiological role of SpoT as a key virulence and stress resistance factor is well established (Fitzsimmons et al., mBio, 2020; Vogt et al., Infect Immun, 2011), structural insight into SpoT is lacking.

[0008] As one of the bacterial pathogens listed in the ESKAPE group, innovative strategies enabling effective treatment of A. baumannii are urgently needed. More specifically, approaches enabling the screening of compounds capable of modulating the activity of the A. baumannii SpoT enzyme would be of great value for the generation of novel antibacterial agents. Summary of the Invention

[0009] We have determined the full-length structure of the Acinetobacter baumannii SpoT enzyme, and more specifically, the complete structure of the bound, active A. baumannii SpoT-ppGpp complex. Obtaining this full-length structure of SpoT is essential for understanding and modulating the stringent response of A. baumannii. This discovery provides important structural insight into the structure of the A. baumannii SpoT enzyme, enabling interpretation of its physiological and microbiological functions at the molecular level. The structural and biochemical data presented herein provide long-missing structural insight into the molecular mechanism of SpoT. We have determined the full-length structure of A. baumannii SpoT (SpoT Ab ) is a monofunctional (p)ppGpp hydrolase and explains why its CTD is an allosteric activator of HD hydrolase function. Full-length HD-active SpoT complexed with a ppGpp substrate. Ab The structure of SpoT reveals a compact monomeric conformation in which all regulatory domains wrap around a Core subdomain that connects the pseudo-SYNTH and TGS domains. This Core is one of the intrinsically disordered regions (IDRs) present in Rel and RelA when they are in their active synthetase state. AbIn this study, Core and TGS cooperate to coordinate and activate the hydrolase domain active site, while simultaneously translating allosteric feedback from other regulatory domains to regulate HD output. We propose a unified conceptual framework that rationalizes the relative balance between the HD and SYNTH activities of the long RSHs Rel, RelA, and SpoT, which are fine-tuned through entropic forces generated by intrinsically disordered regions that act as gatekeepers of enzyme conformation. As an example, but not limited to, compounds that inhibit or even reduce A. baumannii SpoT hydrolase activity lead to the accumulation of toxic ppGpp alarmones and consequent cell death.

[0010] Thus, the present invention relates to the following aspects: Embodiment 1. A method for identifying a compound that modulates A. baumannii SpoT activity, comprising using a three-dimensional structure represented by the set of atomic coordinates set forth in Table 1, or a subset thereof, or using atomic coordinates that deviate from the atomic coordinates of Table 1, or a subset thereof, by a root mean square deviation (RMSD) of residues on protein backbone atoms of 3 Å or less, and evaluating the fit of a candidate compound to said three-dimensional protein structure of A. baumannii SpoT.

[0011] Embodiment 2. The method of embodiment 1, wherein said method is a method for identifying a compound that modulates A. baumannii SpoT hydrolase activity.

[0012] Embodiment 3. The method of embodiment 1 or 2, wherein interaction of the candidate compound with one or more amino acid residues in a region on the surface of the protein defined by the following amino acid residues of a SpoT amino acid sequence, defined by an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:1: Arg45, Lys46, Ser47, Tyr51, His54, His78, Asp79, Ser113, Lys140, Asp143, Asn147, Thr150, Ala153, and Lys158, indicates that the candidate compound is a modulator of SpoT hydrolase activity.

[0013] Embodiment 4. The method of embodiment 1 or 2, wherein said amino acid sequence has at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0014] Aspect 5. The method of any one of Aspects 1 to 4, wherein said amino acid sequence comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:1.

[0015] Aspect 6. The method of any one of Aspects 1 to 5, further comprising determining a score for said candidate compound that modulates A. baumannii SpoT activity, preferably A. baumannii SpoT hydrolase activity, based on the number of interactions with said amino acid residues.

[0016] Aspect 7. The method of any one of Aspects 1-6, further comprising comparing the conformational state of A. baumannii SpoT before and after binding of the candidate compound to A. baumannii SpoT, wherein a change in conformational state indicates that the candidate compound is a true modulator of A. baumannii SpoT activity, and preferably, the conformational state of A. baumannii SpoT before binding of the candidate compound is a conformational state characterized by the atomic coordinates of Table 1.

[0017] Embodiment 8. The method of any one of Embodiments 1 to 7, wherein the method is a method for identifying a compound that partially or fully inhibits (i.e., reduces) A. baumannii SpoT hydrolase activity.

[0018] Embodiment 9. The method according to any one of embodiments 1 to 7, wherein the method is a method for identifying a compound that increases A. baumannii SpoT hydrolase activity.

[0019] Aspect 10. The method of any one of Aspects 1 to 9, further comprising testing the ability of the candidate compound to modulate A. baumannii SpoT hydrolase activity, preferably further comprising testing the ability of the candidate compound to inhibit or increase A. baumannii SpoT hydrolase activity.

[0020] Aspect 11. The method of any one of Aspects 1 to 10, wherein the candidate compound is a compound that interacts with A. baumannii SpoT via an interface between the Core domain and the regulatory C-terminal domain region.

[0021] Embodiment 12. The method of embodiment 11, wherein the candidate compound is a compound that interacts with A. baumannii SpoT through an interface between a Core domain and a regulatory C-terminal domain selected from the group consisting of TGS, HEL, ZFD, RRM, or any combination thereof.

[0022] Aspect 13. A method is a computer-implemented method, the computer having an input device, a processor, a user interface, and an output device, the method comprising: a) generating a three-dimensional structure of the atomic coordinates in Table 1 or a subset thereof; b) matching the structure of step a) with the structure of the candidate compound by computer modeling; c) selecting candidate compounds that possess energetically favorable interactions with the structure of step a). 13. The method according to any one of aspects 1 to 12, comprising:

[0023] Embodiment 14. The method of embodiment 13, wherein said fitting comprises superimposing the structure of step a) with the structure of said candidate compound, and optionally, said fitting comprises superimposing the structure of atomic coordinates corresponding to bound ppGpp with the structure of said candidate compound.

[0024] Embodiment 15. The method of embodiment 13 or 14, wherein said modeling comprises docking modeling.

[0025] Aspect 16. The method of any one of Aspects 13 to 15, wherein the candidate compound of step c) is capable of binding to at least one amino acid residue of the structure of step a) without steric interference.

[0026] Embodiment 17. An in vitro method for identifying compounds that specifically modulate A. baumannii SpoT hydrolase activity, comprising: a) providing a candidate compound; b) providing an A. baumannii SpoT protein or a SpoT-ppGpp complex; c) contacting the candidate compound with the A. baumannii SpoT protein or SpoT-ppGpp complex; d) determining the hydrolase activity of A. baumannii SpoT in the presence and absence of the candidate compound; and e) if a change in hydrolase activity is detected, identifying the candidate compound as a compound that modulates A. baumannii SpoT. A method comprising:

[0027] Aspect 18. The method of Aspect 17, wherein the compound inhibits the hydrolase activity of A. baumannii SpoT or wherein the compound stimulates (i.e., increases) the hydrolase activity of A. baumannii SpoT.

[0028] Embodiment 19. The method of embodiment 17 or 18, wherein the A. baumannii SpoT protein has at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, even more preferably 95% sequence identity to the amino acid sequence of SEQ ID NO: 1, and most preferably the A. baumannii SpoT protein is SEQ ID NO: 1.

[0029] Embodiment 20. The method of any one of embodiments 17 to 19, wherein the A. baumannii SpoT protein or SpoT-ppGpp complex is defined by the atomic coordinates in Table 1.

[0030] Aspect 21. The method according to any one of Aspects 17 to 20, wherein the specific modulation of the hydrolase activity of the SpoT protein occurs through direct binding of the candidate compound to the SpoT protein or to the SpoT-ppGpp complex.

[0031] Embodiment 22. Use of the crystal structure of the A. baumannii SpoT-ppGpp complex, defined by the atomic coordinates set out in Table 1, or a subset thereof, or defined by atomic coordinates that deviate from the atomic coordinates in Table 1, or a subset thereof, by an RMSD on the protein backbone atoms of 3 Å or less, for designing and / or identifying compounds that modulate A. baumannii SpoT hydrolase activity.

[0032] Aspect 23. A computer system, comprising: a) a database containing information including atomic coordinates defined by Table 1, or a subset thereof, stored on a computer-readable storage medium; and b) a user interface for viewing said information A computer system comprising:

[0033] Embodiment 24. Use of a computer system according to embodiment 23 for designing and / or identifying compounds that modulate A. baumannii SpoT activity.

[0034] Embodiment 25. A crystal of an A. baumannii SpoT-ppGpp complex comprising a structure characterized by the atomic coordinates defined in Table 1, or a subset thereof.

[0035] Embodiment 26. The crystal according to embodiment 25, obtained by crystallizing SEQ ID NO: 1.

[0036] Embodiment 27. A crystal according to embodiment 25 or 26, obtained by crystallizing A. baumannii SpoT protein in a solution comprising 0.85 M sodium citrate tribasic dihydrate, 0.1 M Tris pH 8.0, and 0.1 M sodium chloride using space group p21 21 21, and unit cell: 128.791 133.761 211.328 90.00 90.00 90.00, and supplementing the solution with ppGpp prior to harvesting the crystals, preferably at 50 mM.

[0037] Embodiment 28. A computer system intended to generate three-dimensional structural representations of an A. baumannii SpoT protein and / or SpoT-ppGpp complex, a complex of an A. baumannii SpoT protein and a binding compound or modulator, and to analyze or optimize binding of a compound or modulator to said A. baumannii SpoT protein and / or SpoT-ppGpp complex, comprising: (a) Optionally, the coordinates of the A. baumannii SpoT protein structure listed in Table 1, or selected coordinates thereof, varying by no more than 3 Å root mean square deviation of residue backbone atoms; (b) Optionally, the coordinates of the A. baumannii SpoT-ppGp complex structure listed in Table 1 , or selected coordinates thereof, varying by no more than 3 Å root-mean-square deviation of the residue backbone atoms; (c) optionally, coordinates of candidate binding compounds or modulators generated by interpreting X-ray crystallographic, cryo-EM, or NMR data with reference to the coordinates of the A. baumannii SpoT protein structure and / or SpoT-ppGp complex structure listed in Table 1, or selected coordinates thereof, varying by no more than 3 Å root mean square deviation of residue backbone atoms; and (d) Structure factor data derivable from the coordinates in (a), (b), or (c). A system comprising computer readable data including one or more of:

[0038] Embodiment 29. The computer system of embodiment 28, wherein the computer system compares the atomic coordinates of (a) and (c), and if a steric conflict is detected, the candidate compound or modulator is not considered to be a suitable A. baumannii SpoT protein modulator.

[0039] Embodiment 30. The computer system of embodiment 28 or 29, wherein the computer system compares the atomic coordinates of (a) and (c), and if no steric conflict is detected, the candidate compound or modulator is deemed to be a suitable A. baumannii SpoT protein modulator.

[0040] Aspect 31. A computer-readable storage medium comprising a data storage material encoded with computer-readable data, said data comprising: (a) Optionally, the coordinates of the A. baumannii SpoT-ppGp complex listed in Table 1, or selected coordinates thereof, varied by no more than 3 Å root mean square deviation of the residue backbone atoms; (b) Optionally, the coordinates of the A. baumannii SpoT-ppGp complex structure listed in Table 1 , or selected coordinates thereof, varying by no more than 3 Å root-mean-square deviation of the residue backbone atoms; (c) optionally, coordinates of a candidate binding compound or modulator generated by interpreting X-ray crystallographic, cryo-EM, or NMR data with reference to the coordinates of the A. baumannii SpoT-ppGp complex listed in Table 1, or selected coordinates thereof, varying by no more than 3 Å root mean square deviation of residue backbone atoms; and (d) Structure factor data derivable from the coordinates in (a), (b), or (c). 1. A computer-readable storage medium, including one or more of:

[0041] Embodiment 32. A computer-readable storage medium optionally comprising a data storage material encoded with a first set of computer-readable data comprising structural coordinates of an A. baumannii SpoT-ppGpp complex enzyme listed in Table 1 that vary by no more than 3 Å root mean square deviation of residue backbone atoms, or a Fourier transform of at least a portion of selected coordinates thereof, which data, when combined with a second set of machine-readable data comprising an X-ray diffraction pattern of a molecule or molecular complex whose structure is unknown, can be used to determine at least a portion of the structural coordinates corresponding to said second set of machine-readable data using a machine programmed with instructions for using said first set of data and said second set of data.

[0042] Embodiment 33. The computer system of any one of embodiments 28 to 33 or the computer-readable storage medium of embodiment 31 or 32, further comprising a database containing information regarding the three-dimensional structures of candidate compounds or modulators that are small molecules.

[0043] In particular, each of the aspects and embodiments of the invention outlined in this disclosure contemplates the use of the complete set of atomic coordinates of Table 1 included herein, but also contemplates the use of a subset of the atomic coordinates of Table 1, where the subset of coordinates of Table 1 corresponds to the subset of atomic coordinates corresponding to an isolated A. baumannii SpoT enzyme (i.e., SpoT in the active binding conformation of Table 1 without ppGpp), one or more protein domains thereof, or an isolated ppGpp molecule (i.e., ppGpp without SpoT). Each of these subsets, and methods for obtaining them starting from Table 1, are further detailed throughout this disclosure.

[0044] The above and further aspects and preferred embodiments of the present invention are described in the following sections and in the appended claims, the subject matter of which is therefore specifically incorporated into this specification. [Brief explanation of the drawings]

[0045] [Figure 1-1] Full-length monomeric A. baumannii SpoT adopts a compact, "mushroom"-shaped, HD-active τ state. (a) Structure of the "mushroom" SpoTAb-ppGpp complex in the τ state. The domains, from N- to C-terminus, are the NTD domain hydrolase (HD), pseudosynthetase (pseudo-SYNTH) and core, as well as the CTD domain, TGS, helical (HEL), Zn-finger (ZFD), and RNA recognition motif (RRM). The ppGpp alarmone is labeled. (b) Schematic representation of SpoTAb. The "stalk" of the mushroom is formed by the enzymatic HD domain, and the "umbrella" is formed by the regulatory domains: the NTD pseudo-SYNTH domain and the CTD domain. (c) Ribbon representation of the SpoTAb-ppGpp complex. The α6 / α7 motif is held in a suitable position for hydrolysis by the folded Core domain and the TGS β-hairpin, which transmits an allosteric signal from the regulatory domain to the HD. (d) The HD activity of SpoTAb is insensitive to the addition of E. coli 70S ribosomes and is weakly inhibited nonspecifically by both aminoacylated and deacylated E. coli tRNAVal. (e) Analytical size-exclusion chromatography (SEC) of SpoTAb confirms its monomeric nature in solution. [Figure 1-2](f) Experimental X-ray scattering (SAXS) analysis of SpoTAb at 8 mg / mL further confirms the monomeric nature of SpoTAb. Analysis of the normalized Kratky plot (inset) of the SAXS curve reveals a folded, globular shape of SpoTAb. (g) The ab initio envelope of SpoTAb reconstructed from the experimental SAXS data is superimposed on the crystal structure. Comparison of both models shows that in solution, the enzyme adopts the same conformation as observed in the crystal. [Figure 2] A. baumannii SpoT is a Mn2+-dependent (p)ppGpp hydrolase. (a) Surface representation of SpoTAb in the τ state. The active site cavity within the HD domain is enclosed by a dashed line. (b) Close-up of the HD active site of the SpoTAb-ppGpp complex. The acidic half of the interface (residues K140, E82, D83, Y51, and R45) and Mn2+ ions activate water molecules for nucleophilic attack of the pyrophosphate bond of ppGpp, while the basic half of the interface (K46, K158, and R161) stabilizes the 3' and 5' phosphates of the alarmone substrate. (c) Ribbon representation of the active site of SpoTAb, revealing residues involved in ppGpp coordination. (d) Structure of the Mn2+-free N-terminal domain of SpoTAb (SpoTAbNTD). The HD domain is in purple (left part of the structure), and the pseudo-synth is in yellow (right part of the structure). The irregular active site is labeled. (e) Superposition of the HD domain of SpoTAb complexed with ppGpp onto Mn2+-free SpoTAb. Major conformational differences in catalytically important active site residues and structural elements α3, α4, and α8 are highlighted with dashed arrows and shown in bold, respectively. [Figure 3]The CTD controls the hydrolytic activity of SpoT by controlling the equilibrium between the HD-active τ-state conformation and the HD-inactive unfolded conformation. (a, b) SAXS curves of L356D in the τ-state (a) or unfolded state (b). (c) Pseudo-atomic model of the unfolded state of SpoTAb calculated with Dadimodo (Evrard et al., 2011) using the experimental SAXS data in (b). (d) Comparison of experimental SAXS data from the unfolded state of L356D (gray) with the theoretical scattering curve (solid line) of the unfolded state obtained from the Dadimodo model. (e) SAXS curve of RelAAb, consistent with the dimensions of the unfolded state. (f, g) SAXS curves of RelBs in the τ-state (f) or unfolded state (g). (h) Schematic representation of the experimentally observed conformational states and particle dimensions of the long RSH enzyme. [Figure 4] The Core domain of SpoT transmits allosteric signals from the regulatory CTD and pseudo-SYNTH to the enzyme HD domain. (a,b) Schematic representation of the interactions stabilizing the α6-α7 motif in the HD active site (A). The Core encases α7, while the TGS β-hairpin forms a small hydrophobic patch that stabilizes α6. These interactions prevent α6-α7 movement and maintain SpoTAb in a constitutive hydrolase-stimulating state. Key interface residues are shown as sticks and labeled. (b) The experimental SAXS curve of SpoTAb E379K / W382K is consistent with the dimensions of the τ state. Schematic representation of the HD:Core:RRM signaling axis. (c) The structure of the τ state suggests that the RRM is locked in place via supportive interactions provided by the Core and the pseudo-SYNTH, suggesting that additional anchoring of the RRM to the pseudo-SYNTH may further stabilize the τ conformation. (d) The SAXS curve of the SpoTAb I637D / R641D variant, in which substitutions I637D / R641D and I637A / R641A, respectively, promote H-bonding to stabilize the α-helical structure, is consistent with the dimensions of the τ state. [Figure 5]The enzymatic output of semifunctional RelA and SpoT RSH enzymes is evolutionarily tuned through conformational landscape constraints. (a) Regulation of enzymatic output of the ancestral bifunctional Rel[HS]. Upon amino acid starvation, Rel is recruited to starved ribosomal complexes. Ribosome-bound Rel adopts an extended conformation, which relieves the autoinhibitory effect of the CTD region on SYNTH activity. Full activation of SYNTH activity is achieved by binding of (p)ppGpp to an allosteric site within the NTD and the release of SYNTH inhibition by the HD domain. Conversely, upon detachment from the ribosome, the enzyme adopts the τ state. In this conformation, locking of the α6-α7 motif by the CTD organizes HD active site residues to promote HD activity. This, in turn, strongly inhibits SYNTH activity through inter-NTD regulation. Full activation of either SYNTH or HD requires allosteric signaling from the CTD to the NTD enzyme domain. (b,c) The evolution of SpoT as a primarily dedicated hydrolase was accompanied by the loss of allosteric control of the NTD by (p)ppGpp and the ribosome. In the bifunctional SpoT[HS] present in most gammaproteobacteria and betaproteobacteria, the equilibrium is largely shifted toward the HD-active τ state, but the enzyme is capable of inefficient (p)ppGpp synthesis in the unfolded state (B). Hemifunctionalization of SpoT in Moraxellaceae gave rise to the monofunctional hydrolase SpoT[Hs], which naturally exists only in the compact τ state and is synth-inactive. (d) Hemifunctionalization of RelA[hS] in gammaproteobacteria and betaproteobacteria constitutes another extreme example of evolutionary constraints on the conformational dynamics of ancestral Rel[HS]. While losing HD activity, RelA retains all of the allosteric regulatory elements of Rel. Being a dedicated (p)ppGpp synthetase enzyme, RelA away from the ribosome does not adopt the τ state.Instead, it primarily occupies a functionally hindered resting state corresponding to the unwound state of Rel, primed to adopt an elongated ribosome-associated state triggered by the 70S ribosome, uncharged tRNA, and alarmone under stringency. The circles represent catalytic centers in different activation states. [Figure 6] Important amino acid residues for screening candidate compounds. Visualization of the binding pocket of the A. baumannii SpoT enzyme bound to ppGpp. A non-limiting group of important amino acid residues are annotated, which are preferred residues for successful binding of candidate compounds (i.e., candidate A. baumannii SpoT enzyme modulators). DETAILED DESCRIPTION OF THE INVENTION

[0046] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly indicates otherwise.

[0047] The terms "comprising," "comprises," and "consisting of," as used herein, are synonymous with "including," "includes," or "containing," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. These terms also encompass "consisting of" and "consisting essentially of," which have well-established meanings in patent language.

[0048] The recitation of numerical ranges by endpoints includes not only the recited endpoints but also all numbers and fractions subsumed within each range.

[0049] The terms "about" or "approximately," when used herein in reference to a measurable value such as a parameter, amount, duration, and the like, are meant to encompass variation of the specified value and variation from the specified value, for example, variation of ±10% or less from the specified value, preferably variation of ±5% or less, more preferably variation of ±1% or less, and even more preferably variation of ±0.1% or less from the specified value, provided that such variation is appropriate for practicing the invention of the present disclosure. It should be understood that the value to which the modifier "about" refers is itself specifically and preferably disclosed.

[0050] The term "one or more" or "at least one," e.g., one or more members or at least one member of a group of members, is clear in itself, but by way of further illustration, the term specifically encompasses reference to any one of said members, or any two or more of said members (e.g., any three or more, four or more, five or more, six or more, seven or more, etc. of said members, and up to all of said members). In another example, "one or more" or "at least one" can refer to 1, 2, 3, 4, 5, 6, 7, or more.

[0051] The discussion of the background of the invention herein is included to explain the context of the invention and is not an admission that any of the material referred to was published, known, or part of the general knowledge in any country at the priority date of any claim.

[0052] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by an identifying citation. All documents cited herein are incorporated by reference in their entirety. Specifically, the teachings or sections of such documents that are specifically referenced herein are incorporated by reference.

[0053] Unless otherwise defined, all terms (including technical and scientific terms) used in disclosing the present invention have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. For further guidance, definitions of terms are included to better understand the teachings of the present invention. When a particular term is defined in connection with a particular aspect of the present invention or a particular embodiment of the present invention, such meaning is meant to apply throughout the specification, unless otherwise defined, i.e., it is meant to apply in the context of other aspects or embodiments of the present invention.

[0054] In the following sections, various aspects or embodiments of the present invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect or embodiment, unless expressly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature indicated as being preferred or advantageous.

[0055] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with this embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase "one embodiment" or "an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, although they may. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, some embodiments described herein include some features but not others included in other embodiments, meaning that combinations of features from different embodiments form different embodiments within the scope of the present invention and as would be understood by one of ordinary skill in the art. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0056] Amino acids are referred to herein by their full name, their three letter abbreviation, or their one letter abbreviation.

[0057] It will be apparent that terms such as "(candidate) compound", "(candidate) binding compound", "(candidate) ligand", and (candidate) modulator may be used interchangeably to describe the present invention.

[0058] Those skilled in the art are aware of standard molecular biology techniques available in the art (Green and Sambrook, Molecular cloning: a laboratory manual 4 th Ed, Cold Spring Harbor laboratory press, 2012;Ausubel et al., Current protocols in molecular biology, John Wiley and Sons, 1989;Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, 1988;Watson et al., Recombinant DNA, Scientific American Books, New York;Birren et al. Genome Analysis: A Laboratory Manual Series, Vols. 1-4 Cold Spring Harbor laboratory press, New York, 1998).

[0059] The term "RSH enzyme" as used herein is an abbreviation for the group of RelA / SpoT homologous enzymes. RSH enzymes derive their name from their sequence similarity to the RelA and SpoT enzymes of Escherichia coli. RSH enzymes constitute a family of enzymes that synthesize and / or hydrolyze the alarmone ppGpp and play a central role in the bacterial stringent response. So-called "long" RSH enzymes, containing hydrolase and synthetase domains, have been identified in a wide variety of bacteria and plant chloroplasts, but specific RSH enzymes that synthesize or hydrolyze only (p)ppGpp have also been found in different bacteria and animals. In the art, RSH enzymes are classified into three groups based on their activity: long RSH enzymes, small alarmone synthetases (SAS), and small alarmone hydrolases (SAH). These initial groups have been further divided into numerous subgroups (Atkinson et al., Plos One, 2011). Long RSHs contain two catalytic domains ((p)ppGpp hydrolase (HD) domain and (p)ppGpp synthetase (SYN) domain) and a C-terminal protein domain involved in enzyme regulation. In contrast, both SAS and SAH lack a conserved C-terminal regulatory domain. According to the technical field, long RSHs are the most widely distributed and often contain TGS (ThrRS, GTPase, and SpoT) domains and ACT (aspartokinase, chorismate mutase, and TyrA) domains in the C-terminal domain, which may play a role in sensing stress signals, such as starvation signals, and transmitting the signals to the catalytic domain.

[0060] The term "stringent response" is used synonymously in the art with "stringent control" to refer to the stress response mediated by RSH enzymes in response to various stress conditions, including, but not limited to, amino acid starvation, fatty acid limitation, iron limitation, and heat shock. Under such stress conditions, the stringent response mediates a major shift in gene expression from a growth-focused program to a gene expression profile that allows long-term survival in stationary phase after aminoacyl-tRNA pools can no longer support protein synthesis. Thus, the stringent response is a key mediator in the process of bacterial persister cell formation. The stringent response has been widely described in the art (e.g., Traxler et al., Mol Microbiol, 2013). The stringent response is governed by the alarmones guanosine 5',3'-bispyrophosphate and guanosine pentaphosphate (ppGpp and pppGpp, respectively). Accumulation of (p)ppGpp actively inhibits resource-intensive cellular processes such as replication, transcription, and translation. (p)ppGpp has been demonstrated to bind to RNA polymerase proximal to the active site and terminate transcription of stable RNA. Furthermore, (p)ppGpp shortens the half-life of the open complex at most promoters tested in the art, thereby mediating the strong downregulation of promoters with inherently short half-lives, such as promoters of stable RNA genes. Collectively, this stringent response involves a massive downregulation of the translational machinery (Barker et al., J Mol Biol, 2001). Additionally, (p)ppGpp, together with the RNA polymerase-binding transcription factor DksA, has been shown to upregulate the transcription of promoters acting on amino acid biosynthetic genes (Paul et al., PNAS USA, 2005).

[0061] "Persister cells," or "persisters" for short, as used herein, are used to describe a population of bacterial cells that are in or moving toward a metabolically inactive (i.e., dormant) or near-dormant state (so-called stationary phase), characterized by no or very slow growth (Lewis, Nature Reviews Microbiol, 2007). Typically, in an infected organism that is optionally treated with antibiotics, persisters represent a small percentage of the total bacterial population present within the infected organism. Upon termination of antibiotic treatment, persisters emerge from their dormant state and revert to a growth-focused gene expression signature, potentially expanding to full-blown bacterial infection. Due to their slow growth rate, persisters are often described as constituting a subpopulation of bacteria that are increasingly resistant to antibiotics. Persister bacterial cells can result from genetic and / or metabolic changes. Those skilled in the art recognize that the persistence of bacterial cells is associated with the emergence of antibiotic resistance. (Windels et al., Bacterial persistence promotes the evolution of antibiotic resistance, 2019). The production of (p)ppGpp has been linked to the formation of bacterial persister cells (Korch et al., Mol Microbiol, 2003, among others). Persister cells can form within biofilms.

[0062] The term "biofilm" is commonly used in the art to refer to an assembly (i.e., aggregate) of (symbiotic) microorganisms, such as bacteria, in which different cells are attached to one another and, optionally, surfaces are in contact with the cells or parts of cells. Biofilms are further characterized by a viscous extracellular matrix containing extracellular polymeric substances (EPS) produced by the microorganisms of the biofilm, in which the microorganisms are embedded. Biofilms can form in or on both living and non-living surfaces under diverse environments. Biofilms are complex microbiological systems in which the microorganisms contained within the biofilm can be organized into functional units or communities (Lopez et al., Biofilms, Cold Spring Harbor perspectives in biology, 2010).

[0063] The term "alarmone" is known to those skilled in the art and refers to an intracellular signaling molecule that is produced as a result of and in response to environmental stimuli. The main function of alarmones is to regulate gene expression. Typically, alarmone concentrations increase when cells experience stressful environmental factors. (p)ppGpp is considered a canonical example of an alarmone (Hauryliuk et al., Nat Rev Microbiol, 2015). Those skilled in the art understand that the term "(p)ppGpp" encompasses both guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp).

[0064] As indicated above, the inventors' discoveries enable the provision of a screening method for modulating A. baumannii SpoT activity, which is enabled by the atomic coordinates of the A. baumannii SpoT enzyme (more specifically, the SpoT-ppGpp complex) contained in Table 1.

[0065] Accordingly, in a first aspect, the present invention is directed to a method for identifying a compound that modulates A. baumannii SpoT enzymatic activity, the method comprising using a three-dimensional structure represented by the set of atomic coordinates set forth in Table 1, or a subset thereof, or using atomic coordinates that deviate from the atomic coordinates of Table 1, or a subset thereof, by a root mean square deviation (RMSD) of residues on protein backbone atoms of 3 Å or less, and assessing the fit of a candidate compound to said three-dimensional protein structure of A. baumannii SpoT and / or SpoT-ppGpp complex.

[0066] "Acinetobacter baumannii," as used herein, should be construed according to its generally accepted meaning in the art, i.e., an opportunistic Gram-negative bacterial pathogen in humans (Domain: Bacteria; Phylum: Pseudomonadota; Class: Gammaproteobacteria; Order: Pseudomonadales; Family: Moraxellaceae; Genus: Acinetobacter; Species: A. baumannii). "Acinetobacter baumannii SpoT enzyme" refers to "SpoT enzyme," "SpoT," and "SpoT." Ab " is used synonymously with the term "Gpp synthetase," which refers to the bifunctional (p)ppGpp synthetase / guanosine-3',5'-bis(diphosphate) 3'-pyrophosphohydrolase expressed by the bacterium Acinetobacter baumannii.

[0067] "RMSD," "root mean square deviation," or "root mean square deviation of atomic positions," as used herein, refers to a quantitative measure of the similarity between two or more protein structures, and more specifically, refers to a measure of the average distance between the (backbone) atoms of the superimposed proteins. RMSD values ​​are generally calculated according to the formula:

[0068]

number

[0069] The term "atomic coordinates," as used herein, refers to the position of an atom in space, typically represented by a set of X, Y, and Z Cartesian coordinates and the chemical element each atom represents. The atomic coordinates of a particular protein structure are typically combined with an atomic coordinate data file, which may have various data formats, such as the format of Table 1 attached hereto. Other non-limiting data formats include the Protein Data Bank (PDB) format or various text formats. Some variation in atomic coordinates is anticipated, and the claims are intended to encompass such variation. In certain embodiments, the atomic coordinates further comprise additional information. It will be apparent to those skilled in the art that three-dimensional rigid rotation or translation of the atomic coordinates does not alter the structure of the molecule. Because the atomic coordinates disclosed herein are relative collections of points describing a three-dimensional structure, it will be apparent that different sets of coordinates may define similar or identical three-dimensional structures. With this in mind, several computer analysis tools and programs have been developed to assess whether molecular structures have similarity to structures defined by the atomic coordinates or a subset of the atomic coordinates set forth in Table 1 herein. By way of example, and not limitation, a suitable software application for performing such analyses is the Molecular Similarity program in QUANTA (Molecular Simulations Inc., San Diego, CA). The Molecular Similarity program and consort allow for extensive comparisons between different structures, different conformations of the same structure, and different portions of the same structure. This comparison method typically involves calculating one or more optimal translations and rotations required to achieve the absolute minimum RMSD for the fit of a specified pair of equivalent atoms. Thus, the atomic coordinates of the A. baumannii SpoT protein or SpoT-ppGpp complex, or fragments that translate and / or rotate to the atomic coordinates in Table 1, are within the scope of the present invention.

[0070] The methods described herein may be performed using the atomic coordinates set forth in Table 1 (which collectively represent the active state of the A. baumannii SpoT enzyme bound to ppGpp, and are referred to throughout this disclosure as the A. baumannii SpoT-ppGpp complex), or may be performed using a subset thereof (e.g., a subset that defines the structure of active, bound A. baumannii SpoT excluding the ppGpp molecule). The size of the subset is not particularly limited, although one of skill in the art will understand that performance of the methods described herein will benefit from increasing the size of the subset obtained from Table 1. By way of example, and not limitation, the subset may include 20%, preferably 40%, preferably 50%, preferably 60%, preferably 70%, preferably 80%, or preferably 90% of the atomic coordinates set forth in Table 1. The term "subset," as defined herein, refers to a portion of the atomic coordinates in Table 1. By way of example, and not limitation, a possible subset in the context of the present invention is the subset of coordinates defining the isolated ppGpp molecule portion of the A. baumannii SpoT-ppGpp complex (i.e., only the group of atomic coordinates in Table 1 annotated as "G4P" coordinates). An alternative possible subset is the subset of coordinates defining the A. baumannii SpoT enzyme portion of the A. baumannii SpoT-ppGpp complex (i.e., the atomic coordinates in Table 1 excluding those annotated as "G4P"). Thus, the methods described herein can be performed with the set of atomic coordinates shown in Table 1 as a whole. Alternatively, the methods described herein can be performed with the subset of atomic coordinates shown in Table 1 annotated as "G4P" coordinates. Alternatively, the methods described herein may be performed on a subset of the atomic coordinates shown in Table 1 that are not annotated as "G4P" coordinates.

[0071] A further alternative possible subset is a subset of coordinates defining key amino acid residues of the A. baumannii SpoT enzyme in the SpoT-ppGpp complex, such as the key amino acids Arg45, Lys46, Ser47, Tyr51, His54, His78, Asp79, Ser113, Lys140, Asp143, Asn147, Thr150, Ala153, and Lys158.

[0072] In the art, "degree of fit" or "goodness of fit" refers to the likelihood that a particular candidate binding mode represents a favorable binding interaction and allows different ligands to be ranked relative to one another. In certain embodiments, the fit between the three-dimensional A. baumannii SpoT structure (or the three-dimensional SpoT-ppGpp complex structure) and a candidate SpoT modulator is expressed numerically. In alternative embodiments, the fit is represented by a graphical superposition of the A. baumannii SpoT structure (or the SpoT-ppGpp complex) and the compound's structure. In certain embodiments, the fit of a ligand is expressed relative to the fit of known ligands of the A. baumannii SpoT protein. The fit may be expressed as an absolute or relative value, depending on the methodology used to calculate the quantitative score. When the fitness is expressed as an absolute value, this absolute value corresponds to a score assigned to a candidate compound based on the number of in silico interactions predicted to occur with a set of atomic coordinates set forth in Table 1 herein and / or a set of amino acid residues within said region on the surface of a protein described herein. The number of interactions can be one or more, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, more than 10, or all amino acid residues within said region on the surface of a protein defined herein. In certain embodiments, the atomic coordinates set forth in Table 1 and / or the amino acid residues cited herein to constitute a surface region of a protein are further abstracted into a pharmacophore, i.e., a set of molecular features required for molecular recognition of a ligand by a biological macromolecule (here, the candidate compound and the A. baumannii SpoT protein). In certain embodiments, a fitness (i.e., fitness score) of 2.4 is used as a threshold for a candidate compound to be further tested and / or validated. In an alternative embodiment, a fitness score of 3.0 is used.In alternative embodiments, a fit score of 2.4 to 3.0 is used, preferably 2.5 to 3.0, 2.7 to 3.0, or 2.9 to 3.0. In alternative embodiments, a fit score of 2.4 to 2.9 is used, preferably 2.4 to 2.7, or 2.4 to 2.5. In certain embodiments, a variable fit score threshold is used depending on the molecular weight of the candidate compound. In further embodiments, candidate compounds between 301 Da and 330 Da have a fit score threshold of 2.4, candidate compounds between 331 Da and 380 Da have a fit score threshold of 2.5, candidate compounds between 381 Da and 420 Da have a fit score threshold of 2.7, candidate compounds between 421 Da and 490 Da have a fit score threshold of 2.9, and candidate compounds between 491 Da and 540 Da have a fit score threshold of 3.0. When the fit is a relative value, it can be expressed in comparison to a reference compound known to modulate the activity of the A. baumannii SpoT protein. In such embodiments, a candidate compound is considered a true modulator of A. baumannii SpoT if its fit is at least 50%, preferably at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, and most preferably at least 95% relative to the reference compound known to modulate the activity of the A. baumannii SpoT enzyme. From this initial score, it is clear that the fit of multiple ligands can be directly compared. Numerous scoring functions or mechanisms have been described in the art (e.g., Fu and Zhang, Interdiscip Sci, 2019), and it is clear that various scoring functions are suitable for generating a fit between a candidate compound and an A. baumannii SpoT protein. For example, when using the AMBER scoring function (Wang et al., J Comput Chem, 2004), a candidate compound is considered to be a true candidate modulator if a docking score threshold is met. In one particular embodiment, a docking score threshold of -8.9 kcal / mol is used.In certain embodiments, a docking score threshold of -8.9 kcal / mol to -10.5 kcal / mol is used. In further embodiments, a docking score threshold of -9.4 kcal / mol to -10.5 kcal / mol is used. In further embodiments, a docking score threshold of -9.7 kcal / mol to -10.5 kcal / mol is used. In alternative further embodiments, a docking score threshold of -8.9 kcal / mol to -10.3 kcal / mol is used. In further embodiments, a docking score threshold of -8.9 kcal / mol to -9.7 kcal / mol is used. In further embodiments, a docking score threshold of -8.9 kcal / mol to -9.4 kcal / mol is used. In alternative embodiments, a docking score threshold of -10.5 kcal / mol was used. In yet alternative embodiments, a variable docking score threshold was used, preferably based on the molecular weight of the candidate compound. In further embodiments, compounds with a molecular weight between 301 Da and 330 Da are assigned a docking score threshold of -8.9 kcal / mol, compounds with a molecular weight between 331 Da and 380 Da are assigned a docking score of -9.4 kcal / mol, compounds with a molecular weight between 381 Da and 420 Da are assigned a docking score of -9.7 kcal / mol, compounds with a molecular weight between 421 Da and 490 Da are assigned a docking score of -10.3 kcal / mol, and compounds with a molecular weight between 491 and 540 Da are assigned a docking score threshold of -10.5 kcal / mol.

[0073] As defined herein, "in silico analysis" refers to an analysis performed on a computing system or by using a computer simulation system guided by a set of specific instructions, such as a molecular docking computer program or tool. "Molecular docking" refers to a method that allows prediction of the binding and / or preferred orientation of one molecule to another molecule when the two molecules bind to each other to form a stable complex. Thus, molecular docking software is understood to predict the behavior of molecules at the binding site of a target protein. Molecular docking software tools and programs that allow evaluation of the specificity of candidate molecules or compounds for specific targets have been described in the art. Molecular docking software allows for searching for geometric and / or electrostatic complementarity between the binding site surface and the ligand. The molecular docking process can be divided into two main steps: searching and scoring. Numerous examples of various docking tools and programs have been described and are therefore known to those skilled in the art (Pagadala et al., Biophys Rev, 2017). Two main general molecular docking approaches have been described: one is molecular docking, which relies on shape complementarity or geometric matching, and the other relies on simulation of the docking process, in which the ligand-protein pair interaction energy is calculated.

[0074] As used herein, "modulator" refers to a molecule that interacts with (and / or binds to) one or more proteins and affects one or more (enzymatic) activities of said proteins. As used herein, the modulatory effect of the modulators described herein is intended to affect the hydrolase activity of the A. baumannii SpoT protein as defined herein. Thus, the "modulator" discussed herein can refer to a molecule that is a hydrolase activator or a hydrolase inhibitor. Preferred modulators in the context of the present invention are hydrolase inhibitors. Such modulators disrupt the equilibrium between the formation and degradation of ppGpp, causing the accumulation of toxic ppGpp alarmones in A. baumannii bacteria and ultimately cell death. The primary binding site of a modulator is commonly referred to as the orthosteric site, which may be, for example, the active site of an enzyme involved in binding to a substrate. In addition, a modulator may exert its activity by binding to a second binding site, commonly referred to as an allosteric binding site. In the context of the present invention, both orthosteric and allosteric regulators (preferably hydrolase inhibitors) are contemplated. It is understood by those skilled in the art that orthosteric regulators (e.g., orthosteric inhibitors) compete for the binding of A. baumannii SpoT to ppGpp. Commonly contemplated allosteric regulators are further described throughout this specification.

[0075] In the context of the present invention, a modulator is said to be an "inhibitor" if, as a result of interaction between the modulator and a target protein (in the context of the present invention, the A. baumannii SpoT protein), the hydrolase activity of the target protein is at least partially (i.e., to a certain extent) or completely reduced. In the latter case, it is understood that, due to interaction with the modulator, the enzymatic activity of the target protein is reduced to 0% or to an activity level below that measurable by methods available in the art (e.g., Gratani et al., PLoS genet, 2018). "Inhibition," as used herein, refers to the inhibition of a process, a molecular process herein, more specifically, the inhibition of SpoT enzyme hydrolase activity. It will be apparent to those skilled in the art that inhibition can be used synonymously with the term "attenuation." In certain embodiments, the inhibitor selectively inhibits A. baumannii SpoT hydrolase activity. In an alternative embodiment, the inhibitor selectively inhibits A. baumannii SpoT hydrolase activity in addition to the hydrolase and / or synthetase activity of at least one other A. baumannii enzyme. In yet an alternative embodiment, the inhibitor selectively inhibits A. baumannii SpoT hydrolase activity in addition to the hydrolase and / or synthetase activity of at least one other enzyme expressed by a bacterium selected from the group consisting of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.

[0076] Both reversible and irreversible inhibitors are contemplated herein. "Reversible inhibition" and "irreversible inhibition" are terms known to those skilled in the art and are commonly used to further define the type of enzyme inhibitor. The binding of an inhibitor to an enzyme can be either reversible or irreversible. Irreversible inhibitors usually react with the enzyme and induce a chemical change or modification (e.g., by forming a covalent bond). These inhibitors typically modify key amino acid residues required for enzyme activity. In contrast, reversible inhibitors bind non-covalently, and various types of inhibition are described depending on whether these inhibitors bind to the enzyme, the enzyme-substrate complex, or both. Methods for measuring the dissociation constant (Kd) of reversible inhibitors are well known to those skilled in the art (Pollard, Mol Biol Cell, 2010).

[0077] The term "dissociation constant" or "Kd" used herein is an equilibrium constant that quantitatively expresses the tendency of a large entity to reversibly separate or dissociate into smaller components. Dissociation constants are routinely used to quantify the affinity between a ligand and a drug, and are therefore known to those skilled in the art as an indicator of how tightly or strongly a ligand binds to a target protein. The affinity of a ligand for a protein is related to the amount of non-covalent intermolecular interactions between the ligand and the protein, such as hydrogen bonds, electrostatic interactions, hydrophobic interactions, and van der Waals forces. In addition, the concentration of other molecules present in the proximal environment where the ligand-protein interaction occurs can also affect affinity. This observation is known to those skilled in the art as molecular crowding (Rivas et al., Trends Biochem Sci, 2016).

[0078] In certain embodiments where a three-dimensional structure corresponding to a subset of the atomic coordinates set forth in Table 1 is utilized, the subset is selected such that the retained atomic coordinates correspond to the N-terminal catalytic region (NTD) portion of A. baumannii SpoT, optionally supplemented with the atomic coordinates of a ppGpp molecule. In alternative embodiments where a three-dimensional structure corresponding to a subset of the atomic coordinates set forth in Table 1 is utilized, the subset is selected such that the retained atomic coordinates correspond to the C-terminal domain region (CTD) portion of A. baumannii SpoT, optionally supplemented with the atomic coordinates of a ppGpp molecule.

[0079] In embodiments utilizing three-dimensional structures corresponding to a subset of the atomic coordinates set forth in Table 1, the subset is selected such that the retained atomic coordinates correspond to one or more A. baumannii SpoT domains selected from the group consisting of NTD domain hydrolase (HD), pseudosynthetase (pseudo-SYNTH), Core, TGS, helical (Hel), zinc-finger (ZFD), and RNA recognition motif (RRM). Optionally, the subset is selected such that the retained atomic coordinates define an HD domain, thereby corresponding to residues 1-194 of SEQ ID NO:1. Optionally, the subset is selected such that the retained atomic coordinates define a pseudo-SYNTH domain, thereby corresponding to residues 195-332 of SEQ ID NO:1. Optionally, the subset is selected such that the retained atomic coordinates define a Core domain, thereby corresponding to residues 333-380 of SEQ ID NO:1. Optionally, the subset is selected such that the retained atomic coordinates define a TGS domain, thereby corresponding to residues 381-453 of SEQ ID NO: 1. Optionally, the subset is selected such that the retained atomic coordinates define a helical domain, thereby corresponding to residues 457-536 of SEQ ID NO: 1. Optionally, the subset is selected such that the retained atomic coordinates define a ZFD domain, thereby corresponding to residues 560-605 of SEQ ID NO: 1. Optionally, the subset is selected such that the retained atomic coordinates define an RRM domain, thereby corresponding to residues 618-688 of SEQ ID NO: 1. Those skilled in the art will appreciate that each of these subsets (i.e., domains), or groups of these subsets, can form the basis of a method aimed at identifying allosteric modulators of A. baumannii SpoT hydrolase activity (preferably, a method aimed at identifying allosteric inhibitors of A. baumannii SpoT hydrolase activity).

[0080] Optionally, the candidate compound identified by the screening method of the present invention is a small molecule compound.The term "small", as used herein, for example, in the terms "small molecule", or "small compound", or "small candidate (binding) compound", refers to a low molecular weight compound that is organic, inorganic, or organometallic, and has a molecular weight of less than 1000 Da, for example, a molecular weight of less than 900 Da, or less than 750 Da, or even less than 600 Da.The small compound used in the method herein may exist in nature, or may only exist due to chemical synthesis.

[0081] The method of the present invention is a method for identifying compounds that modulate A. baumannii SpoT hydrolase activity. As used herein, the term "hydrolase" refers to a class of enzymes or enzyme domains that utilize water to disrupt or break chemical bonds, generating two separate molecules from one. Therefore, it is clear that hydrolase refers to an enzyme capable of performing hydrolysis. Unless otherwise specified, hydrolase activity herein refers to the hydrolysis of (p)ppGpp, i.e., the removal of the 3' pyrophosphate moiety from (p)ppGpp. Conversely, the term "synthetase," as used herein, refers to an enzyme or enzyme domain that catalyzes a synthetic process. In the context of the present invention, "synthetase activity" refers to the transfer of pyrophosphate from ATP to the 3' site of the ribose of GDP or GTP.

[0082] In one particular embodiment, the amino acid sequence of the A. baumannii SpoT enzyme, when used with the (screening) methods described herein, is SEQ ID NO:1:

[0083] [ka] The amino acid sequence of Acinetobacter baumannii SpoT as defined in claim 1 has at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, and even more preferably at least 95% sequence identity to the amino acid sequence of Acinetobacter baumannii SpoT as defined in claim 1.

[0084] Preferably, the amino acid sequence of the A. baumannii SpoT enzyme comprises, consists essentially of, or consists of SEQ ID NO:1.

[0085] In certain embodiments, interaction of the candidate compound with one or more amino acid residues in a region on the surface of the protein defined by the amino acid residues Arg45, Lys46, Ser47, Tyr51, His54, His78, Asp79, Ser113, Lys140, Asp143, Asn147, Thr150, Ala153, and Lys158 of the SpoT amino acid sequence defined by an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:1 indicates that the candidate compound is a modulator of SpoT hydrolase activity. In a preferred embodiment, interaction of the candidate compound with one or more amino acid residues in a region on the surface of the protein defined by any one or more of the following amino acid residues of an SpoT amino acid sequence defined by an amino acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1: Arg45, Lys46, Ser47, Tyr51, His54, His78, Asp79, Ser113, Lys140, Asp143, Asn147, Thr150, Ala153, and Lys158 indicates that the candidate compound is a modulator of SpoT hydrolase activity. In a further preferred embodiment, interaction of the candidate compound with any one or more amino acid residues in a region on the surface of the protein defined by any one or more of the following amino acid residues: Arg45, Lys46, Ser47, Tyr51, His54, His78, Asp79, Ser113, Lys140, Asp143, Asn147, Thr150, Ala153, and Lys158 of the SpoT amino acid sequence defined by an amino acid sequence comprising, consisting essentially of, or consisting of SEQ ID NO: 1 indicates that the candidate compound is a modulator of SpoT hydrolase activity. The term "region on the surface of the protein," as used herein, is intended to refer to a surface patch defining a binding site that includes the residues listed for said region.

[0086] Optionally, the method includes assessing whether the candidate compound interacts with at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or all (i.e., at least 14) amino acid residues of the group consisting of Arg45, Lys46, Ser47, Tyr51, His54, His78, Asp79, Ser113, Lys140, Asp143, Asn147, Thr150, Ala153, and Lys158 as defined in SEQ ID NO:1. In such embodiments, candidate compounds are considered to be A. baumannii SpoT enzyme modulators at at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or all (i.e., at least 14) amino acid residues of the group consisting of Arg45, Lys46, Ser47, Tyr51, His54, His78, Asp79, Ser113, Lys140, Asp143, Asn147, Thr150, Ala153, and Lys158 as defined in SEQ ID NO:1.

[0087] In certain embodiments, interaction of a candidate modulator with any one or more of the group of amino acids consisting of Arg45, Lys46, Ser47, Tyr51, His54, His78, Asp79, Ser113, Lys140, Asp143, Asn147, Thr150, Ala153, and Lys158 as defined by SEQ ID NO:1 indicates that the candidate modulator is an inhibitor of A. baumannii SpoT hydrolase activity. In an alternative embodiment, interaction of the candidate modulator with any one or more of the group of amino acids consisting of Arg45, Lys46, Ser47, Tyr51, His54, His78, Asp79, Ser113, Lys140, Asp143, Asn147, Thr150, Ala153, and Lys158 as defined by SEQ ID NO:1 indicates that the candidate modulator is an activator of A. baumannii SpoT hydrolase activity.

[0088] Optionally, the screening method described herein may further comprise determining a score for a candidate compound that modulates A. baumannii SpoT activity, preferably A. baumannii SpoT hydrolase activity, based on the number of interactions with Arg45, Lys46, Ser47, Tyr51, His54, His78, Asp79, Ser113, Lys140, Asp143, Asn147, Thr150, Ala153, and Lys158 as defined by SEQ ID NO: 1. In such embodiments, an increased amount of interaction with the amino acid residues contributes to a more favorable score for the candidate compound. The score may be expressed as an absolute value and / or as a relative value compared to one or more reference A. baumannii SpoT modulator molecules. In exemplary embodiments, the score may be a positive integer that is the sum of the number of interactions between the candidate compound and the amino acid residues described herein. In alternative exemplary embodiments, the score may be a percentage, where 0% indicates no interaction between the candidate compound and the A. baumannii SpoT protein, and 100% indicates an interaction with each of the amino acid residues described herein that have been shown to form a relevant portion of, or be part of, the A. baumannii SpoT surface region as defined herein. A candidate compound with a higher score (where the score is linearly correlated with the amount of interaction) clearly indicates that the candidate compound is more likely to be a potent modulator (e.g., inhibitor) of the A. baumannii SpoT protein, compared to a candidate compound with a lower score.

[0089] Optionally, the screening method may use as an input or prerequisite that the candidate compound interacts with the interface between the Core domain and the regulatory CTD region. For example, the method may include an initial step in which only candidate compounds that are known, believed to bind, or predicted to bind to the interface between the Core domain and the regulatory CTD region are retained.

[0090] Optionally, the method further comprises comparing the conformational states of A. baumannii SpoT before and after binding of the candidate compound to A. baumannii SpoT, wherein a change in conformational state indicates that the candidate compound is a true modulator of A. baumannii SpoT hydrolase activity; preferably, the general conformational state of A. baumannii SpoT after candidate binding differs from the atomic coordinates set forth in Table 1 by a root mean square deviation (RMSD) of residues on protein backbone atoms of 3 Å or less, more preferably 2 Å, and even more preferably 1 Å; and most preferably, the general conformational state of A. baumannii SpoT after candidate binding is a conformational state characterized by a subset of the atomic coordinates in Table 1 that define the A. baumannii SpoT enzyme.

[0091] "Conformational change," as described herein, should be understood as a change in the three-dimensional shape of a molecule, in the context of the present invention, A. baumannii SpoT. Conformational changes can be induced by numerous factors, including, but not limited to, temperature, pH, voltage, light, ion concentration, post-translational modification, or binding to another molecule. The conformational changes described in this application are the result, directly or indirectly, of binding to a modulator molecule. Proteins may exhibit different functions and / or interact differently depending on their conformation. In accordance with the present invention, conformational states can, and preferably do, affect the hydrolase activity level of A. baumannii SpoT. In these preferred embodiments, the A. baumannii conformational state is a conformational state characterized by reduced or complete absence of hydrolase activity by the enzyme. In certain embodiments, certain conformations partially or completely inhibit hydrolase and / or synthetase activity. In alternative embodiments, the particular conformation leads to upregulation of hydrolase and / or synthetase activity. When "stabilization" of a conformational state is described in the context of the present invention upon binding to an A. baumannii SpoT modulator, it is intended that the SpoT protein adopts a particular state, including but not limited to an open or closed state, at least over the time window during which the candidate compound-SpoT interaction is occurring.

[0092] In certain embodiments, the method includes detecting any atomic coordinates that differ after binding of a candidate A. baumannii SpoT modulator from the atomic coordinates that characterize the bound, active conformational state of A. baumannii SpoT shown in Table 1.

[0093] Preferably, the method is for identifying compounds that inhibit A. baumannii SpoT hydrolase activity when compared to a reference condition in the absence of the compound. Preferably, the method is for identifying compounds that inhibit A. baumannii SpoT hydrolase activity when compared to a reference condition in the absence of the compound by at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, or more preferably at least 95%. Optionally, the method is for identifying compounds that completely inhibit A. baumannii SpoT hydrolase activity (i.e., 100% inhibition or inhibition to below any detectable activity level).

[0094] Optionally, the method is for identifying a compound that increases A. baumannii SpoT hydrolase activity when compared to a reference condition in the absence of the compound. Preferably, the method is for identifying a compound that increases A. baumannii SpoT hydrolase activity when compared to a reference condition in the absence of the compound, by at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, or more preferably at least 95%. Optionally, the method is for identifying a compound that increases A. baumannii SpoT hydrolase activity (i.e., 100% inhibition or inhibition to below any detectable activity level). In certain embodiments, the method is a method for identifying a compound that increases A. baumannii SpoT hydrolase activity by at least 1.5-fold, preferably at least 2-fold, more preferably at least 5-fold, and more preferably at least 10-fold.

[0095] In certain embodiments, the method further comprises testing the ability of the candidate compound to modulate A. baumannii SpoT hydrolase activity. In certain embodiments, the method comprises testing the candidate compound in vitro and / or in vivo for its ability to inhibit or increase A. baumannii SpoT hydrolase activity, preferably inhibit A. baumannii SpoT hydrolase activity. In certain embodiments, testing the candidate compound comprises testing the compound in competition with one or more native A. baumannii SpoT substrates, such as (p)ppGpp.

[0096] In one illustrative example, testing the hydrolase activity of A. baumannii SpoT in vitro in the presence of a candidate A. baumannii SpoT hydrolase-modulating compound can include contacting the candidate compound with a recombinant A. baumannii SpoT protein and measuring the removal of the 3' pyrophosphate moiety from (p)ppGpp (i.e., monitoring the hydrolysis reaction mediated by A. baumannii SpoT). Similar experimental conditions can be devised for in vivo activity testing. Methods for assessing a wide variety of different enzyme activities are known in the art (Ou et al., Annu Rev Anal Chem, 2018).

[0097] In certain embodiments, the screening methods described herein are computer-implemented methods. In further embodiments, the computer comprises an input device, a processor, a user interface, and an output device. In such embodiments, the method comprises: a) generating a three-dimensional structure of the atomic coordinates of Table 1, or any subset thereof, as described in this disclosure; b) matching the structure of step a) with the structure of the candidate compound by computer modeling; c) selecting a ligand that possesses energetically favorable interactions with the structure of step a). may include:

[0098] In certain embodiments, the method further comprises selecting ligands that possess multiple energetically favorable interactions with the three-dimensional structure, favoring ligands that possess one energetically favorable interaction with the three-dimensional structure. In certain embodiments, the three-dimensional structure was generated using atomic coordinates from at least a subset of the atomic coordinates in Table 1 described herein. In alternative embodiments, the three-dimensional structure was generated using the complete list of atomic coordinates set forth in Table 1.

[0099] The term "energetically favorable interaction," as used herein, refers to any interaction with an interaction energy less than 0 kJ / mol. Alternatively, an energetically favorable interaction can be expressed as an interaction with a negative Gibbs free energy (ΔG) value. Because the degree of protein-ligand association correlates with the magnitude of negative ΔG, ΔG can be considered a determinant of the stability of the protein-ligand complex under investigation, or the binding affinity of the ligand to a given receptor, in this context, the A. baumannii SpoT enzyme. Free energy is a function of the state of the system; therefore, the ΔG value is defined by the initial and final thermodynamic states, regardless of any intermediate states. The concept of energetically favorable interactions is known to those skilled in the art (Du et al., Int J Mol Sci, 2016).

[0100] In certain embodiments, the method comprises superimposing the generated three-dimensional structure of the SpoT enzyme or SpoT-ppGpp complex with a structure of a candidate compound. In further embodiments, the method comprises selecting the most favorable orientation of the structure and the candidate compound from a set of distinct structure-candidate compound superimposed orientations. Thus, in certain embodiments, the method comprises docking modeling or molecular docking. In certain embodiments, the method comprises a computer-implemented step of proposing modifications to the candidate structure to further increase the number of favorable interactions with the generated three-dimensional structure. In further embodiments, the method comprises ranking the resulting collection of candidate compounds based on the number of favorable interactions involving the generated three-dimensional structure, wherein candidate compounds with a greater number of favorable interactions are ranked higher than candidate compounds with fewer favorable interactions.

[0101] The terms "docking modeling" and "molecular docking" refer to one or more quantitative and / or qualitative analyses of molecular structures based on structural information and interaction models. Modeling may refer to any one of a numerically based molecular dynamics model, an interaction computer graphic model, an energy minimization model, a distance geometry model, a molecular mechanics model, or any structure-based constraint model. These exemplary molecular modeling approaches can be utilized with the atomic coordinates or a subset of atomic coordinates described herein in Table 1 to obtain various three-dimensional models and explore the structure of any binding site, such as the binding site of a candidate A. baumannii SpoT modulator. Modeling methods and tools have been developed for designing or selecting chemical molecules that have complementarity to a specific target region (in the context of the present invention, a specific target region of A. baumannii SpoT). In certain embodiments, the chemical molecule (i.e., the candidate compound) has stereochemical complementarity to the target region. In certain embodiments, the candidate compound has general structural similarity to ppGpp. Stereochemical complementarity refers to a situation in which there are several energetically favorable contacts between the candidate compound and the target region of A. baumannii SpoT. Those skilled in the art understand that a certain number of energetically favorable interactions are sufficient to regulate A. baumannii SpoT activity, and therefore it is not a prerequisite that all of the key amino acid residues described herein are involved in energetically favorable interactions. Non-limiting examples of software programs suitable for performing molecular docking analysis are described in detail in the art (Pagadala et al., Biophys Rev, 2017).

[0102] Any computer system described herein, or any computer-implemented method relying on a computer system, may further include a machine learning means for predicting candidate A. baumannii SpoT modulators, such as hydrolase inhibitors, and / or scoring the modulators based on user input of a reference set of candidate compounds or data generated from a previous fitting and / or selection step of the candidate modulators. The combination of machine learning models for in silico screening and prediction of enzyme-binding molecules or modulators is known in the art and is therefore contemplated by the present invention (Li, et al., Molecules, 2019). Non-limiting examples of machine learning models (i.e., machine learning algorithms) include linear regression, logistic regression, decision trees, support vector machines, naive Bayes, k-nearest neighbors (kNN), k-means, random forests, dimensionality reduction algorithms, and gradient boosting algorithms, such as gradient boosting machines (GBM), XGBoost, LightGBM, and CatBoost.

[0103] In certain embodiments, the method includes selecting a candidate compound that can bind to at least one amino acid residue (preferably, multiple amino acid residues) of the generated three-dimensional structure without steric interference. The terms "steric interference," "steric hindrance," and "steric effect" are known to those skilled in the art. Steric interference (also referred to as steric hindrance) is the result of a steric effect and refers to the retardation of a chemical reaction due to steric bulkiness.

[0104] A further aspect herein is an in vitro method for identifying compounds that specifically modulate A. baumannii SpoT hydrolase activity, comprising: a) providing a candidate compound; b) providing an A. baumannii SpoT protein or a SpoT-ppGpp complex; c) contacting the candidate compound with the SpoT protein or the SpoT-ppGpp complex; d) determining the hydrolase activity of A. baumannii SpoT in the presence and absence of the candidate compound; and e) if a change in activity is detected, identifying the candidate compound as a compound that modulates A. baumannii SpoT hydrolase activity. The present invention relates to a method comprising:

[0105] Those skilled in the art will understand that the phrase "specifically modulate" indicates that the compound acts on A. baumannii SpoT hydrolase activity in a manner that directly alters the enzyme hydrolase activity. Thus, this phrase excludes compounds that may indirectly modulate A. baumannii SpoT hydrolase activity, for example, by affecting the viability of the entire organism or affecting the SpoT hydrolase protein expression level. Therefore, preferably, specific modulation of the hydrolase activity of the SpoT protein occurs through direct binding of the candidate compound to the SpoT protein or the SpoT-ppGpp complex.

[0106] Exemplary methods for assessing hydrolase activity are described above. In certain embodiments, the method further comprises selecting additional candidate compounds based on common structural features from a database. In certain embodiments, the methods described herein use recombinant A. baumannii SpoT protein. Means and methods for producing and purifying recombinant proteins have been described in detail in the art (e.g., Graesslund et al., Nat Methods, 2011).

[0107] In certain embodiments, the A. baumannii SpoT protein (optionally in complex with ppGpp) is characterized by amino acids having at least 70% sequence identity to SEQ ID NO: 1. In preferred embodiments, the A. baumannii SpoT protein (optionally in complex with ppGpp) is characterized by amino acids having at least 80% sequence identity to SEQ ID NO: 1, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, and even more preferably at least 95% sequence identity. In a most preferred embodiment, the A. baumannii SpoT protein (optionally in complex with ppGpp) comprises, consists essentially of, or consists of SEQ ID NO: 1. In a preferred embodiment, the A. baumannii SpoT protein and / or the A. baumannii SpoT-ppGpp complex are defined by the atomic coordinates in Table 1.

[0108] In certain embodiments, the method further comprises immobilizing the A. baumannii SpoT protein, SpoT-ppGpp complex, or candidate compound on a solid surface. In further embodiments, the method comprises washing away excess A. baumannii SpoT protein, SpoT-ppGpp complex, or excess candidate compound prior to determining the hydrolase activity. In certain embodiments, the method comprises detecting a change in hydrolase activity by colorimetric or spectrophotometric analysis. In certain embodiments, a change in activity is considered to be an increase in the hydrolase activity of the A. baumannii SpoT protein of at least 10%, preferably 25%, preferably 50%, preferably 75%, or preferably 100% in the presence of a candidate compound, compared to the hydrolase activity when the enzymatic activity of the A. baumannii SpoT protein is assessed in the absence of any (candidate) compound. Alternatively, a change in activity is considered to be an increase in the hydrolase activity of the A. baumannii SpoT protein by at least 1.5-fold, preferably 2-fold, more preferably at least 5-fold, and most preferably at least 10-fold. In an alternative embodiment, a change in activity is considered to be a reduction in the hydrolase activity of the A. baumannii SpoT protein by at least 10%, preferably 25%, preferably 50%, preferably 75%, or preferably 100% in the presence of a candidate compound, when compared to the hydrolase activity when the enzymatic activity of the A. baumannii SpoT protein is assessed in the absence of any (candidate) compound. Alternatively, a change in activity is considered to be a reduction in the hydrolase activity of the A. baumannii SpoT protein by at least 1.5-fold, preferably at least 2-fold, more preferably at least 5-fold, and most preferably at least 10-fold. In certain embodiments, the method identifies candidate compounds capable of inhibiting SpoT hydrolase activity to the extent that it cannot be detected by methods described in the state of the art.In an alternative embodiment, the method identifies candidate compounds capable of stimulating hydrolase activity.

[0109] A further aspect of the present invention relates to the use of the crystal structure of the A. baumannii SpoT protein or SpoT-ppGpp complex defined by the atomic coordinates set forth in Table 1, or a subset thereof as described herein, or by atomic coordinates that deviate from the atomic coordinates of Table 1 or a subset thereof by an RMSD on the protein backbone atoms of 3 Å or less, for designing and / or identifying compounds that modulate (preferably, partially or fully inhibit) A. baumannii SpoT hydrolase activity.

[0110] The term "crystal structure," as used herein, refers to a three-dimensional description of the ordered arrangement or structure of elements such as atoms, ions, or molecules in a crystalline material. Unless otherwise specified, a crystal structure refers to a protein crystal structure obtained by protein crystallography, the process of experimentally forming protein crystals. In a typical protein crystallization process, a protein is dissolved in an aqueous environment containing a sample solution until supersaturation occurs. Various approaches are described in detail in the art, including, but not limited to, vapor diffusion, batch, microdialysis, and liquid-liquid diffusion. Once protein crystals are obtained, various techniques, such as X-ray diffraction, cryo-electron microscopy, or nuclear magnetic resonance, are suitable for determining the protein crystal structure. The term "supersaturation" refers to a state in which a solution contains more dissolved material than can be dissolved in the solvent under normal conditions and is defined in the art as a non-equilibrium state in which a quantity of macromolecule exceeding the solubility limit under specific chemical and physical conditions is still present in the solution (McPherson and Gavira, Struct Biology Commun, 2014). Therefore, protein crystals also contain a large amount of solvent molecules, such as, but not limited to, water. Due to the variety of methodologies used to prepare protein crystals, these crystals also contain various buffers, salts, small binding proteins, and precipitants whose concentrations can vary significantly. Typical crystals range in size from 20 μm to several mm. Crystals ideal for X-ray diffraction analysis are ideally free of cracks and other defects.

[0111] In a further aspect of the present invention, the inventors have discovered that compounds, such as small molecules, that interact with the A. baumannii SpoT protein through the interface between the Core and regulatory domains are particularly interesting for acting as A. baumannii SpoT modulators. Without wishing to be bound by theory, it is hypothesized that pseudo-SYNTH, ZFD, and RRM all subtly up- or down-regulate the HD activity of the A. baumannii SpoT protein by modulating their interaction with Core. The presence of Core and its crosstalk with the HD / pseudo-HD domain likely constitute a universal structural requirement for efficient stabilization of the active state of long RSHs.

[0112] Thus, in certain embodiments, candidate A. baumannii SpoT protein modulators are compounds that bind to the interface between the Core domain and the regulatory domain.

[0113] A further aspect of the present invention relates to a computer system comprising a database containing the atomic coordinates set forth in Table 1, or a subset thereof as described herein, stored on a computer-readable storage medium, and a user interface for viewing this information. Also contemplated are data processing apparatuses, devices, and systems comprising the database containing the atomic coordinates set forth in Table 1, or a subset thereof as described herein, stored on a computer-readable storage medium, and a user interface for viewing this information. The models and atomic coordinates disclosed herein are typically stored on machine-readable or computer-readable media known in the art, non-limiting examples of which include magnetic or optical media, and random access or read-only memory (e.g., tape, diskette, hard disk, CD-ROM and DVD, flash drive or chip, server, and the Internet). In certain embodiments, the computer system comprises means for executing the methods described herein. In certain embodiments, the computer system further comprises an input device for receiving instructions from an operator. In certain embodiments, the computer system includes and / or is connected to a remote data storage system, the remote data storage system being located in a different geographic location than the location of the user interface for viewing information. The data storage system may be located on a network storage medium, such as the Internet, allowing remote access. In certain embodiments, the database included in the computer system is encrypted. In certain embodiments, the computer system has access to at least one database of compound structures, and a user may access the at least one database of compound structures by appropriately instructing the computer system.In certain embodiments, a compound, a list of compounds, or a compound database (also known as a compound library) is loaded into a computer system by an operator. In alternative embodiments, the compound, list of compounds, or compound database is accessible by the computer system from a medium separate from the computer system. In certain embodiments, the computer system comprises a processing unit for evaluating the fit between any compound molecule loaded into the computer system and an A. baumannii SpoT protein and / or SpoT-ppGpp complex. Also contemplated is a computer-readable storage medium comprising instructions that, when executed by a computer, cause the computer to perform any one of the methods disclosed herein.

[0114] Further aspects relate to the use of the computer systems described herein for designing and / or identifying compounds (ligands) that modulate A. baumannii SpoT activity. In certain embodiments, use of the computer system is accomplished by user-input commands. In certain embodiments, the computer system comprises means for selecting candidate A. baumannii SpoT modulators from a list of compounds or a compound library. In certain embodiments, the computer system comprises means for selecting candidate compounds and proposing structural modifications to at least one candidate compound to further increase the number of energetically favorable interactions between the compound and A. baumannii SpoT, and / or means for selecting candidate compounds and proposing structural modifications to at least one candidate compound to reduce or eliminate structural interference between the candidate modulator and one or more residues of A. baumannii SpoT as defined by the atomic coordinates of any one of Table 1. When using the computer system described herein, a user searching for A. baumannii SpoT modulators, who may or may not be an operator of the computer, is provided with an optionally printed list of candidate A. baumannii SpoT modulators (preferably, A. baumannii SpoT hydrolase inhibitors). The computer system provides the user with one or more candidate A. baumannii SpoT modulators (preferably, A. baumannii SpoT hydrolase inhibitors). In certain embodiments, the computer system is configured to specialize in providing the user with candidate compounds that inhibit A. baumannii SpoT hydrolase activity.In alternative embodiments, the computer system may be used to simply provide the user with candidate compounds that upregulate (i.e., increase) A. baumannii SpoT hydrolase activity. In alternative embodiments, the computer system is used to design and / or identify allosteric A. baumannii SpoT modulators. In certain embodiments, the computer system is used to provide a visual representation (i.e., an image of the three-dimensional structure of A. baumannii SpoT) of the candidate A. baumannii SpoT during its interaction with the compound. In certain embodiments, a list of candidate A. baumannii SpoT modulators is generated and stored in an electronic file, optionally sorted according to a scoring system described herein.

[0115] A further aspect of the present invention is directed to a crystal of the A. baumannii SpoT protein and / or SpoT-ppGpp complex, comprising a structure characterized by the atomic coordinates set forth in Table 1, or a subset thereof as described herein. One of skill in the art will understand that a crystal structure characterized by the atomic coordinates set forth in Table 1 corresponds to a SpoT-ppGpp complex and thus represents the active binding state of the enzyme. Optionally, the crystal is obtained by crystallizing a protein comprising SEQ ID NO:1 or by crystallizing the A. baumannii SpoT protein defined by SEQ ID NO:1. Optionally, crystals are obtained by crystallizing A. baumannii SpoT protein in solution using space group p21 21 21, and unit cell: 128.791 133.761 211.328 90.00 90.00 90.00, and supplementing the solution with ppGpp prior to harvesting the crystals, preferably at 50 mM. Optionally, the solution comprises, consists essentially of, or consists of 0.85 M sodium citrate tribasic dihydrate, 0.1 M Tris pH 8.0, and 0.1 M sodium chloride.

[0116] Any crystal structure disclosed herein is said to feature, match, or substantially match a set or subset of atomic coordinates if the structure, or a substantial fragment of the structure, falls within the RMSD limit values ​​disclosed herein. In certain embodiments, at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 90%, and even more preferably at least 95% of the crystal structure have the recited RMSD value. In certain embodiments, "substantially matches" further refers to atoms of amino acid side chains. In this context, a common amino acid side chain is a side chain that is shared between a structure that substantially matches a structure with specific atomic coordinates and the structure defined by said atomic coordinates in Table 1. In one embodiment, the coordinates for the ppGpp binding in the coordinates of the A. baumannii SpoT protein shown in Table 1 can be used to identify the binding pocket of the stabilized conformation (the atomic coordinates corresponding to the ppGpp molecule are those designated by the identifier "G4P"). Alternatively, this coordinate can be removed to easily model new molecules or agents into the conformation of the A. baumannii SpoT protein.

[0117] A further aspect of the present invention is a computer system intended to generate three-dimensional structural representations of the A. baumannii SpoT protein and / or SpoT-ppGpp complex, a complex of the A. baumannii SpoT protein with a binding compound or modulator, and to analyze or optimize binding of a compound or modulator to said A. baumannii SpoT protein and / or SpoT-ppGpp complex, comprising: (a) Optionally, the coordinates of the A. baumannii SpoT protein structure listed in Table 1 (i.e., Table 1 excluding coordinates having the identifier "G4P"), or selected coordinates thereof, that vary by no more than 3 Å of the root mean square deviation of the residue backbone atoms; (b) Optionally, the coordinates of the A. baumannii SpoT-ppGp complex structure listed in Table 1 , or selected coordinates thereof, varying by no more than 3 Å root-mean-square deviation of the residue backbone atoms; (c) optionally, coordinates of candidate binding compounds or modulators generated by interpreting X-ray crystallographic, cryo-EM, or NMR data with reference to the coordinates of the A. baumannii SpoT protein structure and / or SpoT-ppGp complex structure listed in Table 1, or selected coordinates thereof, varying by no more than 3 Å root mean square deviation of residue backbone atoms; and (d) Structure factor data derivable from the coordinates in (a), (b), or (c). The present invention is directed to a system comprising computer readable data including one or more of:

[0118] In certain embodiments, the computer system comprises data including any combination of (a), (b), (c), or (d). In further embodiments, a user can adjust, delete, or add data to the computer system. In certain embodiments, the computer system can receive additional data, adjust data, or delete data associated with (a), (b), (c), or (d). In certain embodiments, a user can access a compound or modulator synthesis protocol through the computer system. In certain embodiments, the computer system guides the user through the synthesis protocol.

[0119] Optionally, a computer system described herein compares the atomic coordinates of (a) and (c), and if a steric conflict is detected, the candidate compound or modulator is not considered to be a suitable A. baumannii SpoT protein modulator. Optionally, a computer system described herein compares the atomic coordinates of (a) and (c), and if a steric conflict is not detected, the candidate compound or modulator is considered to be a suitable A. baumannii SpoT protein modulator.

[0120] Another aspect of the present invention is a computer readable storage medium including a data storage material encoded with computer readable data, the data comprising: (a) Optionally, the coordinates of the A. baumannii SpoT protein structure listed in Table 1 (i.e., Table 1 excluding coordinates having the identifier "G4P"), or selected coordinates thereof, that vary by no more than 3 Å of the root mean square deviation of the residue backbone atoms; (b) Optionally, the coordinates of the A. baumannii SpoT-ppGpp complex structure listed in Table 1, or selected coordinates thereof, varying by no more than 3 Å root-mean-square deviation of the residue backbone atoms; (c) optionally, coordinates of candidate binding compounds or modulators generated by interpreting X-ray crystallographic, cryo-EM, or NMR data with reference to the coordinates of the Rel enzyme structure listed in Table 1, or selected coordinates thereof, varying by no more than 3 Å root mean square deviation of residue backbone atoms; and (d) Structure factor data derivable from the coordinates in (a), (b), or (c). The present invention relates to a computer-readable storage medium including one or more of:

[0121] In certain embodiments, the computer-readable data is encrypted and requires authentication or authorization credentials from a user or another computer-readable storage system to allow the computer system to access the data. In certain embodiments, the computer-readable storage medium is a physical storage medium. In alternative embodiments, the computer-readable storage medium is a non-physical storage medium or a storage medium that is considered to be a non-physical storage medium (i.e., a cloud-based storage medium).

[0122] Another aspect of the present invention relates to a computer-readable storage medium comprising a data storage material encoded with a first set of computer-readable data comprising structural coordinates of an A. baumannii SpoT protein or SpoT-ppGpp complex listed in Table 1, optionally varying by a root mean square deviation of residue backbone atoms of 3 Å or less, or a Fourier transform of at least a portion of selected coordinates thereof, which data, when combined with a second set of machine-readable data comprising an X-ray diffraction pattern of a molecule or molecular complex of unknown structure, can be used to determine at least a portion of the structural coordinates corresponding to the second set of machine-readable data using a machine programmed with instructions for using the first and second sets of data. Fourier transform in the context of the present invention should be interpreted as application of a molecular replacement approach.

[0123] As contemplated herein by the term "Fourier transform," the first step involves calculating a three-dimensional transformation of the molecular model. The weighted reciprocal lattice is then rotated according to this calculated transformation. Fourier transforms in molecular biology (more specifically, structural biology) are described in the art (Rabinovich et al., Acta crystallographica section D biological crystallography, 1998). In certain embodiments, the X-ray diffraction pattern of the molecule or molecular complex of unknown structure is obtained by an apparatus operably linked to the computer storage medium. In alternative embodiments, the X-ray diffraction pattern of the molecule or molecular complex of unknown structure is input into the computer-readable storage medium at the command of a user. In yet another alternative embodiment, the X-ray diffraction pattern of the molecule or molecular complex of unknown structure is retrieved from a publicly accessible database by a computer system equipped with a computer-readable storage medium.

[0124] In certain embodiments, the computer system or computer-readable storage medium described herein further comprises a database containing information regarding the three-dimensional structures of candidate compounds or modulators that are small molecules. In certain embodiments, the computer system or computer-readable storage medium further comprises means for retrieving information from a public information database regarding the three-dimensional structures of candidate compounds or modulators, preferably "small" molecules as defined herein that partially or fully inhibit the hydrolase activity of A. baumannii SpoT, including, but not limited to, PubChem (https: / / pubchem.ncbi.nlm.nih.gov), the Zinc database (https: / / www.zinc.docking.org), and / or MolPort (https: / / www.molport.com). In certain embodiments, the computer system generates information indicating which list or subset of atomic coordinates from Table 1 exhibits or is predicted to exhibit the highest number of energetically favorable interactions with any candidate modulator evaluated by the computer system. In certain embodiments, the user receives an automatically generated list of candidate compounds ranked according to the number of energetically favorable interactions with the A. baumannii SpoT protein defined by each list or subset of atomic coordinates in Table 1. In further embodiments, the computer system provides the user with several common structural groups that can distinguish any combination of candidate modulators, or even hydrolase inhibitors.

[0125] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as set forth below within the spirit and broad scope of the appended claims. Aspects and embodiments of the present invention disclosed herein are further supported by the following non-limiting examples. The following specific experimental examples are provided to support the claimed invention and should not be construed as limiting the scope of the invention. [Example]

[0126] [Example 1] A. baumannii SpoT Ab is a monofunctional hydrolase long RSH The lack of conservation of active site residues important for SYNTH activity suggests that the Moraxallaceae SpoT enzyme, like RelA, has undergone subfunctionalization to become a monofunctional long RSH. Similar to the pseudo-HD domain of RelA, the SYNTH domain region is conserved in Moraxallaceae, possibly as a non-catalytic pseudo-SYNTH domain, suggesting that the SYNTH domain region retains some function in stabilizing or allosterically regulating the HD domain. A. baumannii SpoT in living cells (SpoT Ab To investigate the hydrolytic function of (p)ppGpp, we hypothesized that the hydrolytic activity of SpoT is important for regulating the cellular levels of (p)ppGpp produced by RelA, and therefore, that SpoT regulates the hydrolytic activity of (p)ppGpp produced by RelA. + The present inventors utilized the conditional essentiality of ppGpp in Escherichia coli (Xiao et al., J. Biol. Chem., 1991). 0 The (ΔrelA ΔspoT) E. coli strain was constructed by: i) expressing spoT under the control of the PA1 / O4 / O3 promoter; Aband ii) a pMG25-based plasmid driving IPTG-inducible expression of relA under the control of PBAD. Ec The vector was co-transformed with pMR33 derivatives for arabinose-inducible expression of (p)ppGpp synthetase RelA. Ec Expression of ppGpp 0 The growth of E. coli was strongly inhibited, and this growth was due to the SpoT Ab The ectopic co-expression of SpoT completely rescued the disease. Ab are HD active in a surrogate E. coli host.

[0127] Next, we investigated the (p)ppGpp synthetase activity of SpoT RSH using our dual-plasmid co-expression system. 0 E. coli is auxotrophic for 11 amino acids and SpoT Ec The (p)ppGpp synthetase activity of SpoT is essential for growth of ΔrelA Escherichia coli in minimal medium (Xiao et al., J Biol Chem, 1991). Ec Unlike SpoT Ab ppGpp on M9 minimal medium 0 It was not able to promote the growth of E. coli, which suggests that SpoT Ab These results confirmed that SpoT is synth-inactive. Ab We show that this is a unique monofunctional long RSH that lacks the ability to synthesize (p)ppGpp.

[0128] [Example 2] Full length SpoT Ab has a compact mushroom-like τ-type structure To gain insight into the molecular workings of SpoT, we synthesized full-length catalytically active SpoT in the ppGpp-bound state at 2.9 Å resolution. Ab The X-ray structure of

[0129] The amino acid sequence of the A. baumannii SpoT protein, SEQ ID NO: 1, was used in crystallization experiments. The protein was crystallized at 4°C in 0.85 M sodium citrate tribasic dihydrate, 0.1 M Tris pH 8.0, and 0.1 M sodium chloride (space group: p212121; unit cell: 128.791 133.761 211.328 90.00 90.00 90.00). For soaking, the solution was supplemented with ppGpp at 50 mM along with a cryoprotectant solution prior to crystal harvest.

[0130] This structure reveals a multi-domain structure that differs significantly from those previously observed for long RSH Rel and RelA bound to the ribosome (Arenz et al., Nucleic Acids Res, 2016; Brown et al., Nature, 2016; Loveland et al., Elife, 2016; Pausch et al., Cell Rep, 2020) (Figure 1a-c). Ab The HD, SYNTH, TGS, HEL, ZFD, and RRM domains of SpoT form a mushroom-like tau (τ)-type tetrameric structure (Fig. 1a–c). In this arrangement, the pseudo-SYNTH, TGS, HEL, ZFD, and RRM domains all reside on a single plane, forming a compact, discoid structure that forms the "umbrella" of the "mushroom" (Fig. 1b). The helix-turn-helix subdomain (residues 334–379), which provides the transition between the NTD and CTD regions, resides in the "Core" of the "umbrella" and appears to mediate interactions between all domains of the enzyme. Such an arrangement suggests that the Core, which is disordered in the Rel / RelA structure, stabilizes the discoidal "umbrella" of SpoT (Fig. 1c). Furthermore, the Core is located in the HD domain, which is responsible for the SpoT Ab Finally, the HD protrudes from the plane of the "umbrella" in the opposite direction to the C-terminal RRM domain, forming the "stalk" of the protein structure (Figure 1b-c).

[0131] SpoT AbThe τ-shaped structure of RelA suggests a possible structural mechanism for the autoinhibition of SYNTH activity by the regulatory CTD in both Rel (Mechold et al., J Bacteriol, 2002; Takada et al., Nucleic Acids Res, 2021) and RelA (Svitil et al., J Biol Chem, 1993; Turnbull, Front Microbiol, 2019). While the SYNTH and TGS domains are sequestered within the "umbrella," the HD hydrolase protrudes unconstrained and is primed for (p)ppGpp hydrolysis. The TGS domain specifically binds to the deacylated tRNA CCA-3' end at the A site in the amino acid starvation sensors Rel and RelA (Brown et al., Nature 2016; Loveland et al., Elife 2016; Pausch et al., Cell Rep, 2020; Winther et al., Mol Cell, 2018), and in SpoT Ab In the case of SpoT, it is partially sandwiched between the HD, HEL, and ZFD domains. Ab We detected a mild inhibitory effect of tRNA on hydrolytic activity, but this effect was insensitive to the tRNA aminoacylation state, i.e., nonspecific (Fig. 1d). This was confirmed by the bifunctional E. coli SpoT (SpoT), which was specifically inhibited by deacylated tRNA but not by aminoacylated tRNA. Ec ) contrasts with the HD activity of (Richter, Gen Genet, 1980).

[0132] Our structure reveals that the ZFD and RRM domain sites that mediate rRNA recognition in Rel / RelA (Arenz et al., Nucleic Acids Res, 2016; Brown et al., Nature 2016; Loveland et al., Elife 2016; Pausch et al., Cell Rep, 2020; Winther et al., Mol Cell, 2018) are retained by the Core subdomain, suggesting that in the τ-shaped conformation, the hydrolytically active (HD) domain is ON )SpoT Ab This suggests that the ribosome is incompatible with ribosome binding. In good agreement with this structural prediction, the ribosome is amplified by the Bacillus subtilis REl (Rel Bs ) strongly inhibited the HD activity of SpoT (Takada et al., Nucleic Acids Res, 2021), and the addition of E. coli 70S strongly inhibited the HD activity of SpoT. Ab Therefore, our biochemical results suggest that SpoT Ab This suggests that it is a ribosome-independent enzyme.

[0133] Full-length monofunctional SpoT Ab The τ state of SpoT allows autostimulation of HD activity by the CTD via the Core domain to control (p)ppGpp hydrolysis. Pseudo-SYNTH, ZFD, and RRM all regulate SpoT by modulating their interactions with Core. Ab The presence of Core and its crosstalk with the HD / pseudo-HD domains likely constitute a universal structural requirement for efficiently stabilizing the active state of long RSH. In this sense, the interface between Core and the regulators mediates the regulation of SpoT. Ab Small molecules that interact with the enzyme may inhibit its activity and be used as a starting point for drug design.

[0134] [Example 3] A shorter intrinsically disordered region (IDR) in monofunctional SpoT is associated with specialization for hydrolysis The presence of intrinsically disordered regions (IDRs) located in the α6-α7 loop, the Core subdomain, and the linker between the HEL / ZFD domains in the long RSH RelA and Rel poses experimental challenges for structural studies (Arenz et al., Nucleic Acids Res, 2016; Brown et al., Nature, 2016; Loveland et al., Elife, 2016; Pausch et al., Cell Rep, 2020). The molecular function of these structurally unresolved flexible regions remains unknown. The well-structured τ-state of SpoT Ab Comparison with RelA / Rel bound to partially unstructured ribosomes suggests that the unfolding of the Core and HEL domains constitutes part of a conformational switch that positions the TGS, ZFD, and RRM domains to stimulate the synthetic activity of Rel / RelA upon recruitment to the ribosome.

[0135] The lengths of these disordered or flexible regions are, on average, shorter in monofunctional SpoT and much longer in monofunctional RelA. Bifunctional Rel has interdomain IDRs with sizes between both monofunctional enzymes. In particular, the α6-α7 loop of the HD domain of SpoT[Hs] is one-third the size of RelA, which in turn is twice as long as bifunctional Rel. The same pattern is observed for the IDRs of the other two species (i.e., the Core subdomain and the region connecting the HEL and ZFD domains). This suggests that RelA Ab Compared to SpoT Ab This is consistent with the significantly lower disorder propensity of the Core. We speculate that these IDRs have evolved to tune the HD-to-SYNTH output ratio by stabilizing the τ-state (shorter IDRs) or extended state (longer IDRs) of monofunctional SpoT[Hs] or RelA[hS], respectively.

[0136] [Example 4] SpoT Ab is a monomer Previously, it has been shown that both Rel and RelA are prone to dimerization via their CTDs, which may regulate their enzymatic activity (Pausch et al., Cell Rep, 2020; Gropp et al., J Bacteriol, 2001; Kaspy and Glaser, Front Microbiol, 2020; Yang and Ishiguro, Biochem Cell Biol, 2001). This idea has been the subject of debate, with both genetic experiments (Turnbull et al., Front Microbiol, 2019) and mass spectrometric experiments (Takada et al., Nucleic Acids Res, 2021) suggesting that dimerization is unlikely at physiologically relevant concentrations. Therefore, we used small-angle X-ray scattering (SAXS) coupled with size-exclusion chromatography (SEC) to characterize the dimerization of SpoT in solution. Ab The conformational and oligomeric states of were investigated (Figure 1e–f).

[0137] From the SAXS data, SpoT Ab Both SAXS and SEC revealed that SpoT has a flattened shape, consistent with the structure determined by X-rays. Even at a high concentration of 8 mg / mL, Ab The molecular weight of approximately 90 kDa by SEC and the estimated Mw of approximately 85 kDa and Rg of 34.9 Å by SAXS (Fig. 1e-f) both support the monomeric nature of monomeric SpoT. Ab Furthermore, analysis of the normalized Kratky plot obtained from the scattering curves revealed that the molecular weight of SpoT in solution was 80 kDa. Ab The compact monomeric structure of SpoT is further supported (Fig. 1f), and the ab initio envelope calculated from the experimental SAXS data (Fig. 1g) is consistent with the X-ray determined structure of SpoT. Ab Taken together, these results suggest that in solution, monomeric SpoT AbWe show that the HD domain of the discotic enzyme adopts a conformation that closely resembles the τ conformation observed in crystals where the HD domain protrudes from the discotic enzyme.

[0138] [Example 5] SpoT Ab The enzymatically inactive pseudo-synth is a regulatory domain In the monofunctional stringent regulator RelA, the enzymatically inactive pseudo-HD domain has evolved into a regulatory domain that controls catalysis via an allosteric mechanism within the NTD (Roghanian et al., Mol Cell, 2021; Sinha and Winther, Commun Biol, 2021). This also supports the idea that SpoT is a monofunctional hydrolase in which the pseudo-SYNTH domain has evolved into a strict regulatory / structural domain. Ab This also applies to specialization of SpoT Ab Rel to pseudo-SYNTH domain Tt Superposition of the derived SYNTH domains revealed that SpoT is a soluble form of SpoT, consistent with the various conservation patterns in the G-loop and ATP recognition motifs. Ab Extensive rearrangements of the remaining catalytic domain in GD(T)P are evident. This includes residues that coordinate adenosine and guanosine (R249-N241, R277-E267, and Y329-N304) and most of the phosphate-coordinating groups. Importantly, catalytic residues D272 and Q347 are replaced with S263 and T321, respectively. These substitutions are responsible for the deprotonation and activation of the 3'-OH of GD(T)P, as well as the activation of Mg. 2+ The ITC essentially prevents the binding of SpoT, preventing nucleophilic attack on the β-phosphate of ATP. Ab NTD and RelA Ab NTD As expected, GDP binding by SpoT Ab does not bind to GDP, but RelA Ab binds to GDP with an affinity of 62 μM, which is Ec NTD and Rel Bs NTDThis is similar to our previous estimates (Takada et al., Nucleic Acids Res, 2021; Roghanian et al., Mol Cell, 2021).

[0139] [Example 6] SpoT Ab is not allosterically regulated by the alarmone pppGpp The enzymatic activity of long RSH is regulated by strong allosteric coupling between the HD and SYNTH domains, which leads to antagonistic conformational states (Hogg et al., Cell, 2004; Tamman et al., Nat Chem Biol, 2020; Roghanian et al., Mol Cell, 2021). In Rel / RelA, (p)ppGpp binds to the hinge region connecting the SYNTH and HD / pseudo-HD domains to stimulate SYNTH activity, whereas this regulation is not regulated by the SYNTH domains in SpoT. Ec (Roghanian et al., Mol Cell, 2021). Ab Our structure of τ provides a mechanistic interpretation. In the τ state, the highly structured Core subdomain makes numerous contacts with SYNTH, forming a symmetric domain within SpoT. Ab This provides additional scaffolding to the already more stable version of the HD:SYNTH hinge of RelA. Additionally, the (p)ppGpp binding site contains several key substitutions that would be expected to impair (p)ppGpp binding and alarmone-mediated regulation, specifically at Q203 (a residue involved in ribose coordination and strictly conserved as A in RelA (Roghanian et al., Mol Cell, 2021)) and T209 (a residue involved in phosphate coordination, typically K or R in RelA (Roghanian et al., Mol Cell, 2021)).

[0140] SpoT Ab To directly verify the lack of pppGpp-mediated regulation in β-glucanase, we performed ITC to detect pppGpp and SpoT. AbNTD We characterized the interaction between SpoT and Ab NTD does not bind allosterically to pppGpp. Ec Following the experimental approach previously used for SpoT (Roghanian et al., Mol Cell, 2021), we Ab NTD ( 201 SpoT Ab 211 ) into the allosteric site of A. baumannii RelA ( 236 RElA 246 ) was ported. Ec As in the case of , this resulted in a RelA-like affinity of the chimeric RSH for pppGpp (K = 5.6 μM). Together, these results confirm that the generality of alarmone-mediated control is lost in SpoT and is present only in synth-active Rel / RelA stringent regulators that mediate the acute stringent response during amino acid starvation.

[0141] [Example 7] The bipolar structure of the HD active site is conserved between Rel and SpoT SpoT Ab Inspection of the electron density map of the -ppGpp complex revealed that the alarmones were located on the four SpoTs present in the asymmetric unit of the crystal. Ab The guanine bases of ppGpp (Fig. 2a-c) are highly occupying each molecule. Tt NTD -ppGpp 23 and Rel Tt NTDThis is similar to that observed in the -pppGpp complex (Mojr et al., ACS Chem Biol, 2021). We enzymatically investigated the role of each residue involved in guanine coordination by systematic Ala substitution. Substitution of R45 (stacking guanine) abolished hydrolysis, whereas removal of the van der Waals contact with L154 reduced activity by approximately two-fold, with redundant interaction with K46. Disruption of the guanine hydrogen bond to T150 had only a minor effect. The additional hydrogen bond formed between the guanine carbonyl group and the enzyme backbone is thought to be the primary reason for SpoT's specificity for guanine over adenosine.

[0142] Rel Tt NTD As previously observed for SpoT (Tamman et al., Nat Chem Biol, 2020), Ab The hydrolase active site of ED83 displays a bipolar charge distribution, with a highly basic half mediating stabilization of the 5'- and 3'-polyphosphate groups of the substrate, and the other highly acidic half mediating 3'-pyrophosphate hydrolysis (Figure 2a-b). A more detailed examination of the complex revealed the presence of the Mn ions in the Y51 and 82ED83 active site motifs. 2+ This reveals the important role of SpoT in coordinating and stabilizing the network of water molecules near the sugar phosphate moiety during hydrolysis in cooperation with the cofactor (Fig. 2b-c). Indeed, substitution of Y51, E82, D83, or N147 significantly increased the activity of SpoT. Ab HD is inactive in our enzyme assays. In the positively charged active site, the 5'-polyphosphate is loosely coordinated and exposed to bulk solvent. In contrast, K140 and R144 hold the 3'-pyrophosphate in place during hydrolysis, and Ala substitution of these residues reduces the activity of the enzyme 5- to 10-fold, suggesting that they are critical residues for directing the scissile bond.

[0143] [Example 8] Mn 2+ Ion is SpoTAb Organizing the HD active site Divalent manganese ion Mn in (p)ppGpp pyrophosphate hydrolysis 2+ The key role of Rel (Hogg et al., Cell, 2004; Takada et al., Nucleic Acids Res, 2021; Avarbock et al., Biochemistry, 2000; Van Nerom et al., Acta Crystallogr F Struct Biol Commun, 2019) and SpoT Ec (Heinemeyer et al., Eur J Biochem, 1978). Our isothermal titration calorimetry (ITC) measurements revealed that the unliganded metal-free SpoT Ab NTD Mn with a KD of 35.3 μM 2+ Furthermore, metal-free full-length SpoT has been shown to bind to Ab is completely HD-inactive, but Mn 2+ HD activity is easily restored by the addition of

[0144] Mn 2+ To directly clarify the structural role of SpoT, we investigated the metal-free state of SpoT. Ab NTD The X-ray structure of SpoT was determined (Fig. 2d). Ab Comparison with the structure of the Mn-ppGpp complex reveals that catalysis is involved in Mn 2+ A structural explanation for the essentiality of α3, α4, and α8 is provided, which, in addition to its role in hydrolysis, also contributes to the formation of Mn 2+ The coordination of SpoT brings the two halves of the HD domain together and provides structural support to the active site (Figure 2d-e). Ab The overall topology of the HD domains is 2+ -Ligand type Rel Tt NTD 23 topology, but metal ion removal is similar to SpoT. Ab NTDThe catalytic 78HD79 and 82ED83 motifs are highly misaligned, the loops S110-Y117 and A153-K158 involved in 3'- and 5'-phosphate coordination are disordered, and the guanine coordination loop T44-Y51 adopts a conformation incompatible with base coordination (Fig. 2e). Importantly, all these changes are mediated by the Mn 2+ Upon removal of Rel Tt These observations suggest that evolution as a monofunctional enzyme has led to the development of SpoT. Ab This suggests that the allosteric conformational control between the HD domain and the pseudo-SYNTH domain has been lost.

[0145] [Example 9] The CTD allosterically stimulates the hydrolytic activity of the SpoT NTD To date, our understanding of the function of the CTD region of long RSH has been based solely on studies of Rel and RelA, which have established a role for the CTD in the association of stringent regulators with starved ribosomes, leading to activation of SYNTH activity and autoinhibition of extraribosomal factor SYNTH activity (Arenz et al., Nucleic Acids Res, 2019; Loveland et al., Elife, 2016; Pausch et al., Cell Rep, 2020; Mechold et al., J Bacteriol, 2002; Takada et al., Nucleic Acids Res, 2021). The weak hydrolase activity of CTD-truncated Rel also suggests a possible HD-promoting role of the CTD through intramolecular regulation of hydrolase function (Takada et al., Nucleic Acids Res, 2021; Ronneau et al., Nucleic Acids Res, 2019; Takada et al., Front Microbiol, 2020), suggesting that a similar mechanism may be at work in the case of SpoT.

[0146] To test this hypothesis, we investigated: i) RRM (SpoT Ab 1~614 , amino acids 1–614), ii) RRM and ZFD (SpoT Ab 1~560 ), iii) RRM, ZFD, and HEL(SpoT Ab 1~454 ), iv) All of the CTDs, i.e., RRM, ZFD, HEL, and TGS (SpoT Ab 1~385 ), v) CTD and Core domain (SpoT Ab 1~339 ), and finally, a variant consisting only of the HD domain (SpoT Ab 1~195 ) lacking, SpoT Ab The HD activity of a set of increasingly C-terminally truncated variants of SpoT was characterized both in vitro and in vivo. All of these truncated variants were generated at the endogenous SpoT locus of a ΔrelA Ptac::relA A. baumannii strain, and their ability to grow on complex medium supplemented with IPTG was compared to that of SpoT in vivo. Ab was evaluated as a surrogate for (p)ppGpp hydrolase activity.

[0147] SpoT lacking the RRM or the RRM and ZFD domains Ab Although the variants retained the wild-type ability to sustain bacterial growth (i.e., they could efficiently degrade (p)ppGpp synthesized by RelA), further C-terminal truncations impaired HD function in vivo, as evidenced by significant growth defects. Biochemical assays are consistent with the in vivo data. Truncation of the RRM and ZFD reduces HD activity 5-fold. Further deletion of the TGS-HEL domain dramatically reduces activity 42-fold. Truncation beyond the TGS impaired activity by more than 70-fold, and the isolated HD domain was nearly inactive. Taken together, our results suggest that the CTD region is essential for the functioning of SpoT. AbThese results suggest that the CTD domains function as allosteric activators of the hydrolase function of the NTD. Next, we aimed to elucidate the molecular mechanism of CTD-mediated NTD regulation and assign molecular functions to individual CTD domains.

[0148] [Example 10] The Core domain is the key to controlling the τ state SpoT Ab Both the overall structural arrangement of SpoT and our sequential domain truncation experiments suggest that Core-mediated allosteric crosstalk between the HD and the rest of the enzyme's domains is essential for the enzyme's functionality. To specifically assess the role of individual interdomain interactions, we introduced single-point substitutions at each of the interfaces between Core and the regulatory CTD domain and analyzed SpoT. Ab We measured the hydrolase activity of the variants. The Y375G substitution at the HD:Core:TGS domain reduced activity by 5-fold compared to the wild-type, demonstrating that an intact HD:Core:TGS interface (the structure involved in scaffolding the HD active site) is crucial for HD activity. Substitutions at the ZFD (L373G / D374G) and RRM (A351K) domain interfaces also resulted in significant defects (19-fold and 3-fold reductions, respectively), whereas perturbations at the Core:pseudo-SYNTH domain interface (A348R) only slightly affected hydrolysis. Finally, uncoupling the HD contact from the τ-umbrella via the L356D substitution, located at the interface between the HD Core domain and the α6-α7 motif (Tamman et al., Nat Chem Biol, 2020), dramatically reduced HD activity by 35-fold, suggesting an allosteric signaling pathway between the umbrella and stalk regions of the enzyme. The inventors of the present invention have AbWhen we monitored the thermodynamic stability of these Core variants, we observed that they all exhibited reduced stability and loss of structure compared to the wild-type. This suggests that the increased configurational entropy of Core has an overall effect on the dynamics and compactness of the enzyme. The existence of an allosteric relay mediating CTD-dependent activation of the HD through Core is further supported by the consistent reduction in hydrolysis associated with the aforementioned C-terminal truncations that affect Core feedback to the HD, and the observation that deletion of the HEL and TGS domains results in a 50-fold reduction in activity despite the presence of other regulatory domains (pseudo-SYNTH, ZFD, and RRM).

[0149] We then used SEC-SAXS to investigate the effect of SpoT on the stabilization of the τ state. Ab The role of each contact at the interface between the Core and various domains of SpoT was directly investigated. Ab L356D ) separates this population into two conformational states with significantly different RG (radius of gyration) and particle size (DMAX). Ab L356D In this study, one state is the compact τ shape observed in the crystal structure (Figure 3a), while the other state is more unfolded (RG = 41 Å, DMAX = 130 Å), with dimensions reminiscent of the less compact Rel and RelA, but not as elongated as the ribosome-bound state (Figure 3b). In this unfolded state, the Core and HEL domains appear to transition to a more disordered state consistent with the conformational state of these regions in the fully extended state observed in Rel / RelA (Figure 3c-d), while the other domains maintain their structural integrity. Prompted by this similarity, we next used SAXS to characterize the A. baumannii monofunctional synthetase RelA and the B. subtilis bifunctional RSH RelA. Bs I checked. RelA AbThe dimensions of SpoT (RG = 42 Å, DMAX = 130 Å, Mw = 88 kDa) Ab L356D The dimensions of the unwound part match those of the unwound part, but Rel Bs Both the unwound state and the τ state exist in (Fig. 3e-g).

[0150] Taken together, our results suggest that the Core domain functions as an allosteric relay, transmitting signals from the CTD to the HD. At the structural level, the composition of the Core is key to the conformational state of the enzyme, defined by three major conformations observed in SpoT, Rel, and RelA (Figure 3h). The correlation between reduced HD activity and entropy-increasing substitutions such as A351K, L356D, L371G / D374G, and Y375G supports the idea that structural disorder or increased flexibility of the Core domain (or other IDRs) likely shifts the enzyme's conformational equilibrium away from the τ state. This reduction in activity observed upon disruption of the τ shape is also consistent with the lack of hydrolysis in Rel homologs that undergo an order-to-disorder transition while adapting to the ribosomal A site (Takada et al., Nucleic Acids Res, 2021). In this context, the aforementioned unwound state is likely to be an idle resting state of the long RSH enzyme, where the CTD prevents SYNTH function without activating the HD.

[0151] [Example 11] The TGS domain functions as a scaffold for the HD active site The α6-α7 element is a bifunctional Rel Tt It plays an important role in the allosteric regulation of the opposing activities of Rel (Tamman et al., Nat Chem Biol, 2020). Tt In HD, α6-α7 protrudes away from the catalytic center to accommodate the 3' and 5' polyphosphate groups and act as a catalyst. 82 ED 83 The motif is positioned close to the 3' phosphate, priming the enzyme for hydrolysis. AbIn SpoT, the outward-facing conformation of α6-α7 is further stabilized by the N-terminal region of the TGS and the Core domain, which acts as a clamp to hold α6-α7 in an HD-compatible position, and the HEL domain provides additional support through the Core (Fig. 4a). Ab The dramatic reduction in activity of the variant (Fig. 4d) confirms the functional importance of this stabilizing effect.

[0152] The HD:TGS interface buries the β-hairpin of TGS, an element involved in tRNA recognition in Rel (Pausch et al., Cell Rep, 2020; Takada et al., Nucleic Acids Res, 2021; Takada et al., Front Microbiol, 2020) and RelA (Brown et al., Nature, 2016; Loveland et al., Elife, 2016; Winther et al., Mol Cell, 2018). The β-hairpin directly stacks the α6-α7 element through a small hydrophobic interface formed by W382, Y384, L390, and the R124-E392 salt bridge (Figure 4a). This interface is crossed by the E379K / W382K substitution (SpoT). Ab E379K / W382K Disruption of the HD:TGS interface with Rel reduces the hydrolase activity of the enzyme by 17-fold, suggesting that the HD:TGS interface constitutes an important allosteric signaling pathway. This scaffolding role is complemented by Core, which tightly wraps around α7, thus preventing α6-α7 recoil from the HD active site, which we have previously shown to be a key mechanism for Rel Tt As previously observed in SpoT (Tamman et al., Nat Chem Biol, 2020), this induces NTD opening. Indeed, substitutions at the Core:α6-α7 interface, such as the aforementioned Y375G, also affected hydrolysis. Interestingly, in SpoT Ab E379K / W382K Despite the significant attenuation of HD activity of SpoT, SAXS revealed that Ab E379K / W382Kremains in the τ state (RG = 35 Å, DMAX = 104 Å), suggesting allosteric communication via the HD:Core:TGS axis (Fig. 4b).

[0153] SpoT Ab Considering that SpoT is synth-inactive and not specifically regulated by tRNA or the ribosome (Fig. 1d), the TGS residues involved in tRNA recognition (Rel (Pausch et al., Cell Rep, 2020; Takada et al., Nucleic Acids Res, 2021; Takada et al., Front Microbiol, 2020) and RelA (Brown et al., Nature, 2016; Winther et al., Mol Cell, 2018)) are likely to be involved in tRNA recognition. Ab The key His residues involved in the recognition of the 3' CCA end by SpoT (S407 of SpoT) are Ab (However, bifunctional SpoT Ec It is not surprising that the τ state is present in SpoT (Atkinson et al., PLoS One, 2011). However, the τ state is sterically incompatible with the possibility of tRNA recognition by TGS due to the separation of the β-hairpin and α-helical elements. All these observations suggest that SpoT Ab In this study, TGS is repurposed as a scaffolding domain crucial for sustaining hydrolysis, suggesting that both TGS and Core cooperate to lock α6-α7 in place and stabilize the HD active site. This contrasts with the critical function of Rel / RelA in recognizing uncharged tRNA (Brown et al., Nature 2016; Loveland et al., Elife 2016; Pausch et al., Cell Rep 2020; Takada et al., Nucleic Acids Res 2021, Winther et al., Mol Cell 2018).

[0154] [Example 12] The ZFD and RRM domains are Ab Fine-tuning the hydrolytic activity of With the ZFD and RRM positioned near the disk-shaped umbrella and connecting with the pseudo-SYNTH domain, the resulting interdomain interface likely plays a role not only in τ-state stability but also in allosterically regulating the HD via the HD:pseudo-SYNTH relay. Consistent with this hypothesis, disruptive substitutions at Core:HD(L356D), Core:pseudo-SYNTH:RRM(A351K), and Core:ZFD(L373G / D374G), which reduce τ-state stability, also reduced the HD activity of the enzyme by 35-fold, 3-fold, and 22-fold, respectively. Therefore, we reasoned that substitutions that stabilize the Core:pseudo-SYNTH:RRM and Core:ZFD interfaces would conversely lead to allosteric activation of hydrolysis.

[0155] To test this hypothesis, we introduced substitutions (I637D / R641D) that would increase contact between the RRM and pseudo-SYNTH via hydrogen bonds, and substitutions (D374R) that would increase contact between the Core and ZDF (Fig. 4c). Ab D374R and SpoT Ab I637D / R641D has been shown to be more stable and compact than WT, and SpoT Ab I637D / R641D SAXS measurements of this variant confirmed that it retained the τ state (Figure 4d). As expected, both enzyme variants had increased HD turnover (2.1-fold and 1.6-fold, respectively), and both were significantly higher than wild-type SpoT in vivo. Ab behaves like.

[0156] Taken together, our results demonstrate that HD activity is coupled to the stability of the τ state and that the Core domain functions as an allosteric transducer that allows the catalytic HD to communicate with all regulatory domains. Substitutions or interactions that stabilize the τ state increase hydrolysis, whereas substitutions that destabilize the τ state decrease HD activity.

[0157] [Example 13] Intact τ-type SpoT Ab is required for virulence of A. baumannii Functional (p)ppGpp-mediated signaling plays a key role in antibiotic resistance and virulence of A. baumannii (Perez-Varela et al., J Bacteriol, 2020; Kim et al., Virulence, 2021). Using a honey moth G. mellonella larval infection model, we investigated the role of a mutant spoT gene in the pathogenicity of A. baumannii AB5075. Ab The functionality of the variants was assessed. Only wild-type-like virulence strains had slightly higher HD activity compared to WT SpoT. Ab The strain expressed the D374R variant. Ab D374R The strain rapidly killed 100% of the larvae within the first 2 days, whereas 60% of the larvae were infected with (p)ppGpp 0 The ΔrelA strain survived for 6 days after infection. ΔRRM truncated enzyme SpoT Ab 1~614 Infection with A. baumannii expressing RRM-truncated SpoT resulted in a 25% larval survival rate after 6 days. Ab 1~614 has six times less hydrolase activity than the wild type, and this strain does not exhibit growth defects when cultured on LB plates. Ab 1~454 and SpoT Ab 1~339In A. baumannii strains expressing these SpoT variants, the virulence defect becomes more pronounced. The significant reduction in HD activity associated with these SpoT variants results in 100% survival of infected larvae. Collectively, our results demonstrate that basal levels of HD hydrolase activity are sufficient to sustain bacterial growth under non-stress conditions (on plates and in liquid culture), but that pathogens must regulate HD activity and increase SpoT activity to efficiently achieve successful infection. Ab These results suggest that fully functional TD- and Core-mediated regulation of .

[0158] [Example 14] Consideration of Examples 1 to 13 This study revealed that the full-length monofunctional SpoT Ab The unexpected τ-type structure of SpoT was revealed, which allows the CTD to autostimulate the hydrolase activity of the enzyme. Ab The pseudo-SYNTH domain of the enzyme is a regulatory and structure-stabilizing domain. Together with the TGS, HEL, ZFD, and RRM, the pseudo-SYNTH defines an interaction network that transmits allosteric signals from the CTD to the HD active site via the enzyme's Core to control (p)ppGpp hydrolysis. The Core element is composed of the TGS and Mn 2+ Together, they position the active site residues of the HD in a position suitable for catalysis. Impairment of the function of any of these elements by substitution of a key residue results in a significant defect in hydrolytic activity. In contrast, pseudo-SYNTH, ZFD, and RRM all regulate the interaction with Core, thereby facilitating the catalytic activity of SpoT. AbThe HD domains fine-tune the HD activity of long RSH enzymes. Interestingly, ribosome-associated Rel / RelA (p)ppGpp synthetases lacking Core are nonfunctional and synth-inactive in vivo, whereas minimal enzyme versions with synth activity consist of HD / pseudo-HD, synth, and Core domains (Hogg et al., Cell, 2004; Takada et al., Nucleic Acids Res, 2021; Roghanian et al., Mol Cell, 2021; Ronneau et al., Nucleic Acids Rest, 2019). Thus, the presence of Core and its crosstalk with the HD / pseudo-HD domain likely constitute a universal structural requirement for efficiently stabilizing the active state of long RSH enzymes.

[0159] We propose a unified scheme to rationalize the evolution of enzymatic output of long RSH by fine-tuning the conformational equilibrium between the τ-state, unfolded state, and ribosome-bound state of these enzymes (Figure 5). The very presence of catalytically competent synthetase and hydrolase domains in the bifunctional Rel[HS] and SpoT[HS] requires both the τ-state and the unfolded state as part of the conformational spectrum of these enzymes (Figure 5a-b). The τ-state primes Rel / SpoT for efficient (p)ppGpp hydrolysis, whereas the longer unfolded state sets the enzymes for less efficient (p)ppGpp synthesis. To fully activate synth activity, the enzyme must be further stimulated by starved ribosomes to reach a highly elongated, ribosome-bound state, a transition possible for the amino acid starvation sensor Rel[HS] but not for SpoT, which is not subject to allosteric control by starved ribosomes and ppGpp (Roghanian et al., Mol Cell, 2021) (Figure 5a). In more functionally differentiated enzymes (the dedicated hydrolase Moraxellaceae SpoT[Hs] and the dedicated synthetase RelA[hS]), the unique structural equilibrium is restricted to a subset of conformations accessible to the ancestral bifunctional Rel[HS] (Figure 5c-d). Compared to SpoT[HS], the equilibrium is further shifted in SpoT[Hs] toward the HD-active τ state required for hydrolysis (Figure 5c). In contrast, in RelA[hS], the τ state becomes inaccessible and the enzyme is stabilized in a highly elongated ribosome-bound SYNTH-active state primed for ribosome recruitment ( Figure 5 d).

[0160] The expansion / contraction of disordered regions is likely a molecular driver that fine-tunes the enzymatic output of long RSHs through the restriction of conformational space. Longer IDRs promote the unfolded state of RelA[hS] and increase enzymatic frustration, whereas shorter IDRs promote the compact HD-active τ state in SpoT[Hs]. This genetic fine-tuning of catalytic function is reminiscent of the evolution of human glucocorticoid receptor isoforms (Li et al., Elife, 2017) or UDP-α-d-glucose-6-dehydrogenase (Keul et al., Nature, 2018), which were based on optimizing the length and force generated by intrinsically disordered regions. Such mechanisms appear to evolve as a solution to conformationally heterogeneous proteins in a partially active resting state that are subject to significant energetic and functional frustration.

[0161] The unifying scheme presented here highlights the "hub" nature of SpoT, as well as the regulatory factors of acyl carrier protein (ACP) and RpoD - σ 70 This provides a framework that can be used to theorize how binding partners such as Rsd can modulate their output (Battesti and Bouveret, Mol Microbiol, 2006; Lee et al., Proc Natl Acad Sci USA, 2018), or, in the case of Rel / RelA, how the ribosome impedes hydrolysis by utilizing this extensive allosteric network. NTR and other protein partners of Rel, such as DarB (Ronneau et al., Nucleic Acids Res, 2019; Kruger et al., Nat Commun, 2021), can also modulate the intramolecular allosteric communication between the regulatory domain and the HD by preferring the τ or unwound state, thus regulating the catalytic output of the enzyme.

[0162] [Example 15] Important amino acid residues for candidate compound screening Based on the above findings, we can identify a selection of key residues that are preferred residues for binding by successful candidate compounds (i.e., candidate A. baumannii SpoT enzyme modulators) (Figure 6).

[0163] [Table 1-1]

[0164] [Table 1-2]

[0165] [Table 1-3]

[0166] [Table 1-4]

[0167] [Table 1-5]

[0168] [Table 1-6]

[0169] [Table 1-7]

[0170] [Table 1-8]

[0171] [Table 1-9]

[0172] Table 1-10

[0173] Table 1-11

[0174] Table 1-12

[0175] Table 1-13

[0176] Table 1-14

[0177] Table 1-15

[0178] Table 1-16

[0179] Table 1-17

[0180] Table 1-18

[0181] Table 1-19

[0182] Table 1-20

[0183] Table 1-21

[0184] Table 1-22

[0185] Table 1-23

[0186] Table 1-24

[0187] Table 1-25

[0188] Table 1-26

[0189] Table 1-27

[0190] Table 1-28

[0191] Table 1-29

[0192] Table 1-30

[0193] Table 1-31

[0194] Table 1-32

[0195] Table 1-33

[0196] Table 1-34

[0197] Table 1-35

[0198] Table 1-36

[0199] Table 1-37

[0200] Table 1-38

[0201] Table 1-39

[0202] Table 1-40

[0203] Table 1-41

[0204] Table 1-42

[0205] Table 1-43

[0206] Table 1-44

[0207] Table 1-45

[0208] Table 1-46

[0209] Table 1-47

[0210] Table 1-48

[0211] Table 1-49

[0212] Table 1-50

[0213] Table 1-51

[0214] Table 1-52

[0215] Table 1-53

[0216] Table 1-54

[0217] Table 1-55

[0218] Table 1-56

[0219] Table 1-57

[0220] Table 1-58

[0221] Table 1-59

[0222] Table 1-60

[0223] Table 1-61

[0224] Table 1-62

[0225] Table 1-63

[0226] Table 1-64

[0227] Table 1-65

[0228] Table 1-66

[0229] Table 1-67

[0230] Table 1-68

[0231] Table 1-69

[0232] Table 1-70

[0233] Table 1-71

[0234] Table 1-72

[0235] Table 1-73

[0236] Table 1-74

[0237] Table 1-75

[0238] Table 1-76

[0239] Table 1-77

[0240] Table 1-78

[0241] Table 1-79

[0242] Table 1-80

[0243] Table 1-81

[0244] Table 1-82

[0245] Table 1-83

[0246] Table 1-84

[0247] Table 1-85

[0248] Table 1-86

[0249] Table 1-87

[0250] Table 1-88

[0251] Table 1-89

[0252]

Table 1-90

[0253] Table 1-91

[0254] Table 1-92

[0255] Table 1-93

[0256] Table 1-94

[0257] Table 1-95

[0258] Table 1-96

[0259] Table 1-97

[0260] Table 1-98

[0261] Table 1-99

[0262] Table 1-100

[0263] Table 1-101

[0264] Table 1-102

[0265] Table 1-103

[0266] Table 1-104

[0267] Table 1-105

[0268] Table 1-106

[0269] Table 1-107

[0270] Table 1-108

[0271] Table 1-109

[0272]

Table 1-110

[0273] Table 1-111

[0274] Table 1-112

[0275] Table 1-113

[0276] Table 1-114

[0277] Table 1-115

[0278] Table 1-116

[0279] Table 1-117

[0280] Table 1-118

[0281] Table 1-119

[0282]

Table 1-120

[0283] Table 1-121

[0284] Table 1-122

[0285]

Table 1-123

[0286] Table 1-124

[0287] Table 1-125

[0288] Table 1-126

[0289] Table 1-127

[0290] Table 1-128

[0291] Table 1-129

[0292] Table 1-130

[0293] Table 1-131

[0294] Table 1-132

[0295] Table 1-133

[0296] Table 1-134

[0297] Table 1-135

[0298] Table 1-136

[0299] Table 1-137

[0300] Table 1-138

[0301] Table 1-139

[0302] Table 1-140

[0303] Table 1-141

[0304] Table 1-142

[0305] Table 1-143

[0306] Table 1-144

[0307] Table 1-145

[0308] Table 1-146

[0309] Table 1-147

[0310] Table 1-148

[0311] Table 1-149

[0312]

Table 1-150

[0313] Table 1-151

[0314] Table 1-152

[0315] Table 1-153

[0316] Table 1-154

[0317] Table 1-155

[0318] Table 1-156

[0319] Table 1-157

[0320] Table 1-158

[0321] Table 1-159

[0322] Table 1-160

[0323] Table 1-161

[0324] Table 1-162

[0325] Table 1-163

[0326] Table 1-164

[0327] Table 1-165

[0328] Table 1-166

[0329] Table 1-167

[0330] Table 1-168

[0331] Table 1-169

[0332] Table 1-170

[0333] Table 1-171

[0334] Table 1-172

[0335] Table 1-173

[0336] Table 1-174

[0337] Table 1-175

[0338] Table 1-176

[0339] Table 1-177

[0340] Table 1-178

[0341] Table 1-179

[0342] Table 1-180

[0343] Table 1-181

[0344] Table 1-182

[0345] Table 1-183

[0346] Table 1-184

[0347] Table 1-185

[0348] Table 1-186

[0349] Table 1-187

[0350] Table 1-188

Claims

1. 1. A method for identifying a compound that modulates A. baumannii SpoT activity, comprising using a three-dimensional structure represented by the set of atomic coordinates set forth in Table 1, or a subset thereof, or using atomic coordinates that deviate from the atomic coordinates of Table 1, or a subset thereof, by a root mean square deviation (RMSD) of residues on protein backbone atoms of 3 Å or less, and evaluating the fit of candidate compounds to the three-dimensional protein structure of A. baumannii SpoT.

2. 10. The method of claim 1, wherein the method is a method for identifying compounds that modulate A. baumannii SpoT hydrolase activity.

3. 3. The method of claim 1, wherein interaction of the candidate compound with one or more amino acid residues in a region on the surface of the protein defined by the amino acid residues Arg45, Lys46, Ser47, Tyr51, His54, His78, Asp79, Ser113, Lys140, Asp143, Asn147, Thr150, Ala153, or Lys158 of a SpoT amino acid sequence defined by an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 indicates that the candidate compound is a modulator of SpoT hydrolase activity.

4. 4. The method of any one of claims 1 to 3, wherein the amino acid sequence has at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

1.

5. 5. The method of any one of claims 1 to 4, wherein the amino acid sequence comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:

1.

6. 6. The method of any one of claims 1 to 5, further comprising determining a score for said candidate compound for modulating A. baumannii SpoT activity, preferably A. baumannii SpoT hydrolase activity, based on the number of interactions with said amino acid residues.

7. 7. The method of any one of claims 1 to 6, further comprising comparing the conformational state of A. baumannii SpoT before and after the candidate compound binds to A. baumannii SpoT, wherein a change in conformational state indicates that the candidate compound is a true modulator of A. baumannii SpoT activity, preferably the conformational state of A. baumannii SpoT before the candidate compound binds is a conformational state characterized by the atomic coordinates of Table 1.

8. 8. The method of any one of claims 1 to 7, wherein the method is a method for identifying compounds that reduce A. baumannii SpoT hydrolase activity.

9. 1. A computer-implemented method, the computer including an input device, a processor, a user interface, and an output device, the method comprising: a) generating a three-dimensional structure of the atomic coordinates of Table 1, or a subset thereof; b) matching the structure of step a) with the structure of the candidate compound by computer modeling; c) selecting candidate compounds that possess energetically favorable interactions with the structure of step a). The method according to any one of claims 1 to 8, comprising:

10. 10. The method of claim 9, wherein the fitting comprises superimposing the structure of step a) with the structure of the candidate compound, and optionally, the fitting comprises superimposing the structure of the candidate compound with a structure of atomic coordinates corresponding to bound ppGpp.

11. The method of claim 9 or 10, wherein the candidate compound of step c) is capable of binding to at least one amino acid residue of the structure of step a) without steric interference.

12. Use of the crystal structure of the A. baumannii SpoT-ppGpp complex, defined by the atomic coordinates shown in Table 1, or a subset thereof, or defined by atomic coordinates that deviate from the atomic coordinates in Table 1, or a subset thereof, by an RMSD on the protein backbone atoms of 3 Å or less, to design and / or identify compounds that modulate A. baumannii SpoT hydrolase activity.

13. A crystal of the A. baumannii SpoT-ppGpp complex, including a structure characterized by the atomic coordinates defined in Table 1, or a subset thereof.

14. 1. An in vitro method for identifying compounds that specifically modulate A. baumannii SpoT hydrolase activity, comprising: a) providing a candidate compound; b) providing an A. baumannii SpoT polypeptide or a SpoT-ppGpp complex; c) contacting the candidate compound with the A. baumannii SpoT polypeptide or SpoT-ppGpp complex; d) determining the hydrolase activity of A. baumannii SpoT in the presence and absence of the candidate compound; and e) if a change in hydrolase activity is detected, identifying the candidate compound as a compound that modulates A. baumannii SpoT. A method comprising:

15. 1. A computer system adapted to generate three-dimensional structural representations of an A. baumannii SpoT protein and / or SpoT-ppGpp complex, a complex of an A. baumannii SpoT protein with a binding compound or modulator, and to analyze or optimize binding of a compound or modulator to said A. baumannii SpoT protein and / or SpoT-ppGpp complex, comprising: (a) optionally, the coordinates of the A. baumannii SpoT protein structure listed in Table 1, or selected coordinates thereof, varying by no more than 3 Å root mean square deviation of residue backbone atoms; (b) optionally, the coordinates of the A. baumannii SpoT-ppGp complex structure listed in Table 1, or selected coordinates thereof, varying by no more than 3 Å root mean square deviation of residue backbone atoms; (c) optionally, coordinates of candidate binding compounds or modulators generated by interpreting X-ray crystallographic, cryo-EM, or NMR data with reference to the coordinates of the A. baumannii SpoT protein structure and / or SpoT-ppGp complex structure listed in Table 1, or selected coordinates thereof, that vary by no more than 3 Å root mean square deviation of residue backbone atoms; and (d) Structure factor data derivable from the coordinates of (a), (b), or (c). A system comprising computer readable data including one or more of:

Images