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US-20260128119-A1 - SCREENING METHODS FOR ACINETOBACTER BAUMANNII SPOT ENZYME MODULATORS

US20260128119A1US 20260128119 A1US20260128119 A1US 20260128119A1US-20260128119-A1

Abstract

The present invention concerns screening methods to identify compounds that regulate the activity of Acinetobacter baumannii SpoT enzyme, in particular compounds that are able to partially or completely inhibit the hydrolase activity of said enzyme. The screening methods rely on assessing the degree of fit of candidate compounds with the three-dimensional structure of the A. baumannii SpoT protein and/or A. baumannii SpoT-ppGpp complex represented by a well-defined set of atomic coordinates. The screening methods may further rely on assessing interaction of the candidate compound with one or more amino acid residues of a region on the surface of the SpoT protein.

Inventors

  • Abel GARCIA PINO
  • Cédric Pierre GOVAERTS
  • Hanna AINELO
  • Hedvig TAMMAN
  • Vasili HAURYLIUK

Assignees

  • Université Libre de Bruxelles

Dates

Publication Date
20260507
Application Date
20231020
Priority Date
20221020

Claims (20)

  1. 1 . A method for identifying compounds that modulate A. baumannii SpoT activity comprising the step of employing a three dimensional structure represented by a set of atomic coordinates presented in Table 1 or a subset thereof, or atomic coordinates which deviate from those in Table 1 or a subset thereof by a root mean square deviation (RMSD) of residue over protein backbone atoms by no more than 3 Å and assessing the degree of fit of a candidate compound to said three-dimensional protein structure of A. baumannii SpoT.
  2. 2 . The method according to claim 1 , wherein the method is a method for identifying compounds that modulate A. baumannii SpoT hydrolase activity.
  3. 3 . The method according to claim 1 , wherein interactions of said candidate compound with one or more amino acid residues of a region on the surface of the protein defined by amino acid residues: Arg45, Lys46, Ser47, Tyr51, His54, His78, Asp79, Ser113, Lys140, Asp143, Asn147, Thr150, Ala153, or Lys158 of the SpoT amino acid sequence as defined by an amino acid sequence with at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 indicate the candidate compound is a modulator of SpoT hydrolase activity.
  4. 4 . The method according to claim 1 , wherein the amino acid sequence has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1.
  5. 5 . The method according to claim 1 , 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 according to claim 1 , further comprising determining a score of said candidate compound to modulate A. baumannii SpoT activity based on the number of interactions with said amino acid residues.
  7. 7 . The method according to claim 1 , further comprising comparing the conformational state of A. baumannii SpoT before and after said candidate compound binds to A. baumannii SpoT, wherein a change in conformational state is indicative for the candidate compound to be a bona fide modulator of A. baumannii SpoT activity.
  8. 8 . The method according to claim 1 , wherein the method is a method for identifying compounds that decrease A. baumannii SpoT hydrolase activity.
  9. 9 . The method according to claim 1 , which is a computer-implemented method, said computer comprising an inputting device, a processor, a user interface, and an outputting device, wherein said method comprises the steps of: a) generating a three-dimensional structure of the atomic coordinates of Table 1, or a subset thereof; b) fitting the structure of step a) with the structure of a candidate compound by computational modeling; and c) selecting a candidate compound that possesses energetically favorable interactions with the structure of step a).
  10. 10 . The method according to claim 9 , wherein said fitting comprises superimposing the structure of step a) with the structure of said candidate compound.
  11. 11 . The method according to claim 9 , wherein said candidate compound of step c) can bind to at least 1 amino acid residue of the structure of step a) without steric interference.
  12. 12 . (canceled)
  13. 13 . A crystal of A. baumannii SpoT-ppGpp complex, comprising a structure characterized by the atomic coordinates or a subset thereof as defined in Table 1.
  14. 14 . An in vitro method for identifying a compound which specifically modulates A. baumannii SpoT hydrolase activity, comprising the steps of: a) providing a candidate compound; b) providing an A. baumannii SpoT polypeptide or SpoT-ppGpp complex; c) contacting said candidate compound with said A. baumannii SpoT polypeptide or SpoT-ppGpp complex; d) determining the hydrolase activity of A. baumannii SpoT in the presence and absence of said candidate compound; and e) identifying said candidate compound as a compound which modulates A. baumannii SpoT if a change in hydrolase activity is detected.
  15. 15 . A computer system, intended to generate three dimensional structural representations of an A. baumannii SpoT protein and/or SpoT-ppGpp complex, complexes of A. pbaumannii SpoT protein with binding compounds or modulators for analyzing or optimizing binding of compounds or modulators to said A. baumannii SpoT protein and/or SpoT-ppGpp complex, the system containing computer-readable data comprising one or more of: (a) the coordinates of the A. baumannii SpoT protein structure listed in Table 1, optionally varied by a root mean square deviation of residue backbone atoms of not more than 3 Å, or selected coordinates thereof, (b) the coordinates of the A. baumannii SpoT-ppGpp complex structure listed in Table 1, optionally varied by a root mean square deviation of residue backbone atoms of not more than 3 Å, or selected coordinates thereof; (c) the coordinates of a candidate binding compound or modulator generated by interpreting X-ray crystallographic data, cryo-EM or NMR data by reference to the coordinates of the A. baumannii SpoT protein structure and/or SpoT-ppGpp complex structure, listed in Table 1, optionally varied by a root mean square deviation of residue backbone atoms of not more than 3 Å, or selected coordinates thereof, and (d) structure factor data derivable from the coordinates of (a), (b) or (c).
  16. 16 . The method according to claim 1 , wherein the amino acid sequence has at least 85%-sequence identity to the amino acid sequence of SEQ ID NO: 1.
  17. 17 . The method according to claim 1 , wherein the amino acid sequence has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1.
  18. 18 . The method according to claim 1 , wherein the amino acid sequence has at least 95%-sequence identity to the amino acid sequence of SEQ ID NO: 1.
  19. 19 . The method according to claim 1 , further comprising determining a score of said candidate compound to modulate A. baumannii SpoT hydrolase activity based on the number of interactions with said amino acid residues.
  20. 20 . The method according to claim 7 , wherein the conformational state of A. baumannii SpoT before candidate compound binding is the conformational state characterized by the atomic coordinates of Table 1.

Description

FIELD OF THE INVENTION The invention relates to the elucidation of the Acinetobacter Baumannii SpoT enzyme crystal structure, and screening methods to identify Acinetobacter Baumannii SpoT enzyme binding into the catalytic site of said crystal structure. The invention is of particular interest to the field of molecular biology, more particular in the development of drugs against antibiotic resistant Acinetobacter Baumannii. BACKGROUND OF THE INVENTION The overuse and misuse of antibiotics combined with a lack of progress in the development of new antibacterial drugs have 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 thus bacterial infections are resurging as a prominent threat to human health (Ventola, The antibiotic resistance crisis, Pharmacy and therapeutics, 2015). In the last years, multiple health instances have repeatedly warned about these pathogenic antibiotic (multi)resistant bacteria and the threats they pose to human health (Michael et al., Frontiers in public health, 2013). Six of the most highly virulent and antibiotic resistant bacterial pathogens that can evade or escape commonly used antibiotics due to their increasing multi-drug resistance have been referred to recently as by the acronym “ESKAPE” (group), which consists of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. One mechanism that these bacteria use to survive in presence of antibiotics is by the phenomenon of bacterial tolerance and persistence. Whereas the majority of a bacterial population will proliferate quickly in an infected host organism, a smaller fraction of this population will actively suppress growth. Since the majority of all clinically used antibiotics target rapidly dividing bacteria, the small population of bacteria in the persistence state will not be affected by these drugs and are able to switch back to their normal, non-persistent state post-antibiotic treatment(s). A crucial mediator to obtain the typical phenotype of a tolerant cell (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 levels of (p)ppGpp are tightly regulated by the concerted opposing activities of RelA/SpoT homologue (RSH) enzymes that can both transfer a 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). The RelA-SpoT pair is a product of gene duplication of an ancestral factor—the ribosome-associated bifunctional RSH Rel—and the pair is limited in its taxonomic distribution to Beta- and Gammaproteobacteria (Atkinson et al., PLoS One, 2011; Mittenhuber et al., J Mol Microbiol Biotechnol, 2001). Subfunctionalization—the partitioning of functions between two paralogues that arose through gene duplication⇒appears to have happened at least twice in Gammaproteobacteria. First, relatively soon after the duplication that gave rise to RelA and SpoT, RelA lost its capacity for alarmone hydrolysis, evolving into a monofunctional, synthetase-only (SYNTH-only) RSH. Secondly, as evidenced by a lack of sequence conservation in sites that are critical for nucleotide pyrophosphorylation, during the evolution of the Moraxellaceae lineage of Protobacteria, SpoT has likely lost its synthetase function (Atkinson et al., PLoS One, 2011). This resulted in further specialization into mono-functional (p)ppGpp hydrolase, SpoT[Hs](The uppercase “H” stands for hydrolase competent, while the lowercase “s” indicates “synthetase-incompetent”), as opposed to the bifunctional HD- and SYNTH-competent SpoT[HS] found in other Beta- and Gammaproteobacteria. Notably, recent studies directed to A. baumannii indicated a lack of (p)ppGpp in the ΔrelA strain (i.e. an A. baumannii strain wherein the relA gene is inactivated or deleted), both with and without acute amino acid starvation induced by serine hydroxamate (SHX) (Jung et al., J Antimicrob Chemother, 2020; Perez-Varela et al., J Bacteriol, 2020). These observations are consistent with the hypothesis that RelA is, indeed, the sole source of the alarmone in this bacterium. Furthermore, consistent with the key role of (p)ppGpp-mediated signaling in bacterial virulence and antibiotic tolerance (Kundra et al., Front Microbiol, 2020), the likely ppGpp9 A. baumannii ΔrelA strain displays increased sensitivity to multiple antibiotics (Jung et al., J Antimicrob Chemother, 2020; Perez-Varela et al., J Bacteriol, 2020), decreased virulence in a Galleria mellonella wax moth model and deficiency in switching from the virulent opaque colony variant to the avirulent translucent colony variant (Perez-Varela et al., J Bacteriol, 2020). Rel, RelA and SpoT all share the same conserved d