US-20260124217-A1 - COMPOSITIONS OF BIFUNCTIONAL INHIBITORS OF TETRACYCLINE INACTIVATING ENZYMES AND METHODS OF USE THEREOF

Abstract

Among the various aspects of the present disclosure is the provision of bifunctional inhibitors of tetracycline inactivating enzymes and methods of use thereof. Herein, compositions for inhibitors of tetracycline inactivating enzymes are described. Additionally, a method of treatment for multidrug resistant bacterial infections using inhibitors of tetracycline inactivating enzymes is disclosed.

Inventors

• Timothy Wencewicz
• Gautam Dantas
• Niraj TOLIA
• Emily Williford
• Caitlin DeAngelo
• Kevin Blake
• Hirdesh Kumar

Assignees

• WASHINGTON UNIVERSITY

Dates

Publication Date

20260507

Application Date

20250306

Claims (16)

1. 1 . A bisubstrate tetracycline destructase inhibitor compound comprising a C9-substituted anhydrotetracycline (aTC) analog, the C9-substituted anhydrotetracycline (aTC) analog comprising the structure: wherein R is selected from R Group 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 .
2. 2 . The compound of claim 1 , wherein the compound inhibits both type 1 and type 2 tetracycline destructases (TDases).
3. 3 . The compound of claim 1 , wherein the compound is a competitive inhibitor of TDases.
4. 4 . The compound of claim 1 , wherein the compound binds sites comprising a substrate site and a nicotinamide site.
5. 5 . The compound of claim 4 , wherein the substrate site is a tetracycline (TO) binding site.
6. 6 . The compound of claim 4 , wherein the nicotinamide site is an NADPH binding site.
7. 7 . The compound of claim 1 , wherein the compound inhibits TDase reactions comprising a NADPH oxidation and a FAD reduction.
8. 8 . A method of treating an infection with at least one of multidrug resistant (MDR) Gram-negative bacteria, extensively drug-resistant (XDR) Enterobacteriaceae species, and XDR Acinetobacter species, the method comprising administering, to a patient, a third-generation tetracycline in combination with a bisubstrate tetracycline destructase inhibitor compound.
9. 9 . The method of claim 8 , wherein the bisubstrate tetracycline destructase inhibitor compound comprises a C9-substituted anhydrotetracycline (aTC) analog, the C9-substituted anhydrotetracycline (aTC) analog comprising the structure: wherein R is selected from R Group 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 .
10. 10 . The method of claim 8 , wherein the bisubstrate tetracycline destructase inhibitor compound inhibits both type 1 and type 2 tetracycline destructases (TDases).
11. 11 . The method of claim 8 , wherein the bisubstrate tetracycline destructase inhibitor compound is a competitive inhibitor of TDases.
12. 12 . The method of claim 8 , wherein the bisubstrate tetracycline destructase inhibitor compound binds sites on TDases comprising a substrate site and a nicotinamide site.
13. 13 . The method of claim 12 , wherein the substrate site is a tetracycline (TC) binding site.
14. 14 . The method of claim 12 , wherein the nicotinamide site is an NADPH binding site.
15. 15 . The method of claim 8 , wherein the bisubstrate tetracycline destructase inhibitor compound inhibits TDase reactions comprising a NADPH oxidation and a FAD reduction.
16. 16 . The method of claim 9 , wherein the C9-substituted aTC analog comprises a TDase inhibitory activity and an antibacterial activity.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application Ser. No. 63/561,534 filed on Mar. 5, 2024, which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under AI123394 awarded by the National Institutes of Health. The government has certain rights in the invention. MATERIAL INCORPORATED-BY-REFERENCE Not applicable. FIELD OF THE INVENTION The present disclosure generally relates to compositions of tetracycline destructase inhibitors and methods of use thereof. BACKGROUND OF THE INVENTION Tetracycline (TC) antibiotics are a family of type-II polyketides originally isolated from Streptomyces aureofaciens. TCs have been in clinical use for >70 years as broad-spectrum antibiotics and continue to be used as frontline agents for treating a variety of infections caused by Gram-positive and Gram-negative bacteria. Until recently, it was thought that clinical TC resistance occurs primarily through the expression of efflux pumps and ribosome protection proteins. These resistance mechanisms have been largely overcome in the clinic by the development of last-generation TCs known as glycylcyclines including the FDA-approved drugs tigecycline, eravacycline, and omadacycline. Unfortunately, all known TC antibiotics are susceptible to an emerging third route of clinical resistance: enzymatic inactivation by tetracycline destructase (TDase) enzymes. TDases are members of the class A flavin monooxygenase (FMO) enzyme family. TDases are FAD-dependent and use an NADPH/O2-coupled redox cycle to catalyze the inactivation of TC antibiotics. Oxidation of the bound TC substrate occurs via a C4a-peroxy-flavin intermediate resulting in substrate-dependent oxygen transfer (hydroxylation) and oxygen insertion (Baeyer-Villiger type) reactions. The resulting oxidized TC scaffolds lack antibacterial activity presumably due to a loss of binding affinity for the bacterial ribosome. TDases contain distinct substrate and FAD-binding domains connected via a C-terminal bridge helix. Two distinct types of TDases have <20% sequence homology and cluster by structural features, resistance phenotype, and ecological origin. Type 1 TDases have a constitutively open active site, provide resistance against all known TC antibiotic classes, and are found in human gut commensals and pathogens. Type 2 TDases contain an extra C-terminal helix that ‘gates’ the active site during the catalytic cycle, provide resistance to only first and second-generation TC antibiotics but not glycylcyclines, and are found primarily in environmental microbes. The structural and functional differences of Type 1 and 2 TDases have an important influence on substrate binding mode, flavin dynamics, mechanism of TC inactivation, and inhibition. At this point, Type 1 TDases appear to be the more likely clinical threat but the evolutionary connection between Type 1 and 2 TDases presents a unique opportunity to study TC resistance via enzymatic inactivation. Enzymatic antibiotic inactivation is of particular concern given that this pathogen phenotype depletes the antibiotic challenge for the entire infection environment (including cells not expressing inactivating enzymes). The evolution of antibiotic inactivation enzymes under intense selective pressure is a potential gateway to pan resistance against entire drug classes. The clinical significance and global impact of this resistance mechanism have been fully realized for beta-lactam antibiotics where widespread dissemination of beta-lactamase encoding genes demands the co-administration of a beta-lactamase inhibitor to restore the clinical efficacy of beta-lactam antibiotics. Presumably, TDase inhibitors will be needed in the future given the mobilization and widespread distribution of TDase genes in the environment including clinical and agricultural settings. The emergence of TDases in clinical pathogens is on an upward trajectory and the deployment of new glycylcyclines could exacerbate this trend as has been observed following the release of new broad-spectrum beta-lactam antibiotics. Anhydrotetracycline (aTC), which differs from parent TC by dehydration of the C6 alcohol, is a pan TDase inhibitor and rescues whole cell activity of TC antibiotics in E. coli and Mycobacterium abscessus. The co-crystal structure of aTC bound to Tet(50) reveals a binding mode that is unique from the observed substrate binding mode leading to stabilization of the FAD-cofactor in an unproductive ‘OUT’ conformation that is stabilized through a π-π stacking interaction with the benzylic sidechain of Y267. In addition to the TDase inhibitory activity, aTC alone has inherent antibacterial activity and some general toxicity at the effective concentrations (low μM) due in part to its ability to disrupt cellular membranes. Further, some Type 1 TDases can turnover aTC as a slow subs