The role of adjuvants in overcoming antibacterial resistance due to enzymatic drug modification

Abstract

Antibacterial resistance is a prominent issue with monotherapy often leading to treatment failure in serious infections. Many mechanisms can lead to antibacterial resistance including deactivation of antibacterial agents by bacterial enzymes. Enzymatic drug modification confers resistance to β-lactams, aminoglycosides, chloramphenicol, macrolides, isoniazid, rifamycins, fosfomycin and lincosamides. Novel enzyme inhibitor adjuvants have been developed in an attempt to overcome resistance to these agents, only a few of which have so far reached the market. This review discusses the different enzymatic processes that lead to deactivation of antibacterial agents and provides an update on the current and potential enzyme inhibitors that may restore bacterial susceptibility.

Fig. 1. The β-lactam classes of antibiotics.

Scheme 1. Nucleophilic attack of the hydroxyl group of the PBP active site serine residue on the carbonyl of the β-lactam ring, forming a covalent bond.

Scheme 2. Hydrolysis of cephalosporins by class A β-lactamases. Nucleophilic attack of the hydroxyl group of Ser70 on the amide group of β-lactams results in cleavage of the β-lactam ring.

Fig. 2. Binding and hydrolysis of ampicillin by MBLs (PDB ID: 5O2F). The zinc ions facilitate the nucleophilic attack and help stabilise the tetrahedral intermediate and the hydrolysed product.

Fig. 3. Structures of the six currently approved β-lactamase inhibitors.

Fig. 4. Irreversible binding of clavulanic acid to serine β-lactamase (PDB ID: 6NVU). A The clavulanic acid has taken the place of a β-lactam antibiotic in the active site and the hydrogen bond between the carbonyl of the ring opened acyl–enzyme intermediate ii and serine237 is shown. B Schematic representation of the β-lactamase inhibition, involving nucleophilic attack by the hydroxyl group of serine70 on the carbonyl oxygen of the β-lactam ring, decarboxylation of the initially formed acyl–enzyme intermediate i, and hydrolysis of the imine ii, to form a high-energy acyl–enzyme adduct iii.

Fig. 5. Taniborbactam, the first serine and metallo-β-lactamase inhibitor to enter clinical development, and nacubactam.

Fig. 6. General structure of some clinically used aminoglycosides consisting of free hydroxyl groups, two or more amino groups and an aminocyclitol ring linked to one or more aminosugars. As the hydroxyl and amino groups are involved in binding to rRNA, they are targeted by aminoglycoside modifying enzymes (AMEs).

Fig. 7. Kanamycin A (blue) and acetyl-CoA (orange-red) in the active site of AAC(6′)-Ib (PDB ID 1V0C).

Fig. 8. Structure of the a bisubstrate analogue which mimics the tetrahedral transition state of AAC-catalysed acetylation.

Fig. 9. The fermentation product 7-hydroxytropolone.

Fig. 10. A designed AR in complex with APH(3′)-III (PDB ID 2BKK). The AR protein binds to the C-terminal outside the substrate active site, leading to an alteration in the active site so that the enzyme can no longer modify aminoglycosides (image created using Mol*).

Scheme 3. Deactivation of chloramphenicol by CATI. Once bound, acetyl CoA reaches the enzyme active site through a tunnel. One of the most important residues for catalysis is His193, with the lone pair electrons of the imidazole deprotonating the primary alcohol of chloramphenicol, promoting the attack on the carbonyl of the thioester of acetyl CoA, yielding an acetylated chloramphenicol.

Scheme 4. Other chloramphenicol enzymatic modifications.

Fig. 11. The structures of the macrolide antibiotics.

Fig. 12. Interaction of erythromycin with the nucleotides in its binding site in the protein exit tunnel (PDB ID 1JZY). In ribosomal RNA nucleotide 2058 is an adenine to which the macrolide antibiotic binds strongly. Hydrogen bonds are formed between the 2′,6,11 and 12-hydroxyl groups of the macrolide and adenine bases 2058 and 2509. An electrostatic interaction exists between the conjugate acid of the tertiary amine of the macrolide and the guanine-2505 phosphate. All these interactions play a key role in macrolides binding to this site, with any modifications in structure disrupting the binding process.

Scheme 5. Hydrolysis of macrolides by esterases. Macrolides, such as erythromycin, bind within a buried active site of EreC covered by a glycine and proline rich loop. When the loop is closed, it repositions the ester group of macrolides in close proximity to the catalytic triad, composed of His50, Glu78 and His289. The first step is deprotonation of a water molecule by His50, producing a reactive hydroxide ion which then attacks the carbonyl of the macrolide ester, forming a negatively charged transition-state intermediate which is stabilised by Arg261. The deprotonated hydroxyl group then retrieves the proton previously transferred to His50, releasing the antibiotic in an inactive hydrolysed form.

Fig. 13. 2′-Phosphorylation of erythromycin by an MPH(2′)-I (PDB ID: 5IGP). The nucleotide binds within the hydrophobic N-terminus and interacts with Tyr94 and aspartate residues (Asp200, Asp219, Asp234). The macrolide binding site consists of a hydrophobic region, a negatively charged region and an aspartate residue (Asp200), seen in all MPH subtypes, along with Tyr202, Ala234 and Phe280 residues. Asp200 is thought to position the target 2′-OH of macrolides in an orientation which allows proton transfer during catalysis. For MPHs which utilise GTP, a tyrosine residue (Tyr94) acts as a gatekeeper.

Fig. 14. Examples of chemical structures of tetracyclines. Tetracyclines have a common linear fused tetracyclic nucleus; tigecycline is a third-generation tetracycline with an additional side chain on the D ring.

Fig. 15. X-ray crystal structure of Tet(X) with bound FAD (PDB ID: 2XDO). Tet(X) is composed of two domains stabilised by a C-terminal α-helix and another C-terminal α-helix involved in substrate recognition and binding. The X-ray crystal structure of Tet(X) show that the first domain contains a non-covalently bound FAD that adopts an IN (pointing towards the binding domain) or OUT (pointing away from the binding domain) conformation. The A ring is positioned near the bound FAD molecule and the D ring lies near the C-terminal α-helix. The OUT confirmation is required for the reduction of FAD to FADH2 by NADPH. The reduced form is then able to bind oxygen in the IN confirmation forming a hydroperoxide able to hydroxylate tetracycline at C11. The second domain covers the FAD-binding site while also containing the substrate recognition pocket (image created using Mol*).

Fig. 16. A Hydroxylation of 7-chlortetracycline by Tet(X). B The residues of Tet(X) recognise the A and B ring of tetracyclines and orient it for the hydroxylation at C-11a: FAD represented in green, tetracycline in purple (PDB ID: 2Y6R).

Fig. 17. The structure of rifamycins is composed of a naphthalene core and an ansa bridge, which is a basket-like structure.

Scheme 6. Deactivation of rifamycins by RPH in L. monocytogenes. The enzyme has 3 domains: an N-terminal ATP-grasp domain, a swivel domain containing the catalytic His at the C-terminus and an intermediate domain responsible for binding rifampicin. The rifampicin binding domain contains three residues Val333, Met359 and Val368 forming a hydrophobic patch that stabilises rifampicin. The naphthol ring of rifampicin binds deep in the pocket through van der Waals interactions while the R groups point towards the opening of the pocket and binds to Pro356 and Phe479. The ATP-grasp domain positions the β-phosphate that is then transferred to His825 in the swivel domain. The Glu667–Arg666 residues found in the rifampicin-binding domain then facilitate the transfer of the phosphate group to 21-OH of the polyketide region.

Fig. 18. Deactivation of rifamycins by Rox in N. farcinica (5KOX). Rifampicin (purple) binds perpendicularly to FAD (light blue) with O4 of FAD undergoing hydrogen bonding with the drug. The exact binding interactions differ depending on the species expressing the Rox. For example, in S. venezuelae, Arg213 forms a hydrogen bond with 8-OH whereas, in N. farcinica, the 21-OH forms a hydrogen bond with the Arg43 residue. In N. farcinica, the drug binds to the enzyme through hydrophobic interactions mainly: the polyketide region interacts with the Val205 residue through hydrophobic interactions. In both examples, Rox transfers a hydroxyl group from FAD-OOH to RIF which leads to ring opening, disrupting the ansa bridge structure required for binding.

Fig. 19. Deactivation of isoniazid by NAT (PDB ID: 1W6F). Isoniazid binds to a NAT catalytic triad composed of Cys70, His110 and Asp127. The terminal nitrogen of isoniazid is situated ∼2.4 Å from the γ-S atom of Cys70, making it the most important residue for this catalysis. Isoniazid also undergoes hydrophobic interactions and hydrogen bonding with other residues within the active site. The enzyme is initially acetylated by acetyl CoA and the acetyl group is then transferred onto the hydrazinyl group of isoniazid.

Fig. 20. Chemical structure of 11α-hydroxycinnamosmolide an N-acetyltransferase inhibitor.

Scheme 7. Transfer of an AMP group from ATP to 3-OH of lincosamides by LinB.

Fig. 21. Fosfomycin binding to MurA, which is utilised in the formation of N-acetylmuramic acid, an essential component of the peptidoglycan layer, inhibition by fosfomycin thus leads to cell wall lysis. The catalytic domain of MurA is deep in the cavity of the enzyme's two domains and contains Cys115, which fosfomycin covalently attaches to. Three positively charged residues of MurA, Lys22, Arg120, and Arg397 surround the phosphonate group of fosfomycin creating electrostatic interactions and hydrogen bonds.

Fig. 22. Fosfomycin binding to FosA from K. pneumoniae (PDB ID 5V3D). FosA key residues involved in the binding of fosfomycin include Lys93, Ser97, Ser101, Tyr103, and Arg122. The Mn²⁺ ion acts as a Lewis acid during the nucleophilic attack by glutathione. A K⁺ at the active site does not take part in the catalysis reaction but enhances the electrophilicity of the Mn²⁺ ion, increasing the rate of reaction.

Fig. 23. Crystal structure of FosA3 with inhibitor ANY1 bound (PDB ID 5WEP). ANY1 binds to the active site of FosA by interacting with Lys93, Ser97 and Tyr103.

Fig. 24. Chemical structure of disulfiram. Disulfiram is able to react with BSH, forming a stable adduct and depleting BSH stores.