Sandbox Reserved 1073

Introduction

The Enoyl-ACP Reductase InhA, from Mycobacterium tuberculosis, catalyzes the NADH-dependent reduction of long-chain trans-2-enoyl-ACP fatty acids in the type II fatty acid biosynthesis pathway of M. tuberculosis. InhA is a member of the short chain dehydrogenase/reductase (SDR) family of enzymes. InhA is the only enoyl-ACP reductase found in tuberculosis, making the enzyme a potential drug target.

FAS-II System

Mycolic acids are very long-chain fatty acids (C₆₀ -C₉₀) that are essential components of the mycobacterial cell wall. Mycolic acids are synthesized by at least two known elongation systems, type I and type II fatty acid synthases (FAS-I and FAS-II). The FAS-II system prefers C16 as a starting substrate and can extend up to C56. The FAS-II system utilizes the products from the FAS-I system as primers to extend the chain lengths further. The products of the FAS-II system are the precursors of mycolic acids. Elongation by the FAS-II system occurs by a condensation reactionof acetyl and malonyl substrates, which is achieved in three steps. Step 1 involves transfer of the acyl primer, step 2 involves decarboxylation of the substrate to yield a carbanion, and step 3 involves nucleophilic attack of the carbanion to yield the elongated product.

Mechanism of Action

The InhA gene encodes for the InhA protein. InhA catalyzes the NADH-dependent reduction of the trans double bond between positions C2-C3 of fatty acyl substrates. InhA prefers fatty acyl substrates of C16 or longer, which is consistent of the protein being a member of the FAS-II system. The longer chain length specificity of InhA distinguishes the enzyme from other enoyl-ACP reductase analogues.

Figure 1. Mechanism of InhA protein

Structure

Fatty Acyl Binding Crevice

Catalytic Triad

Hydrogen Bonding Interactions

Clinical Applications

Isoniazid

Isoniazid is a first-line antibiotic that has been used to treat tuberculosis infections for over 50 years. Isoniazid is known to inhibit mycolic acid biosnthesis, which is the function of InhA. The activated form of isoniazid is covalently attached to the nicotinamide ring of NADH. However, Isoniazid is still not an ideal antibiotic because many drug-resistant strains of tuberculosis have shown resistance to this inhibitor. Specifically, the mutation Ser⁹⁴ to Ala of InhA was sufficient enough to have isoniazid resistance.

Figure 5. Isoniazid Mechanism of Action

Other Inhibitors

Drug resistance of M. tuberculosis has become a huge problem for the development of antibiotics. A drug screen of potential inhibitors of InhA (300 compounds), were composed of inhibitors of the Plasmodium falciparum enoyl-reductase, against M. tuberculosis. The enoyl reductases of both bacteria have limited similarities, however two compounds, CD39 and CD117 had activity against drug-susceptible M. tuberculosis. More importantly, both compounds had activity against drug-resistant and multi-drug resistant TB. Treatment of the bacterium with the compounds resulted in the inhibition of mycolic acid and long-chain fatty acid biosynthesis, indicating that these compounds act against enzymes of both the FAS-I and FAS-II system. The benefit of having the compounds have multiple targets is the reduced development of drug resistance, which is the disadvantage of isoniazid. The essential chemical groups that lead to the antimycobacterial properties of the compounds include a thioacetate group, and a t-butyl group.

Figure 6. CD39 Structure

Figure 7. CD117 Structure