Mechanism of thioamide drug action against tuberculosis and leprosy

Abstract

Thioamide drugs, ethionamide (ETH) and prothionamide (PTH), are clinically effective in the treatment of Mycobacterium tuberculosis, M. leprae, and M. avium complex infections. Although generally considered second-line drugs for tuberculosis, their use has increased considerably as the number of multidrug resistant and extensively drug resistant tuberculosis cases continues to rise. Despite the widespread use of thioamide drugs to treat tuberculosis and leprosy, their precise mechanisms of action remain unknown. Using a cell-based activation method, we now have definitive evidence that both thioamides form covalent adducts with nicotinamide adenine dinucleotide (NAD) and that these adducts are tight-binding inhibitors of M. tuberculosis and M. leprae InhA. The crystal structures of the inhibited M. leprae and M. tuberculosis InhA complexes provide the molecular details of target-drug interactions. The purified ETH-NAD and PTH-NAD adducts both showed nanomolar Kis against M. tuberculosis and M. leprae InhA. Knowledge of the precise structures and mechanisms of action of these drugs provides insights into designing new drugs that can overcome drug resistance.

Figure 1.

Chemical structure of ETH, PTH, and INH. Although these prodrugs have similar structures, INH is activated by a catalase-peroxidase, whereas ETH and PTH are activated by a flavin-dependent monooxygenase.

Figure 2.

Active sites of the M. tuberculosis enoyl-acyl ACP reductases bound to inhibitors and the bound inhibitor. (A) The crystal structure of PTH-NAD superimposed onto the simulated annealing omit electron density map contoured at 1 σ. Carbon atoms are gray, oxygen atoms are red, nitrogen atoms are blue, and phosphor atoms are orange. The 2-propyl-isonicotinic acyl group is covalently attached to the 4 position of the nicotinamide ring of NADH in a 4S configuration. (B) Cross section through the surface of the InhA active site with bound INH-NAD. (C) Cross section through the surface of the InhA active site with bound ETH-NAD showing that the 2-ethyl-isonicotinic acyl moiety protrudes into a hydrophobic binding pocket created by the rearrangement of the side chain of Phe¹⁴⁹ (shown behind the transparent surface), which is similar to INH-NAD. (D) Cross section through the surface of the InhA active site with bound PTH-NAD, which has a similar binding mode to INH-NAD and ETH-NAD. The carbon atoms of the adduct inhibitors and Phe¹⁴⁹ are white and yellow, respectively.

Figure 3.

M. tuberculosis InhA with bound inhibitors. (A) Stereo view of the superposition of active sites of the M. tuberculosis InhA:NADH structure and the InhA:ETH-NAD structure, showing the side chain of Phe¹⁴⁹ rotated ∼90° once the ETH-NAD adduct binds to the enzyme. The carbon atoms of residues and NADH in the InhA:NADH structure are cyan. The carbon atoms of residues and ETH-NAD in the InhA:ETH-NAD structure are gold. (B) The stereo view of the active sites of the M. leprae InhA:PTH-NAD structure. The carbon atoms of residues and PTH-NAD adduct are gold and cyan, respectively. Other atoms are colored according to the atom type (red, oxygen atoms; blue, nitrogen atoms; yellow, sulfur atoms; and orange, phosphor atoms).

Figure 4.

Selected interactions between ETH-NAD and the active site of InhA. A conserved water molecule, TIP20, forms a hydrogen bond interaction with the nitrogen atom of the 2-ethyl-isonicotinic acyl moiety of the inhibitor at a distance of 2.9 Å. The other water molecule, TIP2, is in the center of a hydrogen bonding network, which interacts with the oxygen atom of the phosphate group of the adduct and the hydroxyl group of Ser⁹⁴ at distances of 2.7 and 2.9 Å, respectively.

Figure 5.

Possible reaction mechanisms for the activation of ETH and the formation of ETH-NAD. Two plausible mechanisms for the activation of ETH are shown. Either route will lead to the observed ETH-NAD adduct, retaining a tetrahedral carbon at position 4 of the nicotinamide ring.