Fragment-Based Ligand Discovery Applied to the Mycolic Acid Methyltransferase Hma (MmaA4) from Mycobacterium tuberculosis: A Crystallographic and Molecular Modelling Study

Abstract

The mycolic acid biosynthetic pathway represents a promising source of pharmacological targets in the fight against tuberculosis. In Mycobacterium tuberculosis, mycolic acids are subject to specific chemical modifications introduced by a set of eight S-adenosylmethionine dependent methyltransferases. Among these, Hma (MmaA4) is responsible for the introduction of oxygenated modifications. Crystallographic screening of a library of fragments allowed the identification of seven ligands of Hma. Two mutually exclusive binding modes were identified, depending on the conformation of residues 147–154. These residues are disordered in apo-Hma but fold upon binding of the S-adenosylmethionine (SAM) cofactor as well as of analogues, resulting in the formation of the short η1-helix. One of the observed conformations would be incompatible with the presence of the cofactor, suggesting that allosteric inhibitors could be designed against Hma. Chimeric compounds were designed by fusing some of the bound fragments, and the relative binding affinities of initial fragments and evolved compounds were investigated using molecular dynamics simulation and generalised Born and Poisson–Boltzmann calculations coupled to the surface area continuum solvation method. Molecular dynamics simulations were also performed on apo-Hma to assess the structural plasticity of the unliganded protein. Our results indicate a significant improvement in the binding properties of the designed compounds, suggesting that they could be further optimised to inhibit Hma activity.

The pathogenic mechanism of Mycobacterium tuberculosis: implication for new drug development

“…Rifampicin RNA polymerase, β subunit Inhibits RNA synthesis [11,12] fatty acids by the multifunctional fatty acid synthase I (FAS-I), which is then extended to C 48-62 by the FAS-II multienzyme system. At the same time, it is modified by a group of eight S-adenosylmethionine-dependent methyltransferases (SAM-MTs) in two distinct positions (distal and proximal positions on the meromycolic chain) [57,58]. The cis double bonds, which are necessary for the process of decorating, may be converted into cyclopropane by MmaA2 and PcaA [36,59], or converted into a trans double bond by UmaA1 [60], or hydrated into hydroxylated mycolates by MmaA4 [53,57,61,62].…”

“…These ligands have two binding regions (one located in a deep crevice that accommodates substrate/ SAM cofactor, and the other located on the surface of protein), and two different binding modes (Fig. 3d) [58]. Fragment ZT218, ZT260, ZT585, and ZT424 have the same binding mode as the SAM cofactor [58,136].…”

“…3d) [58]. Fragment ZT218, ZT260, ZT585, and ZT424 have the same binding mode as the SAM cofactor [58,136]. However, two fragments ZT275 and ZT320, which are located at the substrate-binding site of MmaA4, induce rearrangement of a segment (residues 147-154 loop) to generate a new conformation, and cause the inability of the cofactor to be compatible with the MmaA4, which indicates that the allosteric inhibitors of MmaA4 can be designed [58].…”

“…Fragment ZT218, ZT260, ZT585, and ZT424 have the same binding mode as the SAM cofactor [58,136]. However, two fragments ZT275 and ZT320, which are located at the substrate-binding site of MmaA4, induce rearrangement of a segment (residues 147-154 loop) to generate a new conformation, and cause the inability of the cofactor to be compatible with the MmaA4, which indicates that the allosteric inhibitors of MmaA4 can be designed [58]. In the complex structures of MmaA4 with ZT275/ZT320, the residue Phe148 of helix η1 is pushed away from its original positions (about 10 Å) in the complex structure of MmaA4-SAM, and the position of adenine moiety of the cofactor is occupied by residues Glu149 and His150 [58].…”

“…However, two fragments ZT275 and ZT320, which are located at the substrate-binding site of MmaA4, induce rearrangement of a segment (residues 147-154 loop) to generate a new conformation, and cause the inability of the cofactor to be compatible with the MmaA4, which indicates that the allosteric inhibitors of MmaA4 can be designed [58]. In the complex structures of MmaA4 with ZT275/ZT320, the residue Phe148 of helix η1 is pushed away from its original positions (about 10 Å) in the complex structure of MmaA4-SAM, and the position of adenine moiety of the cofactor is occupied by residues Glu149 and His150 [58]. A similar conformational change of the helix η1 is also observed in the complex structure of MmaA4-ZT424, and this compound bound to the position of the adenine moiety of SAM cofactor by van der Waals interactions [58].…”