Molecular Pharmaceutics 2015 Jan; 12:898−909.

Molecular Details of INHC10 Binding to wt KatG and Its S315T Mutant.


Teixeira VH1, Ventura C1,2, Leitão R1,3, Ràfols C4, Bosch E4, Martins F*1, Machuqueiro, M1*.


1Centro de Química e Bioquímica and Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal

2Instituto Superior de Educação e Ciências, Alameda das Linhas de Torres 179, 1750 Lisboa, Portugal

3Área Departamental de Engenharia Química, Instituto Superior de Engenharia de Lisboa, Instituto Politécnico de Lisboa, R. Conselheiro Emídio Navarro, 1, 1959-007 Lisboa, Portugal

4Departament de Química Analítica and Institut de Biomedicina (IBUB), Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain USA.



Isoniazid (INH) is still one of the two most effective antitubercular drugs and is included in all recommended multi-therapeutic regimens. To circumvent the increasing resistance of Mycobacterium tuberculosis (Mtb) to INH, mainly associated with mutations in the katG gene, various INH-based compounds have been proposed without much success. It is reported that the KatG enzyme of Mtb has an important role in the activation of INH. However, its S315T mutant, the most frequent cause of INH resistance worldwide, seems to interfere with the conversion of INH to a biologically active form, thus diminishing its activity, allegedly due to a larger steric constraint in the heme access channel. To shed some light into these aspects, a detailed comparative molecular study of the interactions between the wild-type form of the enzyme, or its S315T mutant, and either INH or INH-C10, a newly synthesized acylated INH derivative, was presented. Rather unexpectedly, this INH-C10 compound is six-fold more active than INH in the mutated strain. Interestingly, our in silico results indicate that the aliphatic tail in INH-C10 does bring the compound closer to the heme active site. It seems therefore that INH-C10 is able to counterbalance most of the conformational restrictions introduced by the mutation, which are thought to be responsible for the decrease in INH activity in the mutated strain. Hence, INH-C10 appears to be a very promising lead compound and a new hope in the fight against tuberculosis.

KEYWORDS: ITC; molecular docking; molecular dynamics; new inhibitor; resistance; tuberculosis

PMID: 25590860



Tuberculosis ranks alongside HIV as a major cause of death by a single infectious agent. According to the last WHO report, recently released, 9.6 million people fell ill with TB and 1.5 million died, in 2014. The upsurge of multidrug-resistant (MDR-TB) – 5% of all cases – and extensively drug-resistant tuberculosis (XDR-TB) – 9.7% of MDR-TB cases – makes the quest for new and effective antitubercular drugs crucial and urgent.

In spite of the significant efforts in the last decade or so to develop new drugs and new therapeutic regimens, only one compound with a totally new mechanism of action, bedaquiline, was approved by FDA for the treatment of MDR-TB in adults (Dec. 2012). However, isoniazid (INH), synthesized for the first time in 1952, is still one of the two most effective drugs to treat tuberculosis and remains a key component in all WHO recommended treatments. Yet, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, has been showing an increased resistance to INH. We thus hypothesized that if we departed from INH and made some key modifications in its core structure, we could obtain one or more compounds with improved activity and eventually circumvent resistance. The strategy was to use a QSAR-oriented design to assist us in the synthesis of new, predictively more active, INH derivatives, resorting to different, complementary, and thoroughly validated, QSAR methodologies, namely, Random Forests (RF) and Associative Neural Networks (ASNNs), and also Multiple Linear Regressions (MLR) (1-2).

As a result, several compounds were synthesized and their biological activity evaluated against the wild-type H37Rv and two mutated Mtb strains (1). In this process, five of the new compounds showed measured activities against the H37Rv strain higher than INH and, more significantly, one of them, a INH derivative with a C10 alkyl chain (INH-C10), Figure 1, was found to exhibit a six fold increase in activity against the katG (S315T) mutated strain, by comparison with INH ((1) and Table 1). Based on this latter finding, we posed ourselves the following question: how could one rationalize the behavior of this new compound, INH-C10, vis-à-vis katG S315T, when its structure, with the long alkyl chain, would anticipate larger steric constraints in the access channel to the heme active site and thus a putative increase in resistance?


FM fig1

Figure 1 – Structures of INH and INH-C10


Table 1: Experimental MIC (mM) and ITC kb values. More details in the publication.



It has been described in the literature that the extra methyl group in the S315T mutated strain effectively restricts the accessibility to the active site and the heme group by closing down the dimensions of the narrowest part of the channel from 6 Å in the wt KatG to 4.7 Å in the mutant (3). This difference was inferred from a comparison between two packed X-ray structures (PDB codes 1SJ2 and 2CCD), but no information is available about the conformations of both proteins in solution. Using molecular dynamics simulations, we estimated the volume of the narrowest region of the pocket access tunnel, right next to residue 315, by determining the number of water molecules at a given distance of the central iron atom over time, and found out that the extra methyl group of the mutant has a negligible effect on this access channel. In fact, in the conformational ensembles of both the wt KatG and the S315T mutant forms, we observed either fully opened (Figure 1a,c) or closed (Figure 1b,d) conformations. These results indicate that INH can access the heme site in KatG S315T mutant with similar steric hindrance as in the case of the wt protein.


FM fig2

Figure 2. Open and closed conformations of the heme access tunnel for wt (a,b) and for the S315T mutant (c,d), respectively. The heme group is shown as grey sticks, while Asp137 and Set/Thr315 are shown in beige sticks. Cavity volume is represented by a brown mesh surface.


Yet, an important question remained: how can S315T mutation influence the formation of the INH-NADH adduct? If it is not the access of INH to the heme site that is perturbed, it might be its activation. A reasonable possibility is that the extra methyl group might alter the hydrogen bond network and the electrostatic distribution surrounding the heme group, hence also affecting its redox potential (4). From our results, we noticed two main regions with significant changes in the electrostatic environment, one close to the hydrogen bond network of propionate A, Ser/Thr315, and His276, and another one in the region of Asp137, His108, and the tripeptide adduct MYW. This last region seems to concentrate most of the important changes in the EP surrounding the heme group suggesting an important role in the modulation of the redox potential and hence the activation of this group. These results emphasize and illustrate the role of S315T mutation in modulating the activation of the reactive heme group. It is quite remarkable how a small mutation can have such a significant effect on the electrostatic map of KatG’s heme pocket.



FM fig3

Figure 3. Representative conformations of the largest docking solution cluster with the protein in the resting state. (a) INH and wt protein; (b) INH and S315T mutant; (c) INH-C10 and wt; and (d) INH-C10 and the mutant. The heme is displayed in gray sticks, INH in dark pink, and INH-C10 in cyan. Some important residues are also shown in smaller sticks.


A common approach to evaluate the in silico ligand binding to enzymes is by using molecular docking. We identified several significant changes in the heme site conformational ensembles for both mutant and wt forms and these changes can have an important impact in the binding modes of INH and INH-C10. To assess the effect of these conformational changes, we envisaged a molecular docking protocol with these two molecules interacting with different conformations taken from the MD simulations of wt and S315T forms.

INH seems to have two major modes of binding. In wt KatG, the most predominant corresponds to “ready” conformation with the hydrazine group sitting on top of the heme iron and being stabilized by propionate A (Figure 3a). In the second mode of binding, INH is in a “not ready” conformation with the hydrazine group now facing away from the porphyrin. Interestingly, upon mutation, these two modes of binding change in their relative abundances, and the “not ready” conformation becomes the most abundant (Figure 3b). It seems that the two conformational ensembles (wt and S315T) do have an important influence on the preferred binding mode between INH and KatG.

Our docking studies also showed that INH-C10 has only one preferential mode of binding. Figures 3c and 3d show that the aliphatic tail aligns and interacts with several protein groups at the entrance of the heme site channel, bringing the hydrazine group to sit on top of the porphyrin. This restriction has the effect of largely favoring the mode of binding with a “ready” conformation, independently of the protein form studied (wt or mutated). These results on the role of INH-C10 hydrocarbon tail are in agreement with the ITC measurements results (Table 1), which have shown considerable entropy related increments in the binding constants involving this compound. In the case of the wt KatG, this entropic contribution is not enough to compensate the large decrease in enthalpy. However, in the mutated KatG form, the C10 tail restraint effect has a determinant role in the binding constant increase, which results in the much lower experimental MIC value (Table 1), making the S315T mutated form considerably more sensitive to INH-C10 (1).


The importance of this study is two-fold:

First, it shows that steric hindrance at the access channel to the heme active site cannot be taken as the only factor influencing the binding of these compounds to KatG. In fact, it has been shown that changes in the electrostatic environment around the heme group cannot be overlooked and have indeed a significant role in explaining the unexpected behavior of a bulky INH derivative by comparison with INH against the S315T mutant KatG form.

Second, this work and our former studies show that it is possible to design INH derivatives with improved activity and which are able to elude resistance phenomena in Mycobacterium tuberculosis. INH-C10 seems therefore a promising lead compound to be further used in antitubercular drugs development programs.


  1. Martins F, Santos S, Ventura C, Elvas-Leitão R, Santos L, Vitorino S, Reis M, Miranda V, Correia H F, Aires-de-Sousa J, Kovalishyn V, Latino DARS, Ramos J, Viveiros M 2014 Design, synthesis and biological evaluation of novel isoniazid derivatives with potent antitubercular activity. European Journal of Medicinal Chemistry 81: 119-138
  2. Martins F, Ventura C, Santos S, Viveiros M 2014 QSAR based design of new antitubercular compounds: improved isoniazid derivatives against multidrug-resistant TB. Current Pharmaceutical Design 20: 4427-4454
  3. Zhao X, Yu H, Yu S, Wang F, Sacchettini JC, Magliozzo RS 2006 Hydrogen peroxide-mediated isoniazid activation catalyzed by Mycobacterium tuberculosis catalase-peroxidase (KatG) and its S315T mutant. Biochemistry 45:4131−4140.
  4. Ghiladi RA, Cabelli DE, Ortiz de Montellano PR 2004 Superoxide reactivity of KatG: insights into isoniazid resistance pathways in TB. Journal of the American Chemical Society 126: 4772−4773.


Acknowledgements:  This study was supported by Fundação para a Ciência e a Tecnologia, Portugal, under Projects FCT/PTDC/QUI/67933/2006 and PEst-OE/QUI/UI0612/2013. Authors acknowledge the gift of the enzymes from Xiangbo Zhao and Richard S. Magliozzo, from Brooklyn College, City University of New York, whose work is supported by U.S. Grants NSF:CHE-1058116 and NIH/NIAID 2R56AI060014-06A (RSM). Authors are also grateful to Profs. Richard S. Magliozzo, Miguel Viveiros, Luísa Cyrne, Susana Santos, and M. Soledade Santos for their valuable insights.



Filomena Martins, Ph.D.

Assistant Professor

Dept Química e Bioquímica,

Universidade de Lisboa 1749-016 Lisbon Portugal


Miguel Machuqueiro, Ph.D.


Dept Química e Bioquímica,

Universidade de Lisboa

1749-016 Lisbon Portugal




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