The sharp contrast between the fatty acid biosynthetic pathway in humans (FAS-I) and that found in prokaryotes (FAS-II) has established this bacterial pathway as an attractive target for the design of new antibacterial agents 
. The FAS-II pathway in M.tuberculosis
is involved in the production of mycolic acids, which, along with peptidoglycan and arabinogalactan, are central constituents of the mycobacterium cell wall 
. InhA catalyzes the final, rate-determining step in the fatty acid elongation cycle by converting trans
-2-enoyl-ACP to acyl-ACP in an NADH-dependent reaction 
. This crucial regulatory enzyme is the primary molecular target of isoniazid, which has been used as a frontline anti-tubercular agent for the past 40 years 
. As a pro-drug, the activity of isoniazid is dependent on its activation by KatG, a catalase/peroxidase enzyme. KatG oxidizes isoniazid to an acyl radical that covalently binds to NADH, and functions as a potent inhibitor of InhA. Unfortunately, it is this activation requirement that allows M.tuberculosis
to acquire resistance to the drug. Indeed, mutations in the KatG gene account for around half of all isoniazid-resistant clinical isolates 
. Direct InhA inhibitors that avoid this activation requirement are not susceptible to this resistance mechanism 
. Triclosan and the diazoborines are well-known InhA inhibitors that do not require activation, but unfortunately they are not suitable for human treatment due to their respective poor solubility and toxicity 
. However, a new class of high affinity direct InhA inhibitors, consisting of alkyl diphenyl ethers of triclosan derivatives, have been found to exhibit activity against drug-resistant strains of M.tuberculosis 
. In addition to these alkyl diphenyl ethers, the arylamides 
, indole-piperazines, pyrazole-based inhibitors 
, and indole-based inhibitors 
, have recently been described as other classes of direct InhA inhibitors.
Using a novel computational strategy we have predicted that entacapone and tolcapone will directly inhibit InhA. Our prediction was subsequently confirmed by in vitro
antibacterial and enzyme kinetic assays using Comtan tablets containing the active component entacapone. Thus entacapone and tolcapone are promising lead compounds against drug-resistant strains of M.tuberculosis
. These drugs are currently in clinical use, although the association of tolcapone with hepatotoxicity has caused the drug to be placed under strict regulation in the United States 
. Entacapone, which is not associated with the same hepatotoxic risks, is therefore more attractive as a drug lead. Interestingly, a recent study showed that when patients suffering from Parkinson's disease were treated with rifampin and isoniazid, their condition was observed to improve 
. Since rifampin inhibits DNA-dependent RNA polymerase, this observation implies cross-reactivity between the M.tuberculosis
InhA, the target of isoniazid, and the drug targets of Parkinson's disease, which is consistent with our predictions that InhA inhibitors can also inhibit COMT.
Recent studies have shown that triclosan can trigger the upregulation of M.tuberculosis
detoxification mechanisms that result in its metabolism or efflux from the cell 
. For instance, triclosan has been shown to induce the expression of an aromatic dioxygenase involved in the degradation of arenes. Since triclosan consists of a diphenyl ether structure, it is thought that the induction of this enzyme may serve to degrade, and hence detoxify, triclosan 
. The subsequent modification of triclosan derivatives has led to high affinity alkyl diphenyl ether InhA inhibitors that upregulate neither efflux pumps nor aromatic dioxygenase 
. Although gene transcription studies are required to determine the ability of entacapone and tolcapone to cause upregulation of the aromatic dioxygenase, it is speculated that the strong electron-withdrawing nitrite groups of these drugs may result in significantly lower reduction potentials, therefore making them less prone to oxidation by this enzyme. The narrow ranges of the MIC99
and the IC50
of Comtan support this hypothesis.
From our current experimental results, it is not possible to determine the effect of the inactive ingredients in the Comtan tablets (magnesium stearate, microcrystalline cellulose, hydroxypropyl methylcellulose, yellow iron oxide and red iron oxide, titanium dioxide, sucrose, mannitol, hydrogenated vegetable oil, polysorbate 80, glycerol 85%, croscarmellose sodium) on the growth of M.tuberculosis
. It is unlikely that the major formulations in the Comtan tablets (magnesium stearate, microcrystalline cellulose, hydroxypropyl methylcellulose, iron oxide, titanium dioxide, sucrose) directly affect M.tuberculosis
growth because they are the same as the active ingredients found in anti-tubercular drugs such as Rifater (isoniazid/pyrazinamide/rifampin combination tablet), Rifamate (isoniazid/rifampin tablet), and Priftin, whose active component is a rifamycin derivative (http://www.rxlist.com
). Other ingredients such as polysorbate 80 and croscarmellose sodium are mainly used to enhance the dissolution and stability of entacapone 
. It will be particularly interesting if they are active against the growth of M.tuberculosis
, as they are commonly used food and drug additives. We have observed that a higher concentration of pure entacapone than that which is present in Comtan is required to achieve the same rate of inhibition in the InhA assay. Additional experiments such as X-ray crystallography and mass spectroscopy need to be conducted in order to investigate the precise mechanisms of action of Comtan and entacapone against M.tuberculosis
Although we have demonstrated that Comtan is active against M.tuberculosis in vitro
, further studies are required in order to transform it into an anti-tubercular therapeutic. The active component of entacapone in Comtan has an MIC99
of approximately 80 µg/ml (262 µM), and an estimated IC50
value for InhA inhibition of above 80 µM. However, it exhibits very low cellular toxicity, with no effect on neuroblastoma cell lines that provide an in vitro
model for high throughput toxicity screening 
at concentrations of up to 500 µM 
, thus making the concentration required for M.tuberculosis
inhibition not unreasonable for a lead compound.
The possibility that entacapone inhibits other enzymes besides InhA cannot be excluded. From our initial studies of the ligand binding site similarity network in the M.tuberculosis
structural genome, InhA is one of the most promiscuous proteins, having a similar ligand binding site to more than 20 other enzymes 
. This implies that an InhA inhibitor can potentially interact with multiple targets. If it is proven in future studies that entacapone can inhibit multiple targets simultaneously, the potential of entacapone as an anti-tubercular drug is even more promising, as “dirty” drugs lessen the likelihood of emergent resistance and higher clinical efficacy than exquisitely selective drugs 
A further challenge is transforming entacapone into a nanomolar inhibitor without impacting its ADME/Tox profile. A series of direct InhA inhibitors with IC50
's ranging from 1 nM to greater than 100 µM are available in the RCSB Protein Data Bank (PDB) 
. Thus it is possible to build reliable 3D QSAR models in order to guide the lead optimization process. Since entacapone has been in clinical use for many years, the accumulated knowledge of its pharmacokinetics and pharmacodynamics will be invaluable in predicting ADME/Tox properties of the compound and its derivatives.
The continuing emergence of M.tuberculosis
strains resistant to all existing, affordable drug treatments means that the development of novel, effective and inexpensive drugs is an urgent priority 
. Our chemical systems biology approach to drug discovery revealed that Comtan, with the active component entacapone, shows potential for use as an anti-tubercular drug. Entacapone may adopt different inhibition mechanisms from the first- and second-line drugs that result in MDR and XDR M.tuberculosis
strains. Moreover, it has an excellent safety profile with few side effects, and is commercially available. Therefore, entacapone can potentially be used as a lead compound to develop a new class of anti-tubercular drugs.
By integrating techniques from ligand binding site similarity, small molecule similarity and protein-ligand docking, our chemical systems biology approach is able to model protein-ligand interaction networks on a proteome-wide scale. The systematic use of small molecules to probe biological systems will provide us with valuable clues as to the molecular basis of cellular functions, and at the same time it will shift the conventional one-target-one-drug discovery process to a new multi-target-multi-drug paradigm.