Novel chemotherapeutics for treating multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (MTB) are required to combat the spread of tuberculosis, a disease that kills more than 2 million people annually. Using structure-based drug design, we have developed a series of alkyl diphenyl ethers that are uncompetitive inhibitors of InhA, the enoyl reductase enzyme in the MTB fatty acid biosynthesis pathway. The most potent compound has a Ki' value of 1 nM for InhA and MIC99 values of 2-3 microg mL(-1) (6-10 microM) for both drug-sensitive and drug-resistant strains of MTB. Overexpression of InhA in MTB results in a 9-12-fold increase in MIC99, consistent with the belief that these compounds target InhA within the cell. In addition, transcriptional response studies reveal that the alkyl diphenyl ethers fail to upregulate a putative efflux pump and aromatic dioxygenase, detoxification mechanisms that are triggered by the lead compound triclosan. These diphenyl ether-based InhA inhibitors do not require activation by the mycobacterial KatG enzyme, thereby circumventing the normal mechanism of resistance to the front line drug isoniazid (INH) and thus accounting for their activity against INH-resistant strains of MTB.

To explore the molecular basis for the picomolar affinity of triclosan for FabI, the enoyl reductase enzyme from the type II fatty acid biosynthesis pathway in Escherichia coli, an SAR study has been conducted using a series of triclosan analogues. Triclosan (1) is a slow, tight-binding inhibitor of FabI, interacting specifically with the E.NAD(+) form of the enzyme with a K(1) value of 7 pM. In contrast, 2-phenoxyphenol (2) binds with equal affinity to the E.NAD(+) (K(1) = 0.5 microM) and E.NADH (K(2) = 0.4 microM) forms of the enzyme and lacks the slow-binding step observed for triclosan. Thus, removal of the three triclosan chlorine atoms reduces the affinity of the inhibitor for FabI by 70,000-fold and removes the preference for the E.NAD(+) FabI complex. 5-Chloro-2-phenoxyphenol (3) is a slow, tight-binding inhibitor of FabI and binds to the E.NAD(+) form of the enzyme (K(1) = 1.1 pM) 7-fold more tightly than triclosan. Thus, while the two ring B chlorine atoms are not required for FabI inhibition, replacement of the ring A chlorine increases binding affinity by 450,000-fold. Given this remarkable observation, the SAR study was extended to the 5-fluoro-2-phenoxyphenol (4) and 5-methyl-2-phenoxyphenol (5) analogues to further explore the role of the ring A substituent. While both 4 and 5 are slow, tight-binding inhibitors, they bind substantially less tightly to FabI than triclosan. Compound 4 binds to both E.NAD(+) and E.NADH forms of the enzyme with K(1) and K(2) values of 3.2 and 240 nM, respectively, whereas compound 5 binds exclusively to the E.NADH enzyme complex with a K(2) value of 7.2 nM. Thus, the ring A substituent is absolutely required for slow, tight-binding inhibition. In addition, pK(a) measurements coupled with simple electrostatic calculations suggest that the interaction of the ring A substituent with F203 is a major factor in governing the affinity of analogues 3-5 for the FabI complex containing the oxidized form of the cofactor.

Approximately one-third of the world's population carries Staphylococcus aureus. The recent emergence of extreme drug resistant strains that are resistant to the "antibiotic of last resort", vancomycin, has caused a further increase in the pressing need to discover new drugs against this organism. The S. aureus enoyl reductase, saFabI, is a validated target for drug discovery. To drive the development of potent and selective saFabI inhibitors, we have studied the mechanism of the enzyme and analyzed the interaction of saFabI with triclosan and two related diphenyl ether inhibitors. Results from kinetic assays reveal that saFabI is NADPH-dependent, and prefers acyl carrier protein substrates carrying fatty acids with long acyl chains. On the basis of product inhibition studies, we propose that the reaction proceeds via an ordered sequential ternary complex, with the ACP substrate binding first, followed by NADPH. The interaction of NADPH with the enzyme has been further explored by site-directed mutagenesis, and residues R40 and K41 have been shown to be involved in determining the specificity of the enzyme for NADPH compared to NADH. Finally, in preliminary inhibition studies, we have shown that triclosan, 5-ethyl-2-phenoxyphenol (EPP), and 5-chloro-2-phenoxyphenol (CPP) are all nanomolar slow-onset inhibitors of saFabI. These compounds inhibit the growth of S. aureus with MIC values of 0.03-0.06 microg/mL. Upon selection for resistance, three novel safabI mutations, A95V, I193S, and F204S, were identified. Strains containing these mutations had MIC values approximately 100-fold larger than that of the wild-type strain, whereas the purified mutant enzymes had K i values 5-3000-fold larger than that of wild-type saFabI. The increase in both MIC and K i values caused by the mutations supports the proposal that saFabI is the intracellular target for the diphenyl ether-based inhibitors.

No abstract

In earlier work structure-based design studies resulted in the discovery of alkyl substituted diphenyl ether inhibitors of InhA, the enoyl reductase from Mycobacterium tuberculosis. Compounds such as 5-hexyl-2-phenoxyphenol 19 are nM inhibitors of InhA and inhibit the growth of both sensitive and isoniazid-resistant strains of Mycobacterium tuberculosis with MIC 90 values of 1-2 µg/mL. However, despite their promising in vitro activity, these compounds have ClogP values of over 5. In efforts to reduce the lipophilicity of the compounds, and enhance potentially enhance compound bioavailability, a series of B ring analogues of 19 were synthesized that contained either heterocylic nitrogen rings or phenyl rings having amino, nitro, amide or piperazine functions. Compounds 3c, 3e and 14a show comparable MIC 90 values to that of 19, but have improved ClogP values. KeywordsTuberculosis; Diaryl ether; enoyl reductase; mycolic acids; InhA; isoniazid Tuberculosis (TB) is responsible for more than 1.6 million deaths per annum with 8.8 million new cases being reported each year. These numbers make TB one of the leading infectious causes of death, eclipsed only by AIDS. In addition, according to the World Health Organization, the number of multi-drug-resistant and extensively drug-resistant TB cases is growing with almost a half million new cases being reported each year. Therefore, there is an urgent need to develop novel TB chemotherapeutic agents. 1 *Corresponding authors, PJT: Tel.: (631) 632 7907; Email: peter.tonge@sunysb.edu, RAS: Tel.: (970) 491 1925; Email: richard.slayden@colostate.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. In this study we describe two classes of molecules in which alterations have been made to the diphenyl ether 'B' ring. In one series of compounds we have replaced the B ring with isosteric heterocycles that incorporate nitrogen atoms within the ring, thereby causing little steric perturbation to the overall structure of the molecule (Scheme 2). The second series of compounds have nitro, amino, amide and piperazino functionalities incorporated at the ortho, meta, or para positions of the B ring (Scheme 3 and Scheme 4). This second series of compounds was synthesized not only to improve solubility but also to systematically identify positions on the B ring which could be substituted without diminishing biological activity. NIH Public AccessThe synthesis of the heterocyclic diaryl ether compounds was initiated either by nucleophilic aromatic substitution or by Buchwald-Hartwig cross-coupling of the appropriate nitrogen heterocycle with 4-b...