Fragment Profiling Approach to Inhibitors of the Orphan M. tuberculosis P450 CYP144A1

Abstract

Similarity between the ligand binding profiles of enzymes may aid functional characterization and be of greater relevance to inhibitor development than sequence similarity or structural homology. Fragment screening is an efficient approach for characterization of the ligand binding profile of an enzyme and has been applied here to study the family of cytochrome P450 enzymes (P450s) expressed by Mycobacterium tuberculosis (Mtb). The Mtb P450s have important roles in bacterial virulence, survival, and pathogenicity. Comparing the fragment profiles of seven of these enzymes revealed that P450s which share a similar biological function have significantly similar fragment profiles, whereas functionally unrelated or orphan P450s exhibit distinct ligand binding properties, despite overall high structural homology. Chemical structures that exhibit promiscuous binding between enzymes have been identified, as have selective fragments that could provide leads for inhibitor development. The similarity between the fragment binding profiles of the orphan enzyme CYP144A1 and CYP121A1, a characterized enzyme that is important for Mtb viability, provides a case study illustrating the subsequent identification of novel CYP144A1 ligands. The different binding modes of these compounds to CYP144A1 provide insight into structural and dynamic aspects of the enzyme, possible biological function, and provide the opportunity to develop inhibitors. Expanding this fragment profiling approach to include a greater number of functionally characterized and orphan proteins may provide a valuable resource for understanding enzyme-ligand interactions.

Spectroscopic studies of the cytochrome P450 reaction mechanisms

“…High sensitivity and simple instrumentation, with no special requirements for experimental conditions, make UV-VIS spectroscopy a method of choice for thermodynamic and kinetic studies of cytochromes P450, as well as when employing cryogenic methods, [38,41,59,60,76,95] high pressure, [96,97,98,99] flash photolysis and photoacoustic methods in nanosecond range, [100,101,102,103,104] etc. Optical absorption spectra can be used for monitoring binding mode and for comparing substrate analogs, [105,106,107] or binding of the same substrate to single point mutants. [7,23,108,109] In addition, spectral changes can be used for direct observation of allosteric effects in substrate binding.…”

“…Other examples of NMR application to studies of substrate binding and for structural evaluation of substrate positioning and dynamics in P450 catalytic site have been described. [106,191,192]…”

Potential drug targets in the Mycobacterium tuberculosis cytochrome P450 system

“…A fragment profiling approach was pursued in efforts to obtain a clue to the native substrate, but the results did not yield a specific identity. However, they may be useful if the further development of inhibitors for the enzyme is warranted (57)…”

Bioinformatics in translational drug discovery

“…For example, the family of cytochrome P450 enzymes (P450s) play important roles in Mycobacterium tuberculosis . Using fragment screening, Kavanagh et al [86] identified similarities in the ligand-binding profile of CYP121A1, which is known to be important for M . tuberculosis viability, and the orphan enzyme CYP144A1.…”

“…tuberculosis viability, and the orphan enzyme CYP144A1. An assessment of the similarities and differences in binding between the two enzymes provides insight into both the function of the enzyme and potential inhibitors [86].…”