Biofragments: An Approach towards Predicting Protein Function Using Biologically Related Fragments and its Application to Mycobacterium tuberculosis CYP126

Abstract

We present a novel fragment-based approach that tackles some of the challenges for chemical biology of predicting protein function. The general approach, which we have termed biofragments, comprises two key stages. First, a biologically relevant fragment library (biofragment library) can be designed and constructed from known sets of substrate-like ligands for a protein class of interest. Second, the library can be screened for binding to a novel putative ligand-binding protein from the same or similar class, and the characterization of hits provides insight into the basis of ligand recognition, selectivity, and function at the substrate level. As a proof-of-concept, we applied the biofragments approach to the functionally uncharacterized Mycobacterium tuberculosis (Mtb) cytochrome P450 isoform, CYP126. This led to the development of a tailored CYP biofragment library with notable 3D characteristics and a significantly higher screening hit rate (14 %) than standard drug-like fragment libraries screened previously against Mtb CYP121 and 125 (4 % and 1 %, respectively). Biofragment hits were identified that make both substrate-like type-I and inhibitor-like type-II interactions with CYP126. A chemical-fingerprint-based substrate model was built from the hits and used to search a virtual TB metabolome, which led to the discovery that CYP126 has a strong preference for the recognition of aromatics and substrate-like type-I binding of chlorophenol moieties within the active site near the heme. Future catalytic analyses will be focused on assessing CYP126 for potential substrate oxidative dehalogenation.

Function, essentiality, and expression of cytochrome P450 enzymes and their cognate redox partners in Mycobacterium tuberculosis: are they drug targets?

“…CYP126A1 may have a related function/activity, as it shows a 37%, 36%, and 32% amino acid sequence homology with CYP124A1, CYP125A1, and CYP142A1, respectively (Ouellet et al 2010a ). CYP126A1 showed a strong preference for the binding of aromatics and chlorophenol moieties (Hudson et al 2014 ). In the presence of surrogate redox partners from Spinacea oleracea , CYP124A1, CYP142A1, and CYP125A1 oxidized the cholesterol side chain to carboxylic acid.…”

“…4a, b). HTS to find potent and highly selective inhibitors of CYP126A1 and CYP130A1 has been performed (Podust et al 2009; Hudson et al 2014). In addition, these studies aimed to identify substrates for the orphan CYP126A1 and CYP130A1, which would help to define their physiological roles.…”

Structural Characterization and Ligand/Inhibitor Identification Provide Functional Insights into the Mycobacterium tuberculosis Cytochrome P450 CYP126A1

“…In previous studies, we have taken a fragment-based approach to identify novel small molecule ligands for M. tuberculosis P450s, including CYP121A1, CYP125A1, and CYP126A1 ( 26 – 28 ). In this paper, we present detailed biochemical, spectroscopic, and structural data for CYP126A1, including studies of its binding to novel substrates and inhibitors identified from a library of ∼40,000 compounds structurally related to the World Drug Index ( 29 ).…”

Fragment-Based Approaches to the Development of Mycobacterium tuberculosis CYP121 Inhibitors

“…Plasmid vectors encoding N-terminal His 6 -tagged CYP125 (pET15b/ Rv3545c ) and CYP126 (pET15b/ Rv0778 ) were provided by Dr. Kirsty McLean (Manchester Institute of Biotechnology, University of Manchester). CYP125 7 and CYP126 27 , 68 proteins were expressed and purified in E. coli C41(DE3) cells according to the literature. CYP142, 53 CYP143, and CYP144-TRV 4 proteins were prepared according to the literature and provided by Dr. Kirsty McLean (Manchester Institute of Biotechnology, University of Manchester).…”