A Promiscuous Bacterial P450: The Unparalleled Diversity of BM3 in Pharmaceutical Metabolism

CYP102A1 (BM3) is a catalytically self-sufficient flavocytochrome fusion protein isolated from Bacillus megaterium, which displays similar metabolic capabilities to many drug-metabolizing human P450 isoforms. BM3′s high catalytic efficiency, ease of production and malleable active site makes the enzyme a desirable tool in the production of small molecule metabolites, especially for compounds that exhibit drug-like chemical properties. The engineering of select key residues within the BM3 active site vastly expands the catalytic repertoire, generating variants which can perform a range of modifications. This provides an attractive alternative route to the production of valuable compounds that are often laborious to synthesize via traditional organic means. Exten-sive studies have been conducted with the aim of engineering BM3 to expand metabolite pro-duction towards a comprehensive range of drug-like compounds, with many key examples found both in the literature and in the wider industrial bioproduction setting of desirable oxy-metabolite production by both wild-type BM3 and related variants. This review covers the past and current research on the engineering of BM3 to produce drug metabolites and highlights its crucial role in the future of biosynthetic pharmaceutical production

A Promiscuous Bacterial P450: The Unparalleled Diversity of BM3 in Pharmaceutical Metabolism

From MDPI via Jisc Publications RouterHistory: accepted 2021-10-12, pub-electronic 2021-10-21Publication status: PublishedFunder: Biotechnology and Biological Sciences Research Council; Grant(s): BB/M011208/1CYP102A1 (BM3) is a catalytically self-sufficient flavocytochrome fusion protein isolated from Bacillus megaterium, which displays similar metabolic capabilities to many drug-metabolizing human P450 isoforms. BM3′s high catalytic efficiency, ease of production and malleable active site makes the enzyme a desirable tool in the production of small molecule metabolites, especially for compounds that exhibit drug-like chemical properties. The engineering of select key residues within the BM3 active site vastly expands the catalytic repertoire, generating variants which can perform a range of modifications. This provides an attractive alternative route to the production of valuable compounds that are often laborious to synthesize via traditional organic means. Extensive studies have been conducted with the aim of engineering BM3 to expand metabolite production towards a comprehensive range of drug-like compounds, with many key examples found both in the literature and in the wider industrial bioproduction setting of desirable oxy-metabolite production by both wild-type BM3 and related variants. This review covers the past and current research on the engineering of BM3 to produce drug metabolites and highlights its crucial role in the future of biosynthetic pharmaceutical production

Synthesis and biological evaluation of novel cYY analogues targeting Mycobacterium tuberculosis CYP121A1

The rise in multidrug resistant (MDR) cases of tuberculosis (TB) has led to the need for the development of TB drugs with different mechanisms of action. The genome sequence of Mycobacterium tuberculosis (Mtb) revealed twenty different genes coding for cytochrome P450s. CYP121A1 catalyzes a C-C crosslinking reaction of dicyclotyrosine (cYY) producing mycocyclosin and current research suggests that either mycocyclosin is essential or the overproduction of cYY is toxic to Mtb. A series of 1,4-dibenzyl-2-imidazol-1-yl-methylpiperazine derivatives were designed and synthesised as cYY mimics. The derivatives substituted in the 4-position of the phenyl rings with halides or alkyl group showed promising antimycobacterial activity (MIC 6.25 μg/mL), with the more lipophilic branched alkyl derivatives displaying optimal binding affinity with CYP121A1 (iPr KD = 1.6 μM; tBu KD = 1.2 μM). Computational studies revealed two possible binding modes within the CYP121A1 active site both of which would effectively block cYY from binding

Frequentist Interpretation of Probability

Three series of azole piperazine derivatives that mimic dicyclotyrosine (cYY), the natural substrate of the essential Mycobacterium tuberculosis cytochrome P450 CYP121A1, were prepared and evaluated for binding affinity and inhibitory activity (MIC) against M. tuberculosis. Series A replaces one phenol group of cYY with a C3-imidazole moiety, series B includes a keto group on the hydrocarbon chain preceding the series A imidazole, whilst series C explores replacing the keto group of the piperidone ring of cYY with a CH2-imidazole or CH2-triazole moiety to enhance binding interaction with the heme of CYP121A1. The series displayed moderate to weak type II binding affinity for CYP121A1, with the exception of series B 10a, which displayed mixed type I binding. Of the three series, series C imidazole derivatives showed the best, although modest, inhibitory activity against M. tuberculosis (17d MIC = 12.5 μg/mL, 17a 50 μg/mL). Crystal structures were determined for CYP121A1 bound to series A compounds 6a and 6b that show the imidazole groups positioned directly above the haem iron with binding between the haem iron and imidazole nitrogen of both compounds at a distance of 2.2 Å. A model generated from a 1.5 Å crystal structure of CYP121A1 in complex with compound 10a showed different binding modes in agreement with the heterogeneous binding observed. Although the crystal structures of 6a and 6b would indicate binding with CYP121A1, the binding assays themselves did not allow confirmation of CYP121A1 as the target

Structural characterization of CYP144A1 - a cytochrome P450 enzyme expressed from alternative transcripts in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) causes the disease tuberculosis (TB). The virulent Mtb H37Rv strain encodes 20 cytochrome P450 (CYP) enzymes, many of which are implicated in Mtb survival and pathogenicity in the human host. Bioinformatics analysis revealed that CYP144A1 is retained exclusively within the Mycobacterium genus, particularly in species causing human and animal disease. Transcriptomic annotation revealed two possible CYP144A1 start codons, leading to expression of (i) a "full-length" 434 amino acid version (CYP144A1-FLV) and (ii) a "truncated" 404 amino acid version (CYP144A1-TRV). Computational analysis predicted that the extended N-terminal region of CYP144A1-FLV is largely unstructured. CYP144A1 FLV and TRV forms were purified in heme-bound states. Mass spectrometry confirmed production of intact, His6-tagged forms of CYP144A1-FLV and -TRV, with EPR demonstrating cysteine thiolate coordination of heme iron in both cases. Hydrodynamic analysis indicated that both CYP144A1 forms are monomeric. CYP144A1-TRV was crystallized and the first structure of a CYP144 family P450 protein determined. CYP144A1-TRV has an open structure primed for substrate binding, with a large active site cavity. Our data provide the first evidence that Mtb produces two different forms of CYP144A1 from alternative transcripts, with CYP144A1-TRV generated from a leaderless transcript lacking a 5'-untranslated region and Shine-Dalgarno ribosome binding site

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

The essential enzyme CYP121 is a target for drug development against antibiotic resistant strains of Mycobacterium tuberculosis. A triazol-1-yl phenol fragment 1 was identified to bind to CYP121 using a cascade of biophysical assays. Synthetic merging and optimization of 1 produced a 100-fold improvement in binding affinity, yielding lead compound 2 (KD = 15 μM). Deconstruction of 2 into its component retrofragments allowed the group efficiency of structural motifs to be assessed, the identification of more LE scaffolds for optimization and highlighted binding affinity hotspots. Structure-guided addition of a metal-binding pharmacophore onto LE retrofragment scaffolds produced low nanomolar (KD = 15 nM) CYP121 ligands. Elaboration of these compounds to target binding hotspots in the distal active site afforded compounds with excellent selectivity against human drug-metabolizing P450s. Analysis of the factors governing ligand potency and selectivity using X-ray crystallography, UV-vis spectroscopy, and native mass spectrometry provides insight for subsequent drug development.MEK was supported by a Commonwealth (University of Cambridge) Scholarship awarded in conjunction with the Cambridge Commonwealth Trust and Cambridge Overseas Trust. AGC and KJM were supported by grants from the BBSRC (Grant No: BB/I019669/1 and BB/I019227/1). GGJ received funding from the Ogden Trust and the Isaac Newton Trust administered through the University of Cambridge Bursary Scheme. DSCH was supported by a Croucher Cambridge International Scholarship awarded in conjunction between the Croucher Foundation and the Cambridge Overseas Trust. SAH was supported by an Oliphant Cambridge Australia Scholarship (App No: 10132070) awarded by the Cambridge Commonwealth Trust. The contributions of LBM and LPSC were supported by funds from the Francis Crick Institute, which receives its core funding principally from Wellcome Trust, Cancer Research UK, and the UK Medical Research Council (to LPSC - MC_UP_A253_1111) and funds from FAPESP, CNPq and CAPES-PDSE (to LBM - 2011/21232-1, 140079/2013-0, 99999.003125/2014-09).This is the final version of the article. It first appeared from the American Chemical Society via http://dx.doi.org/10.1021/acs.jmedchem.6b0000

Characterisation of PduS, the pdu Metabolosome Corrin Reductase, and Evidence of Substructural Organisation within the Bacterial Microcompartment

PduS is a corrin reductase and is required for the reactivation of the cobalamin-dependent diol dehydratase. It is one component encoded within the large propanediol utilisation (pdu) operon, which is responsible for the catabolism of 1,2-propanediol within a self-assembled proteinaceous bacterial microcompartment. The enzyme is responsible for the reactivation of the cobalamin coenzyme required by the diol dehydratase. The gene for the cobalamin reductase from Citrobacter freundii (pduS) has been cloned to allow the protein to be overproduced recombinantly in E. coli with an N-terminal His-tag. Purified recombinant PduS is shown to be a flavoprotein with a non-covalently bound FMN that also contains two coupled [4Fe-4S] centres. It is an NADH-dependent flavin reductase that is able to mediate the one-electron reductions of cob(III)alamin to cob(II)alamin and cob(II)alamin to cob(I)alamin. The [4Fe-4S] centres are labile to oxygen and their presence affects the midpoint redox potential of flavin. Evidence is presented that PduS is able to bind cobalamin, which is inconsistent with the view that PduS is merely a flavin reductase. PduS is also shown to interact with one of the shell proteins of the metabolosome, PduT, which is also thought to contain an [Fe-S] cluster. PduS is shown to act as a corrin reductase and its interaction with a shell protein could allow for electron passage out of the bacterial microcompartment

Novel aryl substituted pyrazoles as small molecule inhibitors of cytochrome P450 CYP121A1: Synthesis and antimycobacterial evaluation

Three series of biarylpyrazole imidazole and triazoles are described, which vary in the linker between the biaryl pyrazole and imidazole/triazole group. The imidazole and triazole series with the short −CH2– linker displayed promising antimycobacterial activity, with the imidazole–CH2– series (7) showing low MIC values (6.25–25 μg/mL), which was also influenced by lipophilicity. Extending the linker to −C(O)NH(CH2)2– resulted in a loss of antimycobacterial activity. The binding affinity of the compounds with CYP121A1 was determined by UV–visible optical titrations with KD values of 2.63, 35.6, and 290 μM, respectively, for the tightest binding compounds 7e, 8b, and 13d from their respective series. Both binding affinity assays and docking studies of the CYP121A1 inhibitors suggest type II indirect binding through interstitial water molecules, with key binding residues Thr77, Val78, Val82, Val83, Met86, Ser237, Gln385, and Arg386, comparable with the binding interactions observed with fluconazole and the natural substrate dicyclotyrosine

Design and synthesis of imidazole and triazole pyrazoles as mycobacterium tuberculosis CYP121A1 inhibitors

The emergence of untreatable drug‐resistant strains of Mycobacterium tuberculosis is a major public health problem worldwide, and the identification of new efficient treatments is urgently needed. Mycobacterium tuberculosis cytochrome P450 CYP121A1 is a promising drug target for the treatment of tuberculosis owing to its essential role in mycobacterial growth. Using a rational approach, which includes molecular modelling studies, three series of azole pyrazole derivatives were designed through two synthetic pathways. The synthesized compounds were biologically evaluated for their inhibitory activity towards M. tuberculosis and their protein binding affinity (KD). Series 3 biarylpyrazole imidazole derivatives were the most effective with the isobutyl (10 f) and tert‐butyl (10 g) compounds displaying optimal activity (MIC 1.562 μg/mL, KD 0.22 μM (10 f) and 4.81 μM (10 g)). The spectroscopic data showed that all the synthesised compounds produced a type II red shift of the heme Soret band indicating either direct binding to heme iron or (where less extensive Soret shifts are observed) putative indirect binding via an interstitial water molecule. Evaluation of biological and physicochemical properties identified the following as requirements for activity: LogP >4, H‐bond acceptors/H‐bond donors 4/0, number of rotatable bonds 5–6, molecular volume >340 Å3, topological polar surface area <40 Å2