The catalytic activity and structure of the lipid metabolizing CYP124 cytochrome P450 enzyme from Mycobacterium marinum.

The CYP124 family of cytochrome P450 enzymes, as exemplified by CYP124A1 from Mycobacterium tuberculosis, is involved in the metabolism of methyl branched lipids and cholesterol derivatives. The equivalent enzyme from Mycobacterium marinum was investigated to compare the degree of functional conservation between members of this CYP family from closely related bacteria. We compared substrate binding of each CYP124 enzyme using UV-vis spectroscopy and the catalytic oxidation of methyl branched lipids, terpenes and cholesterol derivatives was investigated. The CYP124 enzyme from M. tuberculosis displayed a larger shift to the ferric high-spin state on binding cholesterol derivatives compared to the equivalent enzyme from M. marinum. The biggest difference was observed with cholesteryl sulfate which induced distinct UV-vis spectra in each CYP124 enzyme. The selectivity for oxidation at the ω-carbon of a branched chain was maintained for all substrates, except cholesteryl sulfate which was not oxidized by either enzyme. The CYP124A1 enzyme from M. marinum, in combination with farnesol and farnesyl acetate, was structurally characterized by X-ray crystallography. These ligand-bound structures of the CYP124 enzyme revealed that the polar component of the substrates bound in a different manner to that of phytanic acid in the structure of CYP124A1 from M. tuberculosis. However, closer to the heme the structures were similar providing an explanation for the high selectivity of the enzyme for terminal methyl C-H bond oxidation. The work here demonstrates that there were differences in the biochemistry of the CYP124 enzymes from these closely related bacteria.

Design, synthesis and molecular modeling study of certain VEGFR-2 inhibitors based on thienopyrimidne scaffold as cancer targeting agents.

Different series of novel thieno [2,3-d]pyrimidine derivative (9a-d,10a-f,l,m and 15a-m) were designed, synthesized and evaluated for their ability to in vitro inhibit VEGFR-2 enzyme. Also, the cytotoxicity of the final compounds was tested against a panel of 60 different human cancer cell lines by NCI. The VEGFR-2 enzyme inhibitory results revealed that compounds 10d, 15d and 15 g are among the most active inhibitors with IC50 values of 2.5, 5.48 and 2.27 µM respectively, while compound 10a remarkably showed the highest cell growth inhibition with mean growth inhibition (GI) percent of 31.57%. It exhibited broad spectrum anti-proliferative activity against several NCI cell lines specifically on human breast cancer (T7-47D) and renal cancer (A498) cell lines of 85.5% and 77.65% inhibition respectively. To investigate the mechanistic aspects underlying the activity, further biological studies like flow cytometry cell cycle together with caspase-3 colorimetric assays were carried on compound 10a. Flow cytometric analysis on both MCV-7 and PC-3 cancer cells revealed that it induced cell-cycle arrest in the G0-G1phase and reinforced apoptosis via activation of caspase-3. Furthermore, molecular modeling studies have been carried out to gain further understanding of the binding mode in the active site of VEGFR-2 enzyme and predict pharmacokinetic properties of all the synthesized inhibitors.

Medicinal Attributes of Thienopyrimidine Based Scaffold Targeting Tyrosine Kinases and Their Potential Anticancer Activities.

Thienopyrimidines (TP), comprising a thiophene ring fused with pyrimidine, are famous bioisosteres to purines, an essential part of the human metabolome. This scaffold has become an interesting structural element in the development of pharmaceutical compounds, due to their wide spectrum applications as cytotoxic agents against different types of human cancer cell lines, cGMP phosphodiesterase inhibitors, and anti-viral, anti-inflammatory, and anti-microbial agents. The structural similarity of this scaffold with adenine made it an excellent moiety to be used in the design of kinase inhibitors. This review focuses on the chemistry of thienopyrimidine derivatives, their potential activities against various kinases, and their structure-activity relationship studies.

Synthesis of steroidal inhibitors for Mycobacterium tuberculosis.

Oxidised derivatives of cholesterol have been shown to inhibit the growth of Mycobacterium tuberculosis (Mtb). The bacteriostatic activity of these compounds has been attributed to their inhibition of CYP125A1 and CYP142A1, two metabolically critical cytochromes P450 that initiate degradation of the sterol side chain. Here, we synthesise and characterise an extensive library of 28 cholesterol derivatives to develop a structure-activity relationship for this class of inhibitors. The candidate compounds were evaluated for MIC with virulent Mtb and in binding studies with CYP125A1 and CYP142A1 from Mtb.

The oxidation of steroid derivatives by the CYP125A6 and CYP125A7 enzymes from Mycobacterium marinum.

The members of the bacterial cytochrome P450 (CYP) monooxygenase family CYP125, catalyze the oxidation of steroid derivatives including cholesterol and phytosterols, as the initial activating step in their catabolism. However, several bacterial species contain multiple genes encoding CYP125 enzymes and other CYP enzymes which catalyze cholesterol/cholest-4-en-3-one hydroxylation. An important question is why these bacterium have more than one enzyme with overlapping substrate ranges capable of catalyzing the terminal oxidation of the alkyl chain of these sterols. To further understand the role of these enzymes we investigated CYP125A6 and CYP125A7 from Mycobacterium marinum with various cholesterol analogues. These have modifications on the A and B rings of the steroid and we assessed the substrate binding and catalytic activity of these with each enzyme. CYP125A7 gave similar results to those reported for the CYP125A1 enzyme from M. tuberculosis. Differences in the substrate binding and catalytic activity with the cholesterol analogues were observed with CYP125A6. For example, while cholesteryl sulfate could bind to both enzymes it was only oxidized by CYP125A6 and not by CYP125A7. CYP125A6 generated higher levels of metabolites with the majority of C-3 and C-7 substituted cholesterol analogues such 7-ketocholesterol. However, 5α-cholestan-3ß-ol was only oxidized by CYP125A7 enzyme. The cholest-4-en-3-one and 7-ketocholesterol-bound forms of the CYP125A6 and CYP125A7 enzymes were modelled using AlphaFold. The structural models highlighted differences in the binding modes of the steroid derivatives within the same enzyme. Significant changes in the binding mode of the steroids between these CYP125 enzymes and other bacterial cholesterol oxidizing enzymes, CYP142A3 and CYP124A1, were also seen. Despite this, all these models predicted the selectivity for terminal methyl hydroxylation, in agreement with the experimental data.

The binding of nitrogen-donor ligands to the ferric and ferrous forms of cytochrome P450 enzymes.

The cytochrome P450 superfamily of heme-thiolate monooxygenase enzymes can catalyse various oxidation reactions. The addition of a substrate or an inhibitor ligand induces changes in the absorption spectrum of these enzymes and UV-visible (UV-vis) absorbance spectroscopy is the most common and readily available technique used to interrogate their heme and active site environment. Nitrogen-containing ligands can inhibit the catalytic cycle of heme enzymes by interacting with the heme. Here we evaluate the binding of imidazole and pyridine-based ligands to the ferric and ferrous forms of a selection of bacterial cytochrome P450 enzymes using UV-visible absorbance spectroscopy. The majority of these ligands interact with the heme as one would expect for type II nitrogen directly coordinated to a ferric heme-thiolate species. However, the spectroscopic changes observed in the ligand-bound ferrous forms indicated differences in the heme environment across these P450 enzyme/ligand combinations. Multiple species were observed in the UV-vis spectra of the ferrous ligand-bound P450s. None of the enzymes gave rise to the isolation of a single species with a Soret band at ∼442-447 nm, indicative of a 6-coordinate ferrous thiolate species with a nitrogen-donor ligand. A ferrous species with Soret band at ∼427 nm coupled with an α-band of increased intensity was observed with the imidazole ligands. With some enzyme-ligand combinations reduction resulted in breaking of the iron­nitrogen bond yielding a 5-coordinate high-spin ferrous species. In other instances, the ferrous form was readily oxidised back to the ferric form on addition of the ligand.

The oxidation of cholesterol derivatives by the CYP124 and CYP142 enzymes from Mycobacterium marinum.

The CYP124 and CYP142 families of bacterial cytochrome P450 monooxygenases (CYPs), catalyze the oxidation of methyl branched lipids, including cholesterol, as one of the initial activating steps in their catabolism. Both enzymes are reported to supplement the CYP125 family of P450 enzymes. These CYP125 enzymes are found in the same bacteria, and are the primary cholesterol/cholest-4-en-3-one metabolizing enzymes. To further understand the role of the CYP124 and CYP142 cytochrome P450s we investigated the Mycobacterium marinum enzymes, MmarCYP124A1 and CYP142A3, with various cholesterol analogs with modifications on the A and B rings of the steroid. We assessed the substrate binding and catalytic activity of each enzyme. Neither enzyme could bind or oxidize cholesteryl acetate or 3,5-cholestadiene, which have modifications at the C3 hydroxyl moiety of cholesterol. The CYP142 enzyme was better able to accommodate and oxidize cholesterol analogs which have changes on the A/B rings including cholesterol-5α,6α-epoxide and diastereomers of 5-cholestan-3-ol. The CYP124 enzyme was more tolerant of changes at C7 of the cholesterol B ring, e.g., 7-ketocholesterol than in the A ring. The selectivity for oxidation at the ω-carbon of a branched chain was observed in all steroids that were oxidized. The 7-ketocholesterol-bound MmarCYP124A1 enzyme from M. marinum, was structurally characterized by X-ray crystallography to 1.81 Å resolution. The 7-ketocholesterol-bound X-ray crystal structure of the MmarCYP124A1 enzyme revealed that the substrate binding mode of this cholesterol derivative was altered compared to those observed with other non-steroidal ligands. The structure provided an explanation for the selectivity of the enzyme for terminal methyl hydroxylation.

The bacterial cytochrome P450 (CYP) CYP125 enzymes can competitively oxidise sitosterol in the presence of cholesterol.

Cholesterol catabolism is an important survival mechanism for the pathogenic Mycobacterium tuberculosis. Various other mycobacteria degrade not only cholesterol but plant sterols such as sitosterol and campesterol. In this work we demonstrate that the cytochrome P450 (CYP) CYP125 enzyme family is capable of sitosterol and campesterol side-chain oxidation and activation in these bacteria. We also show that the CYP142 and CYP124 cholesterol hydroxylating enzyme families are significantly less active for sitosterol hydroxylation compared to CYP125 enzymes.

The Structures of the Steroid Binding CYP142 Cytochrome P450 Enzymes from Mycobacterium ulcerans and Mycobacterium marinum.

The steroid binding CYP142 cytochrome P450 enzymes of Mycobacterium species are involved in the metabolism of cholesterol and its derivatives. The equivalent enzyme from Mycobacterium ulcerans was studied to compare the degree of functional conservation between members of this CYP family. We compared substrate binding of the CYP142A3 enzymes of M. ulcerans and M. marinum and CYP142A1 from M. tuberculosis using UV-vis spectroscopy. The catalytic oxidation of cholesterol derivatives by all three enzymes was undertaken. Both CYP142A3 enzymes were structurally characterized by X-ray crystallography. The amino acid sequences of the CYP142A3 enzymes are more similar to CYP142A1 from M. tuberculosis than CYP142A2 from Mycolicibacterium smegmatis. Both CYP142A3 enzymes have substrate binding properties, which are more resemblant to CYP142A1 than CYP142A2. The cholest-4-en-3-one-bound X-ray crystal structure of both CYP142A3 enzymes were determined at a resolution of <1.8 Å, revealing the substrate binding mode at a high level of detail. The structures of the cholest-4-en-3-one binding CYP142 enzymes from M. ulcerans and M. marinum demonstrate how the steroid binds in the active site of these enzymes. They provide an explanation for the high selectivity of the enzyme for terminal methyl C-H bond oxidation to form 26-hydroxy derivatives. These enzymes in pathogenic Mycobacterium species are candidates for inhibition. The work here demonstrates that similar drug molecules could target these CYP142 enzymes from different species in order to combat Buruli ulcer or tuberculosis.

A comparison of steroid and lipid binding cytochrome P450s from Mycobacterium marinum and Mycobacterium tuberculosis.

The steroid lipid binding cytochrome P450 (CYP) enzymes of Mycobacterium tuberculosis are essential for organism survival through metabolism of cholesterol and its derivatives. The counterparts to these enzymes from Mycobacterium marinum were studied to determine the degree of functional conservation between them. Spectroscopic analyses of substrate and inhibitor binding for the four M. marinum enzymes CYP125A6, CYP125A7, CYP142A3 and CYP124A1 were performed and compared to the equivalent enzymes of M. tuberculosis. The sequence of CYP125A7 from M. marinum was more similar to CYP125A1 from M. tuberculosis than CYP125A6 but both showed differences in the resting heme spin state and in the binding modes and affinities of certain azole inhibitors. CYP125A7 did not show a significant Type II inhibitor-like shift with any of the azoles tested. CYP142A3 bound a similar range of steroids and inhibitors to CYP142A1. However, there were some differences in the extent of the Type I shifts to the high-spin form with steroids and a higher affinity for the azole inhibitors compared to CYP142A1. The two CYP124 enzymes had similar substrate binding properties. M. marinum CYP124 was characterised by X-ray crystallography and displayed strong conservation of active site residues, except near the region where the carboxylate terminus of the phytanic acid substrate would be bound. As these enzymes in M. tuberculosis have been identified as candidates for inhibition the data here demonstrates that alternative strategies for inhibitor design may be required to target CYP family members from distinct pathogenic Mycobacterium species or other bacteria.