Complexes of Trypanosoma cruzi Sterol 14α-Demethylase (CYP51) with Two Pyridine-based Drug Candidates for Chagas Disease

Abstract

Background: Two pyridine derivatives were identified as promising drug candidates in animal models of Chagas disease. Results: They were tested as sterol 14␣-demethylase (CYP51) inhibitors, and x-ray co-structures with T. cruzi CYP51 were determined. Conclusion:The structures explain the potency and selectivity of the compound. Significance: Structural information contributes to a better understanding of P450 inhibition and will facilitate rational design of pathogen-specific drugs.

Human sterol 14α-demethylase as a target for anticancer chemotherapy: towards structure-aided drug design

“…The apparent dissociation constants of the enzyme-substrate complex (K d ) were calculated in Prism 6 software (GraphPad, La Jolla, CA) by fitting the data for the substrate-induced absorbance changes in the difference spectra (A 390 -A 420 ) versus substrate concentration to a one sitetotal binding equation (binding-saturation). Inhibitor binding was monitored as a "type II" spectral response (red shift in the Soret band maximum from 417 to 421-427 nm) (54) reflecting coordination of the basic heterocyclic nitrogen to the P450 heme iron (55). Difference spectra were generated by recording the P450 absorbance in a sample cuvette (5 cm optical path length) versus the absorbance in a reference cuvette, both containing the same amount of the protein.…”

“…In our search for new inhibitors of CYP51s from human pathogens, we concomitantly conducted the control experiments evaluating the inhibitory effects of these compounds on the activity of human CYP51, e.g (51,55,57,60,61). A broad variety of structurally different molecular chemotypes …”

Clinical Candidate VT-1161's Antiparasitic Effect In Vitro , Activity in a Murine Model of Chagas Disease, and Structural Characterization in Complex with the Target Enzyme CYP51 from Trypanosoma cruzi

“…For crystallographic experiments, the T. cruzi CYP51 was truncated to replace the 30-amino-acid membrane anchor sequence at the N terminus (up to residue P31) with the more polar 5-amino-acid sequence fragment MAKKT (32) and purified in three steps, including anion-exchange chromatography on DEAE-Sepharose (GE Healthcare), affinity chromatography on Ni 2ϩ -NTA agarose, and cation-exchange chromatography on the CM Sepharose Fast Flow cation exchanger. Complexes with VT-1161 were obtained by saturating the protein with the inhibitor during the last step of purification (33) by adding a 20 mM stock solution of VT-1161 in dimethyl sulfoxide (DMSO) to the washing and elution buffers at a final concentration of 10 M.…”

“…P450 concentrations were estimated from the Soret band intensity using an absolute molar extinction coefficient ε 417 of 117 mM Ϫ1 cm Ϫ1 (27) or a difference molar extinction coefficient ⌬ ε(450 -490) of 91 mM Ϫ1 cm Ϫ1 for the reduced carbon monoxide difference spectra (34). Titrations with VT-1161 were carried out at a 0.3 M P450 concentration in 5-cm-optical-path-length cuvettes, with ligand binding being monitored as a type II spectral response that reflects the coordination of a basic heterocyclic nitrogen to the P450 heme iron (a red shift in the Soret band maximum from 417 nm to 421-427 nm [35], depending on the basicity of the coordinating nitrogen [33]). Difference spectra were generated by recording the absorbance of P450 in a sample cuvette versus the absorbance in a reference cuvette, both of which contained the same amount of the protein.…”

“…Aliquots of VT-1161 dissolved in DMSO were added to the sample cuvette over a concentration range of 0.1 to 1.0 M, with each titration step being a concentration of 0.1 M. At each step, the corresponding volume of DMSO was added to the reference cuvette. The apparent dissociation constants (K d s) of the CYP51-VT-1161 complex were calculated in GraphPad Prism (version 6) software (GraphPad, La Jolla, CA) by fitting the data for the ligand-induced changes in the absorbance of the difference spectra [⌬(A max Ϫ A min ), where A max is the maximum absorbance and A min is the minimum absorbance] versus the ligand concentration to quadratic equation 1 (tightbinding ligands [33])…”