Galina Lepesheva - Academia.edu (original) (raw)
Papers by Galina Lepesheva
Bioorganic & Medicinal Chemistry Letters
Journal of Medicinal Chemistry
Tetrahedron Letters
The study and development of azole-based CYP51 inhibitors is an active area of research across di... more The study and development of azole-based CYP51 inhibitors is an active area of research across disciplines of biochemistry, pharmacology and infectious disease. Support of in vitro and in vivo studies require the development of robust asymmetric routes to single enantiomer products of this class of compounds. Herein, we describe a scalable and enantioselective synthesis to VNI and VFV, the two potent inhibitors of protozoan sterol 14α-demethylase (CYP51) that are currently under consideration for clinical trials for Chagas disease. A key transformation is the Jacobsen Hydrolytic Kinetic Resolution (HKR) reaction. The utility of the synthetic route is illustrated by the preparation of >25 g quantities of single enantiomers of VNI and VFV.
PLoS neglected tropical diseases, Jan 16, 2017
Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administra... more Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control. Understanding the risks and mechanisms of resistance is therefore of great importance. Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease. Metabolomic analysis demonstrated that ergosterol, the sterol known to bind the drug, is prevalent in wild-type cells, but diminished in the resistant line, where alternative sterols become prevalent. This indicates that the resistance phenotype is related to loss of drug binding. Comparing sequences of the parasites' genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells, but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway, sterol 14α-deme...
Journal of Biological Chemistry, Feb 9, 2006
J Lipid Res, 2012
Sterol 14α-demethylase (CYP51) that catalyzes the removal of the 14α-methyl group from the ster... more Sterol 14α-demethylase (CYP51) that catalyzes the removal of the 14α-methyl group from the sterol nucleus is an essential enzyme in sterol biosynthesis, a primary target for clinical and agricultural antifungal azoles and an emerging target for antitrypanosomal chemotherapy. Here, we present the crystal structure of Trypanosoma (T) brucei CYP51 in complex with the substrate analog 14α-methylenecyclopropyl-Î7-24,25-dihydrolanosterol (MCP). This sterol binds
Advances in Parasitology, 2011
Biochem Soc Trans, 2006
Sterol 14α-demethylases (CYP51) are metabolic cytochromes P450, found in each biological kingdom.... more Sterol 14α-demethylases (CYP51) are metabolic cytochromes P450, found in each biological kingdom. They catalyse a single three-step reaction included in all sterol biosynthetic pathways. Plant CYP51s have strict preference towards their physiological substrate O (obtusifoliol), which is C-4-monomethylated. Natural substrates of animal/fungal CYP51 (lanosterol, 24,25-dihydrolanosterol or 24-methylenelanosterol) are C-4-dimethylated. CYP51 from the pathogenic protozoa TB (Trypanosoma brucei) is the first example of Ospecific sterol 14α-demethylase in non-photosynthetic organisms. Surprisingly, at 83% amino acid identity to the TB orthologue, CYP51 from TC (Trypanosoma cruzi) clearly prefers C-4-dimethylated sterols. Replacement of animal/fungi-like Ile 105 in the B helix of TC CYP51 with phenylalanine, the residue found in this position in all plant and other trypanosome CYP51s, dramatically increases the ability of the enzyme to metabolize O, converting it into a more plant-like sterol 14α-demethylase. A more than 100-fold increase in binding and turnover is observed for the 24-desmethyl analogue of O [N (norlanosterol)], which is found in vivo in procyclic forms of TB and is a good TB CYP51 substrate in vitro. We believe that (i) N is a non-conventional CYP51 substrate, preferred in TB and perhaps other Trypanosomatidae and (ii) functional similarity of TC CYP51 to animal/fungal orthologues is a result of evolutionary convergence (including F105I mutation), leading to different pathways for sterol production in TC versus TB.
Biochem Biophys Res Commun, 2005
Biochimica et biophysica acta, Jan 19, 2015
Cytochrome P450 sterol 14α-demethylase (CYP51) is an essential enzyme for sterol biosynthesis and... more Cytochrome P450 sterol 14α-demethylase (CYP51) is an essential enzyme for sterol biosynthesis and a target for anti-parasitic drug design. However, the design of parasite-specific drugs that inhibit parasitic CYP51 without severe side effects remains challenging. The active site of CYP51 is situated in the interior of the protein. Here, we characterize the potential ligand egress routes and mechanisms in Trypanosoma brucei and human CYP51 enzymes. We performed Random Acceleration Molecular Dynamics simulations of the egress of four different ligands from the active site of models of soluble and membrane-bound T. brucei CYP51 and of soluble human CYP51. In the simulations, tunnel 2f, which leads to the membrane, was found to be the predominant ligand egress tunnel for all the ligands studied. Tunnels S, 1 and W, which lead to the cytosol, were also used in T. brucei CYP51, whereas tunnel 1 was the only other tunnel used significantly in human CYP51. The common tunnels found previousl...
Expert Opinion on Drug Discovery, 2013
Introduction-Endemic in Latin America, Chagas disease is now becoming a serious global health pro... more Introduction-Endemic in Latin America, Chagas disease is now becoming a serious global health problem, and yet has no financial viability for the pharmaceutical industry and remains incurable. In 2012, two antimycotic drugs inhibitors of fungal sterol 14α-demethylase (CYP51)posaconazole and ravuconazole-entered clinical trials. Availability of the X-ray structure of the orthologous enzyme from the causative agent of the disease, protozoan parasite Trypanosoma cruzi, determined in complexes with posaconazole as well as with several experimental protozoaspecific CYP51 inhibitors opens an excellent opportunity to improve the situation. Areas covered-This article summarizes the information available in PubMed and Google on the outcomes of treatment of the chronic Chagas disease. It also outlines the major features of the T. cruzi CYP51 structure and the possible structure-based strategies for rational design of novel T. cruzi specific drugs. Expert opinion-There is no doubt that screenings for alternative drug-like molecules as well as mining the T. cruzi genome for novel drug targets are of great value and might eventually lead to groundbreaking discoveries. However, all newly identified molecules must proceed through the long, expensive and low-yielding drug optimization process, and all novel potential drug targets must be validated in terms of their essentiality and druggability. CYP51 is already a well-validated and highly successful target for clinical and agricultural antifungals. With minimal investments into the final stages of their development/trials, T. cruzi-specific CYP51 inhibitors can provide an immediate treatment for Chagas disease, either on their own or in combination with the currently available drugs.
Biochemical and biophysical research communications, Jan 9, 2005
Sterol 14alpha-demethylase (CYP51) is the most widely distributed of all members of the cytochrom... more Sterol 14alpha-demethylase (CYP51) is the most widely distributed of all members of the cytochrome P450 gene superfamily and the only CYP family found in both prokaryotes and eukaryotes. It is well known as a drug target for microbial pathogenic infections. Studies of CYP51 gene regulation have been carried out primarily in animals because its regulation is similar to those of other genes involved in the cholesterol biosynthetic pathway. The function of CYP51 has been studied widely throughout biology including in animals, plants, yeast/fungi, protozoa, and bacteria. The structure has been determined by X-ray crystallography for the soluble prokaryotic form of CYP51 from Mycobacterium tuberculosis. Together these studies provide the most detailed understanding of any single cytochrome P450 and this minireview summarizes this information.
Biochemistry. Biokhimii͡a, 1998
Optimization of the conditions for heterologous expression of recombinant cytochrome P450scc in E... more Optimization of the conditions for heterologous expression of recombinant cytochrome P450scc in E. coli provided an expression level of about 420 nmoles of cytochrome P450scc per liter of bacterial culture. A new procedure for purification of recombinant protein in substrate-bound high-spin and substrate-free low-spin form is described. Highly purified electrophoretically homogeneous recombinant cytochrome P450scc contains 12.3 and 16.7 nmoles heme per mg protein for substrate-free and substrate-bound forms, respectively. The recombinant and natural cytochrome P450scc from bovine adrenocortical mitochondria were compared functionally and immunochemically. The dissociation constants for the complexes of cytochrome P450scc with cholesterol and adrenodoxin, the efficiency of enzymatic reduction in the reconstituted system (NADPH--adrenodoxin reductase--adrenodoxin), and cholesterol side-chain cleavage activity were determined. It was found that limited proteolysis of the recombinant cy...
Biochemistry. Biokhimii͡a, 1997
Studies have been done to assess the effect of selective chemical modification of Lys338 on the f... more Studies have been done to assess the effect of selective chemical modification of Lys338 on the functional activity and conformational mobility of cytochrome P450scc. It is found that fluorescently labelled cytochrome P450scc retains cholesterol side-chain cleavage activity and the ability for spectral response of type I during interaction with cholesterol and protein-protein interaction with the electron donor, adrenodoxin. Based on the quenching of the FITC fluorescence by iodide after incorporation of the labelled heme protein into an artificial phospholipid membrane, the orientation of Lys338 was determined. By measuring the efficiency of resonance fluorescence energy transfer in an FITC-heme pair during interaction of the labelled cytochrome P450scc with substrate and adrenodoxin as well as under insertion of the heme protein into membrane the changes of inter-molecular distance between Lys338 and heme were registered that indicate a functional importance of conformational mobi...
FEBS Letters, 2014
CYP51 (sterol 14α-demethylase) is an efficient target for clinical and agricultural antifungals a... more CYP51 (sterol 14α-demethylase) is an efficient target for clinical and agricultural antifungals and an emerging target for treatment of Chagas disease, the infection that is caused by multiple strains of a protozoan pathogen Trypanosoma cruzi. Here, we analyze CYP51A from the Y strain T. cruzi. In this protein, proline 355, a residue highly conserved across the CYP51 family, is replaced with serine. The purified enzyme retains its catalytic activity, yet has been found less susceptible to inhibition. These biochemical data are consistent with cellular experiments, both in insect and human stages of the pathogen. Comparative structural analysis of CYP51 complexes with VNI and two derivatives suggests that broad-spectrum CYP51 inhibitors are likely to be preferable as antichagasic drug candidates.
Journal of medicinal chemistry, Jan 12, 2015
3-Nitro-1H-1,2,4-triazole-based amides with a linear, rigid core and 3-nitrotriazole-based flucon... more 3-Nitro-1H-1,2,4-triazole-based amides with a linear, rigid core and 3-nitrotriazole-based fluconazole analogues were synthesized as dual functioning antitrypanosomal agents. Such compounds are excellent substrates for type I nitroreductase (NTR) located in the mitochondrion of trypanosomatids and, at the same time, act as inhibitors of the sterol 14α-demethylase (T. cruzi CYP51) enzyme. Because combination treatments against parasites are often superior to monotherapy, we believe that this emerging class of bifunctional compounds may introduce a new generation of antitrypanosomal drugs. In the present work, the synthesis and in vitro and in vivo evaluation of such compounds is discussed.
Journal of medicinal chemistry, Jan 14, 2014
Chagas disease, which was once thought to be confined to endemic regions of Latin America, has no... more Chagas disease, which was once thought to be confined to endemic regions of Latin America, has now gone global, becoming a new worldwide challenge with no cure available. The disease is caused by the protozoan parasite Trypanosoma cruzi, which depends on the production of endogenous sterols, and therefore can be blocked by sterol 14α-demethylase (CYP51) inhibitors. Here we explore the spectral binding parameters, inhibitory effects on T. cruzi CYP51 activity, and antiparasitic potencies of a new set of β-phenyl imidazoles. Comparative structural characterization of the T. cruzi CYP51 complexes with the three most potent inhibitors reveals two opposite binding modes of the compounds ((R)-6, EC50=1.2 nM, vs (S)-2/(S)-3, EC50=1.0/5.5 nM) and suggests the entrance into the CYP51 substrate access channel and the heme propionate-supporting ceiling of the binding cavity as two distinct areas of the protein that enhance molecular recognition and therefore could be used for the development o...
International Conference on Optical Fibre Sensors in China OFS(C) '91, 1991
ABSTRACT
Bioorganic & Medicinal Chemistry Letters
Journal of Medicinal Chemistry
Tetrahedron Letters
The study and development of azole-based CYP51 inhibitors is an active area of research across di... more The study and development of azole-based CYP51 inhibitors is an active area of research across disciplines of biochemistry, pharmacology and infectious disease. Support of in vitro and in vivo studies require the development of robust asymmetric routes to single enantiomer products of this class of compounds. Herein, we describe a scalable and enantioselective synthesis to VNI and VFV, the two potent inhibitors of protozoan sterol 14α-demethylase (CYP51) that are currently under consideration for clinical trials for Chagas disease. A key transformation is the Jacobsen Hydrolytic Kinetic Resolution (HKR) reaction. The utility of the synthetic route is illustrated by the preparation of >25 g quantities of single enantiomers of VNI and VFV.
PLoS neglected tropical diseases, Jan 16, 2017
Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administra... more Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control. Understanding the risks and mechanisms of resistance is therefore of great importance. Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease. Metabolomic analysis demonstrated that ergosterol, the sterol known to bind the drug, is prevalent in wild-type cells, but diminished in the resistant line, where alternative sterols become prevalent. This indicates that the resistance phenotype is related to loss of drug binding. Comparing sequences of the parasites' genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells, but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway, sterol 14α-deme...
Journal of Biological Chemistry, Feb 9, 2006
J Lipid Res, 2012
Sterol 14α-demethylase (CYP51) that catalyzes the removal of the 14α-methyl group from the ster... more Sterol 14α-demethylase (CYP51) that catalyzes the removal of the 14α-methyl group from the sterol nucleus is an essential enzyme in sterol biosynthesis, a primary target for clinical and agricultural antifungal azoles and an emerging target for antitrypanosomal chemotherapy. Here, we present the crystal structure of Trypanosoma (T) brucei CYP51 in complex with the substrate analog 14α-methylenecyclopropyl-Î7-24,25-dihydrolanosterol (MCP). This sterol binds
Advances in Parasitology, 2011
Biochem Soc Trans, 2006
Sterol 14α-demethylases (CYP51) are metabolic cytochromes P450, found in each biological kingdom.... more Sterol 14α-demethylases (CYP51) are metabolic cytochromes P450, found in each biological kingdom. They catalyse a single three-step reaction included in all sterol biosynthetic pathways. Plant CYP51s have strict preference towards their physiological substrate O (obtusifoliol), which is C-4-monomethylated. Natural substrates of animal/fungal CYP51 (lanosterol, 24,25-dihydrolanosterol or 24-methylenelanosterol) are C-4-dimethylated. CYP51 from the pathogenic protozoa TB (Trypanosoma brucei) is the first example of Ospecific sterol 14α-demethylase in non-photosynthetic organisms. Surprisingly, at 83% amino acid identity to the TB orthologue, CYP51 from TC (Trypanosoma cruzi) clearly prefers C-4-dimethylated sterols. Replacement of animal/fungi-like Ile 105 in the B helix of TC CYP51 with phenylalanine, the residue found in this position in all plant and other trypanosome CYP51s, dramatically increases the ability of the enzyme to metabolize O, converting it into a more plant-like sterol 14α-demethylase. A more than 100-fold increase in binding and turnover is observed for the 24-desmethyl analogue of O [N (norlanosterol)], which is found in vivo in procyclic forms of TB and is a good TB CYP51 substrate in vitro. We believe that (i) N is a non-conventional CYP51 substrate, preferred in TB and perhaps other Trypanosomatidae and (ii) functional similarity of TC CYP51 to animal/fungal orthologues is a result of evolutionary convergence (including F105I mutation), leading to different pathways for sterol production in TC versus TB.
Biochem Biophys Res Commun, 2005
Biochimica et biophysica acta, Jan 19, 2015
Cytochrome P450 sterol 14α-demethylase (CYP51) is an essential enzyme for sterol biosynthesis and... more Cytochrome P450 sterol 14α-demethylase (CYP51) is an essential enzyme for sterol biosynthesis and a target for anti-parasitic drug design. However, the design of parasite-specific drugs that inhibit parasitic CYP51 without severe side effects remains challenging. The active site of CYP51 is situated in the interior of the protein. Here, we characterize the potential ligand egress routes and mechanisms in Trypanosoma brucei and human CYP51 enzymes. We performed Random Acceleration Molecular Dynamics simulations of the egress of four different ligands from the active site of models of soluble and membrane-bound T. brucei CYP51 and of soluble human CYP51. In the simulations, tunnel 2f, which leads to the membrane, was found to be the predominant ligand egress tunnel for all the ligands studied. Tunnels S, 1 and W, which lead to the cytosol, were also used in T. brucei CYP51, whereas tunnel 1 was the only other tunnel used significantly in human CYP51. The common tunnels found previousl...
Expert Opinion on Drug Discovery, 2013
Introduction-Endemic in Latin America, Chagas disease is now becoming a serious global health pro... more Introduction-Endemic in Latin America, Chagas disease is now becoming a serious global health problem, and yet has no financial viability for the pharmaceutical industry and remains incurable. In 2012, two antimycotic drugs inhibitors of fungal sterol 14α-demethylase (CYP51)posaconazole and ravuconazole-entered clinical trials. Availability of the X-ray structure of the orthologous enzyme from the causative agent of the disease, protozoan parasite Trypanosoma cruzi, determined in complexes with posaconazole as well as with several experimental protozoaspecific CYP51 inhibitors opens an excellent opportunity to improve the situation. Areas covered-This article summarizes the information available in PubMed and Google on the outcomes of treatment of the chronic Chagas disease. It also outlines the major features of the T. cruzi CYP51 structure and the possible structure-based strategies for rational design of novel T. cruzi specific drugs. Expert opinion-There is no doubt that screenings for alternative drug-like molecules as well as mining the T. cruzi genome for novel drug targets are of great value and might eventually lead to groundbreaking discoveries. However, all newly identified molecules must proceed through the long, expensive and low-yielding drug optimization process, and all novel potential drug targets must be validated in terms of their essentiality and druggability. CYP51 is already a well-validated and highly successful target for clinical and agricultural antifungals. With minimal investments into the final stages of their development/trials, T. cruzi-specific CYP51 inhibitors can provide an immediate treatment for Chagas disease, either on their own or in combination with the currently available drugs.
Biochemical and biophysical research communications, Jan 9, 2005
Sterol 14alpha-demethylase (CYP51) is the most widely distributed of all members of the cytochrom... more Sterol 14alpha-demethylase (CYP51) is the most widely distributed of all members of the cytochrome P450 gene superfamily and the only CYP family found in both prokaryotes and eukaryotes. It is well known as a drug target for microbial pathogenic infections. Studies of CYP51 gene regulation have been carried out primarily in animals because its regulation is similar to those of other genes involved in the cholesterol biosynthetic pathway. The function of CYP51 has been studied widely throughout biology including in animals, plants, yeast/fungi, protozoa, and bacteria. The structure has been determined by X-ray crystallography for the soluble prokaryotic form of CYP51 from Mycobacterium tuberculosis. Together these studies provide the most detailed understanding of any single cytochrome P450 and this minireview summarizes this information.
Biochemistry. Biokhimii͡a, 1998
Optimization of the conditions for heterologous expression of recombinant cytochrome P450scc in E... more Optimization of the conditions for heterologous expression of recombinant cytochrome P450scc in E. coli provided an expression level of about 420 nmoles of cytochrome P450scc per liter of bacterial culture. A new procedure for purification of recombinant protein in substrate-bound high-spin and substrate-free low-spin form is described. Highly purified electrophoretically homogeneous recombinant cytochrome P450scc contains 12.3 and 16.7 nmoles heme per mg protein for substrate-free and substrate-bound forms, respectively. The recombinant and natural cytochrome P450scc from bovine adrenocortical mitochondria were compared functionally and immunochemically. The dissociation constants for the complexes of cytochrome P450scc with cholesterol and adrenodoxin, the efficiency of enzymatic reduction in the reconstituted system (NADPH--adrenodoxin reductase--adrenodoxin), and cholesterol side-chain cleavage activity were determined. It was found that limited proteolysis of the recombinant cy...
Biochemistry. Biokhimii͡a, 1997
Studies have been done to assess the effect of selective chemical modification of Lys338 on the f... more Studies have been done to assess the effect of selective chemical modification of Lys338 on the functional activity and conformational mobility of cytochrome P450scc. It is found that fluorescently labelled cytochrome P450scc retains cholesterol side-chain cleavage activity and the ability for spectral response of type I during interaction with cholesterol and protein-protein interaction with the electron donor, adrenodoxin. Based on the quenching of the FITC fluorescence by iodide after incorporation of the labelled heme protein into an artificial phospholipid membrane, the orientation of Lys338 was determined. By measuring the efficiency of resonance fluorescence energy transfer in an FITC-heme pair during interaction of the labelled cytochrome P450scc with substrate and adrenodoxin as well as under insertion of the heme protein into membrane the changes of inter-molecular distance between Lys338 and heme were registered that indicate a functional importance of conformational mobi...
FEBS Letters, 2014
CYP51 (sterol 14α-demethylase) is an efficient target for clinical and agricultural antifungals a... more CYP51 (sterol 14α-demethylase) is an efficient target for clinical and agricultural antifungals and an emerging target for treatment of Chagas disease, the infection that is caused by multiple strains of a protozoan pathogen Trypanosoma cruzi. Here, we analyze CYP51A from the Y strain T. cruzi. In this protein, proline 355, a residue highly conserved across the CYP51 family, is replaced with serine. The purified enzyme retains its catalytic activity, yet has been found less susceptible to inhibition. These biochemical data are consistent with cellular experiments, both in insect and human stages of the pathogen. Comparative structural analysis of CYP51 complexes with VNI and two derivatives suggests that broad-spectrum CYP51 inhibitors are likely to be preferable as antichagasic drug candidates.
Journal of medicinal chemistry, Jan 12, 2015
3-Nitro-1H-1,2,4-triazole-based amides with a linear, rigid core and 3-nitrotriazole-based flucon... more 3-Nitro-1H-1,2,4-triazole-based amides with a linear, rigid core and 3-nitrotriazole-based fluconazole analogues were synthesized as dual functioning antitrypanosomal agents. Such compounds are excellent substrates for type I nitroreductase (NTR) located in the mitochondrion of trypanosomatids and, at the same time, act as inhibitors of the sterol 14α-demethylase (T. cruzi CYP51) enzyme. Because combination treatments against parasites are often superior to monotherapy, we believe that this emerging class of bifunctional compounds may introduce a new generation of antitrypanosomal drugs. In the present work, the synthesis and in vitro and in vivo evaluation of such compounds is discussed.
Journal of medicinal chemistry, Jan 14, 2014
Chagas disease, which was once thought to be confined to endemic regions of Latin America, has no... more Chagas disease, which was once thought to be confined to endemic regions of Latin America, has now gone global, becoming a new worldwide challenge with no cure available. The disease is caused by the protozoan parasite Trypanosoma cruzi, which depends on the production of endogenous sterols, and therefore can be blocked by sterol 14α-demethylase (CYP51) inhibitors. Here we explore the spectral binding parameters, inhibitory effects on T. cruzi CYP51 activity, and antiparasitic potencies of a new set of β-phenyl imidazoles. Comparative structural characterization of the T. cruzi CYP51 complexes with the three most potent inhibitors reveals two opposite binding modes of the compounds ((R)-6, EC50=1.2 nM, vs (S)-2/(S)-3, EC50=1.0/5.5 nM) and suggests the entrance into the CYP51 substrate access channel and the heme propionate-supporting ceiling of the binding cavity as two distinct areas of the protein that enhance molecular recognition and therefore could be used for the development o...
International Conference on Optical Fibre Sensors in China OFS(C) '91, 1991
ABSTRACT