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1.
J Med Chem ; 64(17): 12790-12807, 2021 09 09.
Article in English | MEDLINE | ID: mdl-34414766

ABSTRACT

Phenotypic whole cell high-throughput screening of a ∼150,000 diverse set of compounds against Mycobacterium tuberculosis (Mtb) in cholesterol-containing media identified 1,3-diarylpyrazolyl-acylsulfonamide 1 as a moderately active hit. Structure-activity relationship (SAR) studies demonstrated a clear scope to improve whole cell potency to MIC values of <0.5 µM, and a plausible pharmacophore model was developed to describe the chemical space of active compounds. Compounds are bactericidal in vitro against replicating Mtb and retained activity against multidrug-resistant clinical isolates. Initial biology triage assays indicated cell wall biosynthesis as a plausible mode-of-action for the series. However, no cross-resistance with known cell wall targets such as MmpL3, DprE1, InhA, and EthA was detected, suggesting a potentially novel mode-of-action or inhibition. The in vitro and in vivo drug metabolism and pharmacokinetics profiles of several active compounds from the series were established leading to the identification of a compound for in vivo efficacy proof-of-concept studies.


Subject(s)
Antitubercular Agents/pharmacology , Cell Wall/metabolism , Mycobacterium tuberculosis/drug effects , Sulfonamides/pharmacology , Antitubercular Agents/chemical synthesis , Antitubercular Agents/chemistry , Drug Discovery , Hep G2 Cells , Humans , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Mycobacterium tuberculosis/metabolism , Structure-Activity Relationship , Sulfonamides/chemistry
3.
J Med Chem ; 63(21): 13013-13030, 2020 11 12.
Article in English | MEDLINE | ID: mdl-33103428

ABSTRACT

A series of 2,4-disubstituted imidazopyridines, originating from a SoftFocus Kinase library, was identified from a high throughput phenotypic screen against the human malaria parasite Plasmodium falciparum. Hit compounds showed moderate asexual blood stage activity. During lead optimization, several issues were flagged such as cross-resistance against the multidrug-resistant K1 strain, in vitro cytotoxicity, and cardiotoxicity and were addressed through structure-activity and structure-property relationship studies. Pharmacokinetic properties were assessed in mice for compounds showing desirable in vitro activity, a selectivity window over cytotoxicity, and microsomal metabolic stability. Frontrunner compound 37 showed good exposure in mice combined with good in vitro activity against the malaria parasite, which translated into in vivo efficacy in the P. falciparum NOD-scid IL-2Rγnull (NSG) mouse model. Preliminary mechanistic studies suggest inhibition of hemozoin formation as a contributing mode of action.


Subject(s)
Antimalarials/chemistry , Hemeproteins/antagonists & inhibitors , Imidazoles/chemistry , Plasmodium falciparum/physiology , Protozoan Proteins/antagonists & inhibitors , Pyridines/chemistry , Animals , Antimalarials/metabolism , Antimalarials/pharmacology , Antimalarials/therapeutic use , Disease Models, Animal , Half-Life , Hemeproteins/metabolism , Imidazoles/metabolism , Imidazoles/pharmacology , Imidazoles/therapeutic use , Life Cycle Stages/drug effects , Malaria/drug therapy , Malaria/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Mice, SCID , Microsomes, Liver/metabolism , Plasmodium falciparum/drug effects , Plasmodium falciparum/metabolism , Protozoan Proteins/metabolism , Pyridines/metabolism , Pyridines/pharmacology , Pyridines/therapeutic use , Structure-Activity Relationship
4.
ACS Infect Dis ; 6(7): 1951-1964, 2020 07 10.
Article in English | MEDLINE | ID: mdl-32470286

ABSTRACT

Phenotypic whole-cell screening against Mycobacterium tuberculosis (Mtb) in glycerol-alanine-salts supplemented with Tween 80 and iron (GASTE-Fe) media led to the identification of a 2-aminoquinazolinone hit compound, sulfone 1 which was optimized for solubility by replacing the sulfone moiety with a sulfoxide 2. The synthesis and structure-activity relationship (SAR) studies identified several compounds with potent antimycobacterial activity, which were metabolically stable and noncytotoxic. Compound 2 displayed favorable in vitro properties and was therefore selected for in vivo pharmacokinetic (PK) studies where it was found to be extensively metabolized to the sulfone 1. Both derivatives exhibited promising PK parameters; however, when 2 was evaluated for in vivo efficacy in an acute TB infection mouse model, it was found to be inactive. In order to understand the in vitro and in vivo discrepancy, compound 2 was subsequently retested in vitro using different Mtb strains cultured in different media. This revealed that activity was only observed in media containing glycerol and led to the hypothesis that glycerol was not used as a primary carbon source by Mtb in the mouse lungs, as has previously been observed. Support for this hypothesis was provided by spontaneous-resistant mutant generation and whole genome sequencing studies, which revealed mutations mapping to glycerol metabolizing genes indicating that the 2-aminoquinazolinones kill Mtb in vitro via a glycerol-dependent mechanism of action.


Subject(s)
Antitubercular Agents , Mycobacterium tuberculosis , Animals , Antitubercular Agents/pharmacology , Drug Design , Mice , Structure-Activity Relationship
5.
ACS Omega ; 5(12): 6967-6982, 2020 Mar 31.
Article in English | MEDLINE | ID: mdl-32258933

ABSTRACT

A phenotypic whole cell high-throughput screen against the asexual blood and liver stages of the malaria parasite identified a benzimidazole chemical series. Among the hits were the antiemetic benzimidazole drug Lerisetron 1 (IC50 NF54 = 0.81 µM) and its methyl-substituted analogue 2 (IC50 NF54 = 0.098 µM). A medicinal chemistry hit to lead effort led to the identification of chloro-substituted analogue 3 with high potency against the drug-sensitive NF54 (IC50 NF54 = 0.062 µM) and multidrug-resistant K1 (IC50 K1 = 0.054 µM) strains of the human malaria parasite Plasmodium falciparum. Compounds 2 and 3 gratifyingly showed in vivo efficacy in both Plasmodium berghei and P. falciparum mouse models of malaria. Cardiotoxicity risk as expressed in strong inhibition of the human ether-a-go-go-related gene (hERG) potassium channel was identified as a major liability to address. This led to the synthesis and biological assessment of around 60 analogues from which several compounds with improved antiplasmodial potency, relative to the lead compound 3, were identified.

7.
J Med Chem ; 61(20): 9371-9385, 2018 10 25.
Article in English | MEDLINE | ID: mdl-30256636

ABSTRACT

A lead-optimization program around a 2,6-imidazopyridine scaffold was initiated based on the two early lead compounds, 1 and 2, that were shown to be efficacious in an in vivo humanized Plasmodium falciparum NODscidIL2Rγnull mouse malaria infection model. The observation of atypical dose-response curves when some compounds were tested against multidrug resistant malaria parasite strains guided the optimization process to define a chemical space that led to typical sigmoidal dose-response and complete kill of multidrug resistant parasites. After a structure and property analysis identified such a chemical space, compounds were prepared that displayed suitable activity, ADME, and safety profiles with respect to cytotoxicity and hERG inhibition.


Subject(s)
Antimalarials/chemistry , Antimalarials/pharmacology , Drug Resistance, Multiple/drug effects , Imidazoles/chemistry , Imidazoles/pharmacology , Plasmodium falciparum/drug effects , Pyridines/chemistry , Pyridines/pharmacology , Absorption, Physicochemical , Animals , Antimalarials/metabolism , Antimalarials/pharmacokinetics , Dose-Response Relationship, Drug , Imidazoles/metabolism , Imidazoles/pharmacokinetics , Mice , Pyridines/metabolism , Pyridines/pharmacokinetics , Structure-Activity Relationship , Tissue Distribution
8.
Article in English | MEDLINE | ID: mdl-29941635

ABSTRACT

The 2-aminopyridine MMV048 was the first drug candidate inhibiting Plasmodium phosphatidylinositol 4-kinase (PI4K), a novel drug target for malaria, to enter clinical development. In an effort to identify the next generation of PI4K inhibitors, the series was optimized to improve properties such as solubility and antiplasmodial potency across the parasite life cycle, leading to the 2-aminopyrazine UCT943. The compound displayed higher asexual blood stage, transmission-blocking, and liver stage activities than MMV048 and was more potent against resistant Plasmodium falciparum and Plasmodium vivax clinical isolates. Excellent in vitro antiplasmodial activity translated into high efficacy in Plasmodium berghei and humanized P. falciparum NOD-scid IL-2Rγ null mouse models. The high passive permeability and high aqueous solubility of UCT943, combined with low to moderate in vivo intrinsic clearance, resulted in sustained exposure and high bioavailability in preclinical species. In addition, the predicted human dose for a curative single administration using monkey and dog pharmacokinetics was low, ranging from 50 to 80 mg. As a next-generation Plasmodium PI4K inhibitor, UCT943, based on the combined preclinical data, has the potential to form part of a single-exposure radical cure and prophylaxis (SERCaP) to treat, prevent, and block the transmission of malaria.

9.
J Med Chem ; 61(9): 4213-4227, 2018 05 10.
Article in English | MEDLINE | ID: mdl-29665687

ABSTRACT

Optimization of a chemical series originating from whole-cell phenotypic screening against the human malaria parasite, Plasmodium falciparum, led to the identification of two promising 2,6-disubstituted imidazopyridine compounds, 43 and 74. These compounds exhibited potent activity against asexual blood stage parasites that, together with their in vitro absorption, distribution, metabolism, and excretion (ADME) properties, translated to in vivo efficacy with clearance of parasites in the PfSCID mouse model for malaria within 48 h of treatment.


Subject(s)
Drug Discovery , Imidazoles/chemistry , Imidazoles/pharmacokinetics , Malaria/drug therapy , Plasmodium falciparum/physiology , Pyridines/chemistry , Pyridines/pharmacokinetics , Animals , Disease Models, Animal , Drug Stability , ERG1 Potassium Channel/metabolism , Humans , Imidazoles/metabolism , Imidazoles/therapeutic use , Malaria/genetics , Malaria/metabolism , Mice , Pyridines/metabolism , Pyridines/therapeutic use , Solubility , Structure-Activity Relationship , Tissue Distribution , Water/chemistry
10.
J Med Chem ; 60(24): 10118-10134, 2017 12 28.
Article in English | MEDLINE | ID: mdl-29148755

ABSTRACT

A BioFocus DPI SoftFocus library of ∼35 000 compounds was screened against Mycobacterium tuberculosis (Mtb) in order to identify novel hits with antitubercular activity. The hits were evaluated in biology triage assays to exclude compounds suggested to function via frequently encountered promiscuous mechanisms of action including inhibition of the QcrB subunit of the cytochrome bc1 complex, disruption of cell-wall homeostasis, and DNA damage. Among the hits that passed this screening cascade, a 6-dialkylaminopyrimidine carboxamide series was prioritized for hit to lead optimization. Compounds from this series were active against clinical Mtb strains, while no cross-resistance to conventional antituberculosis drugs was observed. This suggested a novel mechanism of action, which was confirmed by chemoproteomic analysis leading to the identification of BCG_3193 and BCG_3827 as putative targets of the series with unknown function. Initial structure-activity relationship studies have resulted in compounds with moderate to potent antitubercular activity and improved physicochemical properties.


Subject(s)
Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Mycobacterium tuberculosis/drug effects , Structure-Activity Relationship , Administration, Oral , Animals , Antitubercular Agents/chemical synthesis , Blood Proteins/metabolism , Drug Stability , High-Throughput Screening Assays , Humans , Male , Mice, Inbred C57BL , Microsomes, Liver/drug effects , Mycobacterium tuberculosis/isolation & purification , Proteomics/methods , Pyrimidines/chemistry , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology
11.
J Med Chem ; 59(21): 9890-9905, 2016 11 10.
Article in English | MEDLINE | ID: mdl-27748596

ABSTRACT

Introduction of water-solubilizing groups on the 5-phenyl ring of a 2-aminopyrazine series led to the identification of highly potent compounds against the blood life-cycle stage of the human malaria parasite Plasmodium falciparum. Several compounds displayed high in vivo efficacy in two different mouse models for malaria, P. berghei-infected mice and P. falciparum-infected NOD-scid IL-2Rγnull mice. One of the frontrunners, compound 3, was identified to also have good pharmacokinetics and additionally very potent activity against the liver and gametocyte parasite life-cycle stages.


Subject(s)
Antimalarials/pharmacology , Life Cycle Stages/drug effects , Malaria/drug therapy , Parasitic Diseases, Animal/drug therapy , Plasmodium berghei/drug effects , Plasmodium falciparum/drug effects , Pyrazines/pharmacology , Animals , Antimalarials/chemistry , Antimalarials/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Ether-A-Go-Go Potassium Channels/metabolism , Hep G2 Cells , Humans , Mice , Mice, SCID , Microsomes, Liver/chemistry , Microsomes, Liver/metabolism , Molecular Structure , Parasitic Diseases, Animal/parasitology , Parasitic Sensitivity Tests , Plasmodium berghei/growth & development , Plasmodium falciparum/growth & development , Pyrazines/chemistry , Pyrazines/metabolism , Solubility , Structure-Activity Relationship , Water/chemistry
12.
J Med Chem ; 57(6): 2789-98, 2014 Mar 27.
Article in English | MEDLINE | ID: mdl-24568587

ABSTRACT

A novel class of imidazopyridazines identified from whole cell screening of a SoftFocus kinase library was synthesized and evaluated for antiplasmodial activity against K1 (multidrug resistant strain) and NF54 (sensitive strain). Structure-activity relationship studies led to the identification of highly potent compounds against both strains. Compound 35 was highly active (IC50: K1 = 6.3 nM, NF54 = 7.3 nM) and comparable in potency to artesunate, and 35 exhibited 98% activity in the in vivo P. berghei mouse model (4-day test by Peters) at 4 × 50 mg/kg po. Compound 35 was also assessed against P. falciparum in the in vivo SCID mouse model where the efficacy was found to be more consistent with the in vitro activity. Furthermore, 35 displayed high (78%) rat oral bioavailability with good oral exposure and plasma half-life. Mice exposure at the same dose was 10-fold lower than in rat, suggesting lower oral absorption and/or higher metabolic clearance in mice.


Subject(s)
Antimalarials/chemical synthesis , Antimalarials/pharmacology , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Plasmodium/drug effects , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Kinases/chemistry , Pyridazines/chemical synthesis , Pyridazines/pharmacology , Animals , Antimalarials/pharmacokinetics , Biological Availability , Drug Design , Drug Resistance , Drug Stability , Gene Library , Half-Life , High-Throughput Screening Assays , Malaria/drug therapy , Malaria/parasitology , Malaria, Falciparum/drug therapy , Malaria, Falciparum/psychology , Mice , Mice, SCID , Parasitic Sensitivity Tests , Plasmodium berghei , Plasmodium falciparum/drug effects , Rats , Structure-Activity Relationship
13.
J Med Chem ; 57(3): 1014-22, 2014 Feb 13.
Article in English | MEDLINE | ID: mdl-24446664

ABSTRACT

A novel series of 2,4-diaminothienopyrimidines with potential as antimalarials was identified from whole-cell high-throughput screening of a SoftFocus ion channel library. Synthesis and structure-activity relationship studies identified compounds with potent antiplasmodial activity and low in vitro cytotoxicity. Several of these analogues exhibited in vivo activity in the Plasmodium berghei mouse model when administered orally. However, inhibition of the hERG potassium channel was identified as a liability for this series.


Subject(s)
Antimalarials/chemical synthesis , Pyrimidines/chemical synthesis , Thiophenes/chemical synthesis , Administration, Oral , Animals , Antimalarials/chemistry , Antimalarials/pharmacology , Cell Line , Databases, Chemical , Drug Resistance, Multiple , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , High-Throughput Screening Assays , Humans , Malaria/drug therapy , Malaria/parasitology , Male , Mice , Microsomes, Liver/metabolism , Plasmodium berghei , Plasmodium falciparum/drug effects , Pyrimidines/chemistry , Pyrimidines/pharmacology , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Thiophenes/chemistry , Thiophenes/pharmacology
14.
J Med Chem ; 56(21): 8860-71, 2013 Nov 14.
Article in English | MEDLINE | ID: mdl-24099149

ABSTRACT

Replacement of the pyridine core of antimalarial 3,5-diaryl-2-aminopyridines led to the identification of a novel series of pyrazine analogues with potent oral antimalarial activity. However, other changes to the pyridine core and replacement or substitution of the 2-amino group led to loss of antimalarial activity. The 3,5-diaryl-2-aminopyrazine series showed impressive in vitro antiplasmodial activity against the K1 (multidrug resistant) and NF54 (sensitive) strains of Plasmodium falciparum in the nanomolar IC50 range of 6-94 nM while also demonstrating good in vitro metabolic stability in human liver microsomes. In the Plasmodium berghei mouse model, this series generally exhibited good efficacy at low oral doses. One of the frontrunner compounds, 4, displayed potent in vitro antiplasmodial activity with IC50 values of 8.4 and 10 nM against the K1 and NF54 strains, respectively. When evaluated in P. berghei -infected mice, compound 4 was completely curative at an oral dose of 4 × 10 mg/kg.


Subject(s)
Aminopyridines/pharmacology , Antimalarials/pharmacology , Plasmodium berghei/drug effects , Plasmodium falciparum/drug effects , Administration, Oral , Aminopyridines/administration & dosage , Aminopyridines/chemistry , Animals , Antimalarials/administration & dosage , Antimalarials/chemistry , CHO Cells , Cricetulus , Disease Models, Animal , Dose-Response Relationship, Drug , Humans , Mice , Microsomes, Liver/chemistry , Microsomes, Liver/metabolism , Molecular Structure , Parasitic Sensitivity Tests , Rats , Structure-Activity Relationship
15.
J Med Chem ; 55(24): 11022-30, 2012 Dec 27.
Article in English | MEDLINE | ID: mdl-23189922

ABSTRACT

In an effort to address potential cardiotoxicity liabilities identified with earlier frontrunner compounds, a number of new 3,5-diaryl-2-aminopyridine derivatives were synthesized. Several compounds exhibited potent antiplasmodial activity against both the multidrug resistant (K1) and sensitive (NF54) strains in the low nanomolar range. Some compounds displayed a significant reduction in potency in the hERG channel inhibition assay compared to previously reported frontrunner analogues. Several of these new analogues demonstrated promising in vivo efficacy in the Plasmodium berghei mouse model and will be further evaluated as potential clinical candidates. The SAR for in vitro antiplasmodial and hERG activity was delineated.


Subject(s)
Aminopyridines/chemical synthesis , Antimalarials/chemical synthesis , Administration, Oral , Aminopyridines/chemistry , Aminopyridines/pharmacology , Animals , Antimalarials/chemistry , Antimalarials/pharmacology , Drug Resistance, Multiple , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Humans , Malaria/drug therapy , Mice , Microsomes, Liver/metabolism , Plasmodium berghei , Plasmodium falciparum/drug effects , Solubility , Structure-Activity Relationship
16.
J Med Chem ; 55(7): 3479-87, 2012 Apr 12.
Article in English | MEDLINE | ID: mdl-22390538

ABSTRACT

A novel class of orally active antimalarial 3,5-diaryl-2-aminopyridines has been identified from phenotypic whole cell high-throughput screening of a commercially available SoftFocus kinase library. The compounds were evaluated in vitro for their antiplasmodial activity against K1 (chloroquine and drug-resistant strain) and NF54 (chloroquine-susceptible strain) as well as for their cytotoxicity. Synthesis and structure-activity studies identified a number of promising compounds with selective antiplasmodial activity. One of these frontrunner compounds, 15, was equipotent across the two strains (K1 = 25.0 nM, NF54 = 28.0 nM) and superior to chloroquine in the K1 strain (chloroquine IC(50) K1 = 194.0 nM). Compound 15 completely cured Plasmodium berghei-infected mice with a single oral dose of 30 mg/kg. Dose-response studies generated ED(50) and ED(90) values of 0.83 and 1.74 mg/kg for 15 in the standard four-dose Peters test. Pharmacokinetic studies in the rat indicated that this compound has good oral bioavailability (51% at 20 mg/kg) and a reasonable half-life (t(1/2) ∼ 7-8 h).


Subject(s)
Aminopyridines/chemical synthesis , Antimalarials/chemical synthesis , Administration, Oral , Aminopyridines/pharmacokinetics , Aminopyridines/pharmacology , Animals , Antimalarials/pharmacokinetics , Antimalarials/pharmacology , Biological Availability , Cell Line , Chloroquine/pharmacology , Cytochrome P-450 Enzyme Inhibitors , Drug Resistance , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Female , Humans , Isoenzymes/antagonists & inhibitors , Malaria/drug therapy , Mice , Microsomes, Liver/metabolism , Plasmodium berghei , Plasmodium falciparum/drug effects , Rabbits , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship
17.
Biol Chem ; 393(12): 1547-54, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23667908

ABSTRACT

Somatic angiotensin I-converting enzyme (ACE)has two homologous active sites (N and C domains) that show differences in various biochemical properties.In a previous study, we described the use of positionals canning synthetic combinatorial (PS-SC) libraries of fluorescence resonance energy transfer (FRET) peptides to define the ACE C-domain versus N-domain substrate specificity and developed selective substrates for the C-domain(Bersanetti et al., 2004). In the present work, we used the results from the PS-SC libraries to define the N-domain preferences and designed selective substrates for this domain. The peptide Abz-GDDVAK(Dnp)-OH presented the most favorable residues for N-domain selectivity in the P 3 to P 1 ' positions. The fluorogenic analog Abz-DVAK(Dnp)-OH (Abz = ortho -aminobenzoic acid; Dnp = 2,4-dinitrophenyl)showed the highest selectivity for ACE N-domain( k cat /K m = 1.76 µ m -1 · s -1) . Systematic reduction of the peptide length resulted in a tripeptide that was preferentially hydrolyzed by the C-domain. The binding of Abz-DVAK(Dnp)-OH to the active site of ACE N-domain was examined using a combination of conformational analysis and molecular docking. Our results indicated that the binding energies for the N-domain-substrate complexes were lower than those for the C-domain-substrate, suggesting that the former complexes are more stable.


Subject(s)
Fluorescence Resonance Energy Transfer , Peptides/chemistry , Peptides/metabolism , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Amino Acid Sequence , Catalytic Domain , Fluorescence Resonance Energy Transfer/methods , Humans , Molecular Sequence Data , Peptide Library , Protein Binding , Protein Structure, Tertiary , Substrate Specificity , Thermodynamics
18.
J Med Chem ; 54(21): 7713-9, 2011 Nov 10.
Article in English | MEDLINE | ID: mdl-21966980

ABSTRACT

An aminomethylthiazole pyrazole carboxamide lead 3 with good in vitro antiplasmodial activity [IC(50): 0.08 µM (K1, chloroquine and multidrug resistant strain) and 0.07 µM (NF54, chloroquine sensitive strain)] and microsomal metabolic stability was identified from whole cell screening of a SoftFocus kinase library. Compound 3 also exhibited in vivo activity in the P. berghei mouse model at 4 × 50 mg/kg administration via the oral route, showing 99.5% activity and 9 days survival and showed low in vitro cytotoxicity. Pharmacokinetic studies in rats revealed good oral bioavailability (51% at 22 mg/kg) with a moderate rate of absorption, reasonable half-life (t(1/2) 3 h), and high volume of distribution with moderately high plasma and blood clearance after IV administration. Toward toxicity profiling, 3 exhibited moderate potential to inhibit CYP1A2 (IC(50) = 1.5 µM) and 2D6 (IC(50) = 0.4 µM) as well as having a potential hERG liability (IC(50) = 3.7 µM).


Subject(s)
Antimalarials/chemical synthesis , Thiazoles/chemical synthesis , Administration, Oral , Animals , Antimalarials/pharmacokinetics , Antimalarials/pharmacology , Biological Availability , Cytochrome P-450 CYP1A2 Inhibitors , Drug Interactions , Drug Resistance , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Humans , In Vitro Techniques , Injections, Intravenous , Malaria/drug therapy , Male , Mice , Microsomes/metabolism , Parasitic Sensitivity Tests , Plasmodium berghei , Plasmodium falciparum/drug effects , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Thiazoles/pharmacokinetics , Thiazoles/pharmacology
19.
Biol Chem ; 390(9): 931-40, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19558329

ABSTRACT

Dipeptidyl carboxypeptidase from Escherichia coli (EcDcp) is a zinc metallopeptidase with catalytic properties closely resembling those of angiotensin I-converting enzyme (ACE). However, EcDcp and ACE are classified in different enzyme families (M3 and M2, respectively) due to differences in their primary sequences. We cloned and expressed EcDcp and studied in detail the enzyme's S(3) to S(1)' substrate specificity using positional-scanning synthetic combinatorial (PS-SC) libraries of fluorescence resonance energy transfer (FRET) peptides. These peptides contain ortho-aminobenzoic acid (Abz) and 2,4-dinitrophenyl (Dnp) as donor/acceptor pair. In addition, using FRET substrates developed for ACE [Abz-FRK(Dnp)P-OH, Abz-SDK(Dnp)P-OH and Abz-LFK(Dnp)-OH] as well as natural ACE substrates (angiotensin I, bradykinin, and Ac-SDKP-OH), we show that EcDcp has catalytic properties very similar to human testis ACE. EcDcp inhibition studies were performed with the ACE inhibitors captopril (K(i)=3 nM) and lisinopril (K(i)=4.4 microM) and with two C-domain-selective ACE inhibitors, 5-S-5-benzamido-4-oxo-6-phenylhexanoyl-L-tryptophan (kAW; K(i)=22.0 microM) and lisinopril-Trp (K(i)=0.8 nM). Molecular modeling was used to provide the basis for the differences found in the inhibitors potency. The phylogenetic relationship of EcDcp and related enzymes belonging to the M3 and M2 families was also investigated and the results corroborate the distinct origins of EcDcp and ACE.


Subject(s)
Endopeptidases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Peptidyl-Dipeptidase A/metabolism , Recombinant Proteins/metabolism , Animals , Endopeptidases/classification , Endopeptidases/genetics , Enzyme Activation/drug effects , Escherichia coli Proteins/classification , Escherichia coli Proteins/genetics , Humans , Hydrogen-Ion Concentration , Peptidyl-Dipeptidase A/genetics , Phylogeny , Protein Structure, Secondary , Recombinant Proteins/classification , Recombinant Proteins/genetics , Sodium Chloride/pharmacology , Substrate Specificity
20.
J Virol ; 82(9): 4656-9, 2008 May.
Article in English | MEDLINE | ID: mdl-18305051

ABSTRACT

The foot-and-mouth disease virus (FMDV) leader proteinase (L(pro)) self-processes inefficiently at the L(pro)/VP4 cleavage site LysLeuLys*GlyAlaGly (* indicates cleaved peptide bond) when the leucine at position P2 is replaced by phenylalanine. Molecular modeling and energy minimization identified the L(pro) residue L143 as being responsible for this discrimination. The variant L(pro) L143A self-processed efficiently at the L(pro)/VP4 cleavage site containing P2 phenylalanine, whereas the L143M variant did not. L(pro) L143A self-processing at the eIF4GII sequence AspPheGly*ArgGlnThr was improved but showed more-extensive aberrant processing. Residue 143 in L(pro) is occupied only by leucine and methionine in all sequenced FMDV serotypes, implying that these bulky side chains are one determinant of the restricted specificity of L(pro).


Subject(s)
Endopeptidases/genetics , Endopeptidases/metabolism , Foot-and-Mouth Disease Virus/enzymology , Amino Acid Sequence , Animals , Hydrolysis , Leucine , Models, Molecular , Mutation, Missense , Substrate Specificity/genetics
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