RESUMO
Emtricitabine (FTC) and lamivudine (3TC), containing an oxathiolane ring with unnatural (-)-stereochemistry, are widely used nucleoside reverse transcriptase inhibitors (NRTIs) in anti-HIV therapy. Treatment with FTC or 3TC primarily selects for the HIV-1 RT M184V/I resistance mutations. Here we provide a comprehensive kinetic and structural basis for inhibiting HIV-1 RT by (-)-FTC-TP and (-)-3TC-TP and drug resistance by M184V. (-)-FTC-TP and (-)-3TC-TP have higher binding affinities (1/Kd) for wild-type RT but slower incorporation rates than dCTP. HIV-1 RT ternary crystal structures with (-)-FTC-TP and (-)-3TC-TP corroborate kinetic results demonstrating that their oxathiolane sulfur orients toward the DNA primer 3'-terminus and their triphosphate exists in two different binding conformations. M184V RT displays greater (>200-fold) Kd for the L-nucleotides and moderately higher (>9-fold) Kd for the D-isomers compared to dCTP. The M184V RT structure illustrates how the mutation repositions the oxathiolane of (-)-FTC-TP and shifts its triphosphate into a non-productive conformation.
Assuntos
Farmacorresistência Viral , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Nucleotídeos/química , Inibidores da Transcriptase Reversa/química , Alelos , Substituição de Aminoácidos , Bases de Dados Genéticas , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/enzimologia , Humanos , Testes de Sensibilidade Microbiana , Mutação , Nucleotídeos/farmacologia , Inibidores da Transcriptase Reversa/farmacologiaRESUMO
We hereby disclose the discovery of inhibitors of CaMKII (7h and 7i) that are highly potent in rat ventricular myocytes, selective against hERG and other off-target kinases, while possessing good CaMKII tissue isoform selectivity (cardiac γ/δ vs. neuronal α/ß). In vitro and in vivo ADME/PK studies demonstrated the suitability of these CaMKII inhibitors for PO (7h rat Fâ¯=â¯73%) and IV pharmacological studies.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
Idelalisib (also known as GS-1101, CAL-101, IC489666, and Zydelig) is a PI3Kδ inhibitor that has recently been approved for the treatment of several hematological malignancies. Given its use in human diseases, we needed a clear picture of how idelalisib binds to and inhibits PI3Kδ. Our data show that idelalisib is a potent and selective inhibitor of the kinase activity of PI3Kδ. A kinetic characterization clearly demonstrated ATP-competitive inhibition, and several additional biochemical and biophysical assays showed that the compound binds reversibly and noncovalently to the kinase. A crystal structure of idelalisib bound to the p110δ subunit of PI3Kδ furthers our understanding of the binding interactions that confer the potency and selectivity of idelalisib.
Assuntos
Fosfatidilinositol 3-Quinases/química , Purinas/química , Quinazolinonas/química , Trifosfato de Adenosina/química , Androstadienos/química , Animais , Ligação Competitiva , Domínio Catalítico , Classe I de Fosfatidilinositol 3-Quinases , Classe Ia de Fosfatidilinositol 3-Quinase/química , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Cinética , Camundongos , Modelos Moleculares , Inibidores de Fosfoinositídeo-3 Quinase , Ligação Proteica , WortmaninaRESUMO
As a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied the in vitro pharmacology of a novel C-nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concentration [EC50], 0.048 to 0.68 µM). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC50, 1.5 µM). The active 5'-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, with Ki/Km values of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, respectively. With its unique dual substitutions of 1'-CN and 2'-C-Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistance in vitro. Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymatic assays (>30-fold). Consistent with its in vitro profile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clinical trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability.
Assuntos
Antivirais/química , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Nucleosídeos/química , Nucleosídeos/farmacologia , Pró-Fármacos/farmacologia , Replicação Viral/efeitos dos fármacos , Antivirais/efeitos adversos , Linhagem Celular Tumoral , Sobrevivência Celular , Células Hep G2 , Humanos , Nucleosídeos/efeitos adversos , Pró-Fármacos/efeitos adversos , Pró-Fármacos/química , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well understood. Here, using a panel of NS5B proteins with mutations in key regulatory motifs of NS5B--the C-terminal tail and ß-loop--in conjunction with a diverse set of NS5B allosteric inhibitors, we show that thumb site II inhibitors possess a distinct mechanism of action. A combination of enzyme activity studies and direct binding assays reveals that these inhibitors require both regulatory elements to maintain the polymerase inhibitory activity. Removal of either element has little impact on the binding affinity of thumb site II inhibitors, but significantly reduces their potency. NS5B in complex with a thumb site II inhibitor displays a characteristic melting profile that suggests stabilization not only of the thumb domain but also the whole polymerase. Successive truncations of the C-terminal tail and/or removal of the ß-loop lead to progressive destabilization of the protein. Furthermore, the thermal unfolding transitions characteristic for thumb site II inhibitor-NS5B complex are absent in the inhibitor-bound constructs in which interactions between C-terminal tail and ß-loop are abolished, pointing to the pivotal role of both regulatory elements in communication between domains. Taken together, a comprehensive picture of inhibition by compounds binding to thumb site II emerges: inhibitor binding provides stabilization of the entire polymerase in an inactive, closed conformation, propagated via coupled interactions between the C-terminal tail and ß-loop.
Assuntos
Sítio Alostérico/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hepacivirus/enzimologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Motivos de Aminoácidos , Domínio Catalítico , Estabilidade Enzimática , Furanos/farmacologia , Modelos Moleculares , Deleção de Sequência , Tiofenos/farmacologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Apolipoprotein A-I (ApoA-I), a primary protein component of high-density lipoprotein (HDL), plays an important role in cholesterol metabolism mediating the formation of HDL and the efflux of cellular cholesterol from macrophage foam cells in arterial walls. Lipidation of ApoA-I is mediated by adenosine triphosphate (ATP) binding cassette A1 (ABCA1). Insufficient ABCA1 activity may lead to increased risk of atherosclerosis due to reduced HDL formation and cholesterol efflux. The standard radioactive assay for measuring cholesterol transport to ApoA-I has low throughput and poor dynamic range, and it fails to measure phospholipid transfer. We describe the development of two sensitive, nonradioactive high-throughput assays that report on the lipidation of ApoA-I: a homogeneous assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) and a discontinuous assay that uses the label-free Epic platform. The TR-FRET assay employs HiLyte Fluor 647-labeled ApoA-I with N-terminal biotin bound to streptavidin-terbium. When fluorescent ApoA-I was incorporated into HDL, TR-FRET decreased proportionally to the increase in the ratio of lipids to ApoA-I, demonstrating that the assay was sensitive to the amount of lipid bound to ApoA-I. In the Epic assay, biotinylated ApoA-I was captured on a streptavidin-coated biosensor. Measured resonant wavelength shift was proportional to the amount of lipids associated with ApoA-I, indicating that the assay senses ApoA-I lipidation.
Assuntos
Apolipoproteína A-I/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Ensaios de Triagem em Larga Escala/métodos , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/agonistas , Transportadores de Cassetes de Ligação de ATP/metabolismo , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Aterosclerose/metabolismo , Aterosclerose/prevenção & controle , Biotina , Células Cultivadas , Colesterol/metabolismo , Corantes Fluorescentes/química , Humanos , Metabolismo dos Lipídeos , Lipídeos , Lipoproteínas HDL/metabolismo , Macrófagos/metabolismo , EstreptavidinaRESUMO
GS-9148 ([5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl]-phosphonic acid) is a dAMP (2'-deoxyadenosine monophosphate) analog that maintains its antiviral activity against drug-resistant HIV. Crystal structures for HIV-1 reverse transcriptase (RT) bound to double-stranded DNA, ternary complexes with either GS-9148-diphosphate or 2'-deoxyadenosine triphosphate (dATP), and a post-incorporation structure with GS-9148 translocated to the priming site were obtained to gain insight into the mechanism of RT inhibition. The binding of either GS-9148-diphosphate or dATP to the binary RT-DNA complex resulted in the fingers subdomain closing around the incoming substrate. This produced up to a 9 A shift in the tips of the fingers subdomain as it closed toward the palm and thumb subdomains. GS-9148-diphosphate shows a similar binding mode as dATP in the nucleotide-binding site. Residues whose mutations confer resistance to nucleotide/nucleoside RT inhibitors, such as M184, Y115, L74, and K65, show little to no shift in orientation whether GS-9148-diphosphate or dATP is bound. One difference observed in binding is the position of the central ring. The dihydrofuran ring of GS-9148-diphosphate interacts with the aromatic side chain of Y115 more than does the ribose ring of dATP, possibly picking up a favorable pi-pi interaction. The ability of GS-9148-diphosphate to mimic the active-site contacts of dATP may explain its effective inhibition of RT and maintained activity against resistance mutations. Interestingly, the 2'-fluoro moiety of GS-9148-diphosphate was found in close proximity to the Q151 side chain, potentially explaining the observed moderately reduced susceptibly to GS-9148 conferred by Q151M mutation.
Assuntos
DNA/química , DNA/metabolismo , Guanosina/análogos & derivados , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/metabolismo , Cristalografia por Raios X , Farmacorresistência Viral , Guanosina/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF 3Phe), (13)C/(15)N-labeled p-methoxyphenylalanine (OMePhe), and (15)N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF 3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in (1)H-(15)N HSQC, (1)H-(13)C HSQC, and (19)F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF 3Phe mutants of an active site tyrosine inhibited binding; incorporating (15)N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.