RESUMO
Acinetobacter baumannii, a commonly multidrug-resistant Gram-negative bacterium responsible for large numbers of bloodstream and lung infections worldwide, is increasingly difficult to treat and constitutes a growing threat to human health. Structurally novel antibacterial chemical matter that can evade existing resistance mechanisms is essential for addressing this critical medical need. Herein, we describe our efforts to inhibit the essential A. baumannii lipooligosaccharide (LOS) ATP-binding cassette (ABC) transporter MsbA. An unexpected impurity from a phenotypic screening was optimized as a series of dimeric compounds, culminating with 1 (cerastecin D), which exhibited antibacterial activity in the presence of human serum and a pharmacokinetic profile sufficient to achieve efficacy against A. baumannii in murine septicemia and lung infection models.
RESUMO
Carbapenem-resistant Acinetobacter baumannii infections have limited treatment options. Synthesis, transport and placement of lipopolysaccharide or lipooligosaccharide (LOS) in the outer membrane of Gram-negative bacteria are important for bacterial virulence and survival. Here we describe the cerastecins, inhibitors of the A. baumannii transporter MsbA, an LOS flippase. These molecules are potent and bactericidal against A. baumannii, including clinical carbapenem-resistant Acinetobacter baumannii isolates. Using cryo-electron microscopy and biochemical analysis, we show that the cerastecins adopt a serpentine configuration in the central vault of the MsbA dimer, stalling the enzyme and uncoupling ATP hydrolysis from substrate flipping. A derivative with optimized potency and pharmacokinetic properties showed efficacy in murine models of bloodstream or pulmonary A. baumannii infection. While resistance development is inevitable, targeting a clinically unexploited mechanism avoids existing antibiotic resistance mechanisms. Although clinical validation of LOS transport remains undetermined, the cerastecins may open a path to narrow-spectrum treatment modalities for important nosocomial infections.
Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Antibacterianos , Proteínas de Bactérias , Lipopolissacarídeos , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/metabolismo , Lipopolissacarídeos/metabolismo , Animais , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/tratamento farmacológico , Camundongos , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Transporte Biológico , Testes de Sensibilidade Microbiana , Humanos , Microscopia Crioeletrônica , Carbapenêmicos/farmacologia , Carbapenêmicos/metabolismo , Modelos Animais de Doenças , Feminino , Transportadores de Cassetes de Ligação de ATPRESUMO
All herpesviruses encode a conserved DNA polymerase that is required for viral genome replication and serves as an important therapeutic target. Currently available herpesvirus therapies include nucleoside and non-nucleoside inhibitors (NNI) that target the DNA-bound state of herpesvirus polymerase and block replication. Here we report the ternary complex crystal structure of Herpes Simplex Virus 1 DNA polymerase bound to DNA and a 4-oxo-dihydroquinoline NNI, PNU-183792 (PNU), at 3.5 Å resolution. PNU bound at the polymerase active site, displacing the template strand and inducing a conformational shift of the fingers domain into an open state. These results demonstrate that PNU inhibits replication by blocking association of dNTP and stalling the enzyme in a catalytically incompetent conformation, ultimately acting as a nucleotide competing inhibitor (NCI). Sequence conservation of the NCI binding pocket further explains broad-spectrum activity while a direct interaction between PNU and residue V823 rationalizes why mutations at this position result in loss of inhibition.
Assuntos
DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/genética , Herpesviridae/efeitos dos fármacos , Herpesviridae/enzimologia , Antivirais/farmacologia , Sítios de Ligação , DNA Polimerase Dirigida por DNA/metabolismo , Farmacorresistência Viral/efeitos dos fármacos , Exodesoxirribonucleases , Nucleotídeos , Quinolinas/farmacologia , Proteínas Virais , Replicação ViralRESUMO
By employing a phenotypic screen, a set of compounds, exemplified by 1, were identified which potentiate the ability of histone deacetylase inhibitor vorinostat to reverse HIV latency. Proteome enrichment followed by quantitative mass spectrometric analysis employing a modified analogue of 1 as affinity bait identified farnesyl transferase (FTase) as the primary interacting protein in cell lysates. This ligand-FTase binding interaction was confirmed via X-ray crystallography and temperature dependent fluorescence studies, despite 1 lacking structural and binding similarity to known FTase inhibitors. Although multiple lines of evidence established the binding interaction, these ligands exhibited minimal inhibitory activity in a cell-free biochemical FTase inhibition assay. Subsequent modification of the biochemical assay by increasing anion concentration demonstrated FTase inhibitory activity in this novel class. We propose 1 binds together with the anion in the active site to inhibit farnesyl transferase. Implications for phenotypic screening deconvolution and HIV reactivation are discussed.
RESUMO
A second-generation small molecule P2X3 receptor antagonist has been developed. The lead optimization strategy to address shortcomings of the first-generation preclinical lead compound is described herein. These studies were directed towards the identification and amelioration of preclinical hepatobiliary findings, reducing potential for drug-drug interactions, and decreasing the projected human dose of the first-generation lead.
Assuntos
Analgésicos/uso terapêutico , Benzamidas/uso terapêutico , Dor/tratamento farmacológico , Antagonistas do Receptor Purinérgico P2X/uso terapêutico , Piridinas/uso terapêutico , Receptores Purinérgicos P2X3/metabolismo , Analgésicos/síntese química , Analgésicos/química , Analgésicos/farmacocinética , Animais , Benzamidas/síntese química , Benzamidas/química , Benzamidas/farmacocinética , Cães , Desenho de Fármacos , Interações Medicamentosas , Glucuronosiltransferase/antagonistas & inibidores , Meia-Vida , Hiperbilirrubinemia/prevenção & controle , Estrutura Molecular , Antagonistas do Receptor Purinérgico P2X/síntese química , Antagonistas do Receptor Purinérgico P2X/química , Antagonistas do Receptor Purinérgico P2X/farmacocinética , Piridinas/síntese química , Piridinas/química , Piridinas/farmacocinética , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Conditions for facile solution-phase amide conjugation of amine-modified siRNA with a diverse set of carboxylic acid partners using the coupling reagent HATU are described. These conditions eliminate the need for isolated activated esters and allow for rapid access to conjugates with a wide range of lipophilicity and functionality in good yield.
Assuntos
Reagentes de Ligações Cruzadas/química , RNA Interferente Pequeno/química , Amidas/síntese química , Aminas/química , Ácidos Carboxílicos/química , Métodos , SoluçõesRESUMO
In our ongoing efforts to develop CGRP receptor antagonists for the treatment of migraine, we aimed to improve upon telecagepant by targeting a compound with a lower projected clinical dose. Imidazoazepanes were identified as potent caprolactam replacements and SAR of the imidazole yielded the tertiary methyl ether as an optimal substituent for potency and hERG selectivity. Combination with the azabenzoxazinone spiropiperidine ultimately led to preclinical candidate 30 (MK-2918).
Assuntos
Azepinas/síntese química , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Imidazóis/síntese química , Imidazóis/farmacologia , Analgésicos não Narcóticos/síntese química , Analgésicos não Narcóticos/química , Analgésicos não Narcóticos/farmacologia , Animais , Azepinas/química , Azepinas/farmacologia , Disponibilidade Biológica , Caprolactama/química , Células Cultivadas , Cães , Humanos , Imidazóis/química , Concentração Inibidora 50 , Macaca mulatta , Transtornos de Enxaqueca/tratamento farmacológico , Estrutura Molecular , Ratos , Relação Estrutura-AtividadeRESUMO
Calcitonin gene-related peptide (CGRP) is a neuropeptide that plays a key role in the pathophysiology of migraine headache. MK-0974 (telcagepant) is a potent and selective antagonist of the human and rhesus CGRP receptors and is currently in Phase III clinical studies for the acute treatment of migraine. The pharmacology of MK-0974 has been studied extensively, but there has not been a thorough characterization of its binding properties. Here, we characterize the binding of a tritiated analog of MK-0974 on human neuroblastoma (SK-N-MC) membranes and rhesus cerebellum. [(3)H]MK-0974 displayed reversible and saturable binding to both SK-N-MC membranes and rhesus cerebellum with a K(D) of 1.9 nM and 1.3 nM, respectively. Agonists and antagonists of the CGRP receptor displaced [(3)H]MK-0974 in a concentration-dependent manner in competition binding experiments. Both CGRP and adrenomedullin demonstrated biphasic competition while MK-0974 and the peptide antagonist CGRP(8-37) displaced [(3)H]MK-0974 in a monophasic fashion. In competitive binding studies with [(3)H]MK-0974 and CGRP, the fraction of high-affinity binding was reduced significantly by incubating the membranes with GTPgammaS. In kinetic binding experiments, the off-rate of [(3)H]MK-0974 was determined to be 0.51 min(-1) with a half-life of 1.3 min. In conclusion, the radioligand [(3)H]MK-0974 has proven to be a useful tool for studying the binding characteristics of MK-0974 and has broadened our understanding of this promising molecule.
Assuntos
Azepinas/metabolismo , Azepinas/uso terapêutico , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Imidazóis/metabolismo , Imidazóis/uso terapêutico , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Animais , Azepinas/química , Azepinas/farmacologia , Ligação Competitiva , Humanos , Imidazóis/química , Imidazóis/farmacologia , Cinética , Macaca mulatta , Ligação Proteica , Trítio/químicaRESUMO
Two novel routes have been developed to the (3 R,6 S)-3-amino-6-(2,3-difluorophenyl)-1-(2,2,2-trifluoroethyl)azepan-2-one 2 of the CGRP receptor antagonist clinical candidate telcagepant (MK-0974, 1). The first employs a ring-closing metathesis of the styrene 7 as the key reaction, while the second makes use of a highly diastereoselective Hayashi-Miyaura Rh-catalyzed arylboronic acid addition to nitroalkene 16. The latter route has been implemented to produce multigram quantities of telcagepant for extensive preclinical evaluation.
Assuntos
Azepinas/síntese química , Imidazóis/síntese química , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de CalcitoninaRESUMO
In our effort to find potent, orally bioavailable CGRP receptor antagonists for the treatment of migraine, a novel series based on a pyridinone template was investigated. After optimizing the privileged structure and the placement of the attached phenyl ring, systematic SAR was carried out on both the N-alkyl and C-5 aryl substituents. Several analogs with good potency and pharmacokinetic profiles were identified.
Assuntos
Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Piridonas/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Meia-Vida , Piridonas/administração & dosagem , Piridonas/química , Piridonas/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
Calcitonin gene-related peptide (CGRP) is a potent neuropeptide that plays a key role in the pathophysiology of migraine headache. CGRP levels in the cranial circulation are increased during a migraine attack, and CGRP itself has been shown to trigger migraine-like headache. The correlation between CGRP release and migraine headache points to the potential utility of CGRP receptor antagonists as novel therapeutics in the treatment of migraine. Indeed, clinical proof-of-concept in the acute treatment of migraine was demonstrated with an intravenous formulation of the CGRP receptor antagonist BIBN4096BS (olcegepant). Here we report on the pharmacological characterization of the first orally bioavailable CGRP receptor antagonist in clinical development, MK-0974 [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide]. In vitro, MK-0974 is a potent antagonist of the human (K(i) = 0.77 nM) and rhesus (K(i) = 1.2 nM) CGRP receptors but displays >1500-fold lower affinity for the canine and rat receptors as determined via (125)I-human CGRP competition binding assays. A rhesus pharmacodynamic assay measuring capsaicin-induced changes in forearm dermal blood flow via laser Doppler imaging was utilized to determine the in vivo activity of CGRP receptor antagonism. MK-0974 produced a concentration-dependent inhibition of dermal vasodilation, generated by capsaicin-induced release of endogenous CGRP, with plasma concentrations of 127 and 994 nM required to block 50 and 90% of the blood flow increase, respectively. In conclusion, MK-0974 is a highly potent, selective, and orally bioavailable CGRP receptor antagonist, which may be valuable in the acute treatment of migraine.
Assuntos
Azepinas/administração & dosagem , Azepinas/química , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Imidazóis/administração & dosagem , Imidazóis/química , Transtornos de Enxaqueca/tratamento farmacológico , Administração Oral , Animais , Linhagem Celular , Cães , Relação Dose-Resposta a Droga , Feminino , Humanos , Macaca mulatta , Masculino , Transtornos de Enxaqueca/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismoRESUMO
Calcitonin gene-related peptide (CGRP) has been implicated in the pathogenesis of migraine. Herein we describe optimization of CGRP receptor antagonists based on an earlier lead structure containing a (3R)-amino-(6S)-phenylcaprolactam core. Replacement of the phenylimidazolinone with an azabenzimidazolone gave stable derivatives with lowered serum shifts. Extensive SAR studies of the C-6 aryl moiety revealed the potency-enhancing effect of the 2,3-difluorophenyl group, and trifluoroethylation of the N-1 amide position resulted in improved oral bioavailabilities, ultimately leading to clinical candidate 38 (MK-0974).
Assuntos
Azepinas/síntese química , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Imidazóis/síntese química , Transtornos de Enxaqueca/tratamento farmacológico , Administração Oral , Animais , Azepinas/farmacocinética , Azepinas/farmacologia , Disponibilidade Biológica , Células CACO-2 , Permeabilidade da Membrana Celular , Cães , Humanos , Imidazóis/farmacocinética , Imidazóis/farmacologia , Macaca mulatta , Ratos , Fluxo Sanguíneo Regional/efeitos dos fármacos , Pele/irrigação sanguínea , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Calcitonin gene-related peptide (CGRP) has been implicated in the pathogenesis of migraine. Replacements for the benzodiazepine core of an earlier lead structure 1 including 5-, 6-, and 7-membered lactams were explored. Within the 7-membered ring scaffold, phenyl substitution at various positions afforded the potent (3R)-amino-(6S)-phenyl caprolactam template. The phenylimidazolinone privileged structure gave additional potency enhancements, as 24 showed good potency in both CGRP binding (K(i)=2 nM) and cAMP (IC(50)=4 nM) assays and was orally bioavailable in rats (27%).
Assuntos
Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Caprolactama/farmacologia , Transtornos de Enxaqueca/tratamento farmacológico , Administração Oral , Animais , Caprolactama/síntese química , Caprolactama/química , Química Farmacêutica/métodos , Desenho de Fármacos , Humanos , Imidazóis/farmacologia , Concentração Inibidora 50 , Cinética , Modelos Químicos , RatosRESUMO
A series of novel diaryl ether lactams have been identified as very potent dual inhibitors of protein farnesyltransferase (FTase) and protein geranylgeranyltransferase I (GGTase-I), enzymes involved in the prenylation of Ras. The structure of the complex formed between one of these compounds and FTase has been determined by X-ray crystallography. These compounds are the first reported to inhibit the prenylation of the important oncogene Ki-Ras4B in vivo. Unfortunately, doses sufficient to achieve this endpoint were rapidly lethal.