RESUMEN
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.
RESUMEN
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.
Asunto(s)
Infecciones por Acinetobacter , Acinetobacter baumannii , Antibacterianos , Proteínas Bacterianas , Lipopolisacáridos , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/metabolismo , Lipopolisacáridos/metabolismo , Animales , Infecciones por Acinetobacter/microbiología , Infecciones por Acinetobacter/tratamiento farmacológico , Ratones , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Transporte Biológico , Pruebas de Sensibilidad Microbiana , Humanos , Microscopía por Crioelectrón , Carbapenémicos/farmacología , Carbapenémicos/metabolismo , Modelos Animales de Enfermedad , Femenino , Transportadoras de Casetes de Unión a ATPRESUMEN
Herein we report the development of an automated deoxygenative C(sp2)-C(sp3) coupling of aryl bromide with alcohols to enable parallel medicinal chemistry. Alcohols are among the most diverse and abundant building blocks, but their usage as alkyl precursors has been limited. Although metallaphotoredox deoxygenative coupling is becoming a promising strategy to form C(sp2)-C(sp3) bond, the reaction setup limits its widespread application in library synthesis. To achieve high throughput and consistency, an automated workflow involving solid-dosing and liquid-handling robots has been developed. We have successfully demonstrated this high-throughput protocol is robust and consistent across three automation platforms. Furthermore, guided by cheminformatic analysis, we examined alcohols with comprehensive chemical space coverage and established a meaningful scope for medicinal chemistry applications. By accessing the rich diversity of alcohols, this automated protocol has the potential to substantially increase the impact of C(sp2)-C(sp3) cross-coupling in drug discovery.
RESUMEN
Mutations in the lysosomal enzyme glucocerebrosidase (GCase, GBA1 gene) are the most common genetic risk factor for developing Parkinson's disease (PD). GCase metabolizes the glycosphingolipids glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Mutations in GBA1 reduce enzyme activity and the resulting accumulation of glycosphingolipids may contribute to the underlying pathology of PD, possibly via altering lysosomal function. While reduction of GCase activity exacerbates α-synuclein (α-syn) aggregation, it has not been determined that this effect is the result of altered glycosphingolipid levels and lysosome function or some other effect of altering GCase. The glycosphingolipid GlcCer is synthesized by a single enzyme, glucosylceramide synthase (GCS), and small molecule inhibitors (GCSi) reduce cellular glycosphingolipid levels. In the present studies, we utilize a preformed fibril (PFF) rodent primary neuron in vitro model of α-syn pathology to investigate the relationship between glycosphingolipid levels, α-syn pathology, and lysosomal function. In primary cultures, pharmacological inhibition of GCase and D409V GBA1 mutation enhanced accumulation of glycosphingolipids and insoluble phosphorylated α-syn. Administration of a novel small molecule GCSi, benzoxazole 1 (BZ1), significantly decreased glycosphingolipid concentrations in rodent primary neurons and reduced α-syn pathology. BZ1 rescued lysosomal deficits associated with the D409V GBA1 mutation and α-syn PFF administration, and attenuated α-syn induced neurodegeneration of dopamine neurons. In vivo studies revealed BZ1 had pharmacological activity and reduced glycosphingolipids in the mouse brain to a similar extent observed in neuronal cultures. These data support the hypothesis that reduction of glycosphingolipids through GCS inhibition may impact progression of synucleinopathy and BZ1 is useful tool to further examine this important biology.
Asunto(s)
Benzoxazoles/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Glucosilceramidasa/genética , Glucosiltransferasas/antagonistas & inhibidores , Glicoesfingolípidos/metabolismo , Lisosomas/efectos de los fármacos , Sinucleinopatías/metabolismo , alfa-Sinucleína/efectos de los fármacos , Animales , Neuronas Dopaminérgicas/metabolismo , Técnicas In Vitro , Lisosomas/metabolismo , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Cultivo Primario de Células , Agregado de Proteínas , Ratas , Sinucleinopatías/genética , alfa-Sinucleína/metabolismoRESUMEN
The measurement of receptor occupancy (RO) using positron emission tomography (PET) has been instrumental in guiding discovery and development of CNS directed therapeutics. We and others have investigated muscarinic acetylcholine receptor 4 (M4) positive allosteric modulators (PAMs) for the treatment of symptoms associated with neuropsychiatric disorders. In this article, we describe the synthesis, in vitro, and in vivo characterization of a series of central pyridine-related M4 PAMs that can be conveniently radiolabeled with carbon-11 as PET tracers for the in vivo imaging of an allosteric binding site of the M4 receptor. We first demonstrated its feasibility by mapping the receptor distribution in mouse brain and confirming that a lead molecule 1 binds selectively to the receptor only in the presence of the orthosteric agonist carbachol. Through a competitive binding affinity assay and a number of physiochemical properties filters, several related compounds were identified as candidates for in vivo evaluation. These candidates were then radiolabeled with 11C and studied in vivo in rhesus monkeys. This research eventually led to the discovery of the clinical radiotracer candidate [11C]MK-6884.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Piridinas/farmacología , Receptor Muscarínico M4/agonistas , Animales , Células CHO , Radioisótopos de Carbono/química , Radioisótopos de Carbono/farmacología , Cricetulus , Humanos , Macaca mulatta , Agonistas Muscarínicos/química , Enfermedades Neurodegenerativas/diagnóstico por imagen , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Tomografía de Emisión de Positrones , Piridinas/química , Receptor Muscarínico M4/metabolismoRESUMEN
Herein we describe the discovery and optimization of a new series of 2,3-disubstituted and 2,3,6-trisubstituted muscarinic acetylcholine receptorâ 4 (M4 ) positive allosteric modulators (PAMs). Iterative libraries enabled rapid exploration of one-dimensional structure-activity relationships (SAR) and identification of potency-enhancing heterocycle and N-alkyl pyrazole substituents. Further optimization led to identification of the potent, receptor-subtype-selective, brain-penetrant tool compound 24 (7-[3-[1-[(1-fluorocyclopentyl)methyl]pyrazol-4-yl]-6-methyl-2-pyridyl]-3-methoxycinnoline). It is efficacious in preclinical assays that are predictive of antipsychotic effects, producing dose-dependent reversal of amphetamine-induced hyperlocomotion in rats and mice, but not in M4 knockout mice. Cholinergic-related adverse effects observed in rats treated with 24 at unbound plasma concentrations more than 3-fold higher than an efficacious dose in the hyperlocomotion assay were fewer and less severe than those observed in rats treated with the nonselective M4 agonist xanomeline, suggesting a receptor-subtype-selective PAM has the potential for an improved safety profile.
Asunto(s)
Descubrimiento de Drogas , Piridinas/química , Piridinas/farmacología , Receptor Muscarínico M4/efectos de los fármacos , Regulación Alostérica , Animales , Humanos , Ratas , Receptor Muscarínico M4/metabolismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Analgésicos/uso terapéutico , Benzamidas/uso terapéutico , Dolor/tratamiento farmacológico , Antagonistas del Receptor Purinérgico P2X/uso terapéutico , Piridinas/uso terapéutico , Receptores Purinérgicos P2X3/metabolismo , Analgésicos/síntesis química , Analgésicos/química , Analgésicos/farmacocinética , Animales , Benzamidas/síntesis química , Benzamidas/química , Benzamidas/farmacocinética , Perros , Diseño de Fármacos , Interacciones Farmacológicas , Glucuronosiltransferasa/antagonistas & inhibidores , Semivida , Hiperbilirrubinemia/prevención & control , Estructura Molecular , Antagonistas del Receptor Purinérgico P2X/síntesis química , Antagonistas del Receptor Purinérgico P2X/química , Antagonistas del Receptor Purinérgico P2X/farmacocinética , Piridinas/síntesis química , Piridinas/química , Piridinas/farmacocinética , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Studies on human genetics have suggested that inhibitors of the Nav1.7 voltage-gated sodium channel hold considerable promise as therapies for the treatment of chronic pain syndromes. Herein, we report novel, peripherally-restricted benzoxazolinone aryl sulfonamides as potent Nav1.7 inhibitors with excellent selectivity against the Nav1.5 isoform, which is expressed in the heart muscle. Elaboration of initial lead compound 3d afforded exemplar 13, which featured attractive physicochemical properties, outstanding lipophilic ligand efficiency and pharmacological selectivity against Nav1.5 exceeding 1000-fold. Key structure-activity relationships associated with oral bioavailability were leveraged to discover compound 17, which exhibited a comparable potency/selectivity profile as well as full efficacy following oral administration in a preclinical model indicative of antinociceptive behavior.
Asunto(s)
Analgésicos/farmacología , Benzoxazoles/farmacología , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Dolor/tratamiento farmacológico , Sulfonamidas/farmacología , Administración Oral , Analgésicos/administración & dosificación , Analgésicos/química , Animales , Benzoxazoles/administración & dosificación , Benzoxazoles/química , Disponibilidad Biológica , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Formaldehído/administración & dosificación , Humanos , Ratones , Estructura Molecular , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Dolor/inducido químicamente , Ratas , Relación Estructura-Actividad , Sulfonamidas/administración & dosificación , Sulfonamidas/químicaRESUMEN
3-Hydroxy-4-pyridinones and 5-hydroxy-4-pyrimidinones were identified as inhibitors of catechol-O-methyltransferase (COMT) in a high-throughput screen. These heterocyclic catechol mimics exhibit potent inhibition of the enzyme and an improved toxicity profile versus the marketed nitrocatechol inhibitors tolcapone and entacapone. Optimization of the series was aided by X-ray cocrystal structures of the novel inhibitors in complex with COMT and cofactors SAM and Mg(2+). The crystal structures suggest a mechanism of inhibition for these heterocyclic inhibitors distinct from previously disclosed COMT inhibitors.
RESUMEN
Reduced dopamine neurotransmission in the prefrontal cortex has been implicated as causal for the negative symptoms and cognitive deficit associated with schizophrenia; thus, a compound which selectively enhances dopamine neurotransmission in the prefrontal cortex may have therapeutic potential. Inhibition of catechol-O-methyltransferase (COMT, EC 2.1.1.6) offers a unique advantage, since this enzyme is the primary mechanism for the elimination of dopamine in cortical areas. Since membrane bound COMT (MB-COMT) is the predominant isoform in human brain, a high throughput screen (HTS) to identify novel MB-COMT specific inhibitors was completed. Subsequent optimization led to the identification of novel, non-nitrocatechol COMT inhibitors, some of which interact specifically with MB-COMT. Compounds were characterized for in vitro efficacy versus human and rat MB and soluble (S)-COMT. Select compounds were administered to male Wistar rats, and ex vivo COMT activity, compound levels in plasma and cerebrospinal fluid (CSF), and CSF dopamine metabolite levels were determined as measures of preclinical efficacy. Finally, novel non-nitrocatechol COMT inhibitors displayed less potent uncoupling of the mitochondrial membrane potential (MMP) compared to tolcapone as well as nonhepatotoxic entacapone, thus mitigating the risk of hepatotoxicity.
Asunto(s)
Antipsicóticos/farmacocinética , Inhibidores de Catecol O-Metiltransferasa , Catecol O-Metiltransferasa/metabolismo , Inhibidores Enzimáticos/farmacología , Animales , Antipsicóticos/síntesis química , Benzofenonas/química , Benzofenonas/farmacología , Biomarcadores , Western Blotting , Catecol O-Metiltransferasa/aislamiento & purificación , Membrana Celular/enzimología , Membrana Celular/metabolismo , Dopamina/metabolismo , Inhibidores Enzimáticos/química , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Masculino , Metaloproteinasas de la Matriz/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Nitrofenoles/química , Nitrofenoles/farmacología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Proteínas Recombinantes/química , Esquizofrenia/tratamiento farmacológico , Especificidad por Sustrato , TolcaponaRESUMEN
INTRODUCTION: An (18)F-labeled positron emission tomography (PET) tracer for amyloid plaque is desirable for early diagnosis of Alzheimer's disease, particularly to enable preventative treatment once effective therapeutics are available. Similarly, such a tracer would be useful as a biomarker for enrollment of patients in clinical trials for evaluation of antiamyloid therapeutics. Furthermore, changes in the level of plaque burden as quantified by an amyloid plaque PET tracer may provide valuable insights into the effectiveness of amyloid-targeted therapeutics. This work describes our approach to evaluate and select a candidate PET tracer for in vivo quantification of human amyloid plaque. METHODS: Ligands were evaluated for their in vitro binding to human amyloid plaques, lipophilicity and predicted blood-brain barrier permeability. Candidates with favorable in vitro properties were radiolabeled with (18)F and evaluated in vivo. Baseline PET scans in rhesus monkey were conducted to evaluate the regional distribution and kinetics of each tracer using tracer kinetic modeling methods. High binding potential in cerebral white matter and cortical grey matter was considered an unfavorable feature of the candidate tracers. RESULTS: [(18)F]MK-3328 showed the most favorable combination of low in vivo binding potential in white matter and cortical grey matter in rhesus monkeys, low lipophilicity (Log D=2.91) and high affinity for human amyloid plaques (IC(50)=10.5±1.3 nM). CONCLUSIONS: [(18)F]MK-3328 was identified as a promising PET tracer for in vivo quantification of amyloid plaques, and further evaluation in humans is warranted.
Asunto(s)
Encéfalo/metabolismo , Radioisótopos de Flúor/farmacocinética , Oxazoles/farmacocinética , Placa Amiloide/metabolismo , Piridinas/farmacocinética , Radiofármacos , Enfermedad de Alzheimer/diagnóstico por imagen , Animales , Autorradiografía , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Humanos , Concentración 50 Inhibidora , Macaca mulatta , Placa Amiloide/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Radiofármacos/síntesis química , Radiofármacos/farmacocinética , Distribución TisularRESUMEN
The Merck Fragment Library was screened versus acid-sensing ion channel 3 (ASIC3), a novel target for the treatment of pain. Fragment hits were optimized using two strategies, and potency was improved from 0.7 mM to 3 µM with retention of good ligand efficiency and incorporation of reasonable physical properties, off-target profile, and rat pharmacokinetics.
Asunto(s)
Descubrimiento de Drogas , Fenómenos Electrofisiológicos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Canales Iónicos Sensibles al Ácido , Animales , Estructura Molecular , Fragmentos de Péptidos , Ratas , Bibliotecas de Moléculas Pequeñas , Canales de SodioRESUMEN
5-Fluoro-2-aryloxazolo[5,4-b]pyridines were synthesized and investigated as potential (18)F containing ß-amyloid PET ligands. In competition binding assays using human AD brain homogenates, compounds 14b, 16b, and 17b were identified as having favorable potency versus human ß-amyloid plaque and were radiolabeled for further evaluation in in vitro binding and in vivo PET imaging experiments. These studies led to the identification of 17b (MK-3328) as a candidate PET ligand for the clinical assessment of ß-amyloid plaque load.
RESUMEN
[reaction: see text] Additions of chiral allenylzinc and indium reagents to N-Boc alaninal were examined as a possible route to a C20-C26 segment of superstolide A. Allenylzinc reagents, prepared in situ by palladiozincation of (R)- and (S)-5-pivalyloxy-3-butyn-2-ol mesylate, showed excellent reagent control to afford the anti,syn and anti,anti diastereomers as the nearly exclusive adducts.