RESUMO
The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1-3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.
Assuntos
Guanosina , Nucleotidiltransferases , Adenosina , Animais , Interferons , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Transdução de SinaisRESUMO
This paper describes the structure-activity-relationships of novel fluoroalkyl substituents at the C2 position of iminothiazine dioxide beta secretase inhibitors. Key discoveries include reduced amidine basicity and its effect on Pgp, cell potency, and efficacy in various preclinical in vivo efficacy animal models. Findings from these structure-activity-relationships are discussed.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Óxidos/farmacologia , Tiazinas/farmacologia , Administração Oral , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Células HEK293 , Humanos , Estrutura Molecular , Óxidos/administração & dosagem , Óxidos/química , Ratos , Relação Estrutura-Atividade , Tiazinas/administração & dosagem , Tiazinas/químicaRESUMO
Fungi have historically been the source of numerous important medicinal compounds, but full exploitation of their genetic potential for drug development has been hampered in traditional discovery paradigms. Here we describe a radically different approach, top-down drug discovery (TD3), starting with a massive digital search through a database of over 100,000 fully genomicized fungi to identify loci encoding molecules with a predetermined human target. We exemplify TD3 by the selection of cyclin-dependent kinases (CDKs) as targets and the discovery of two molecules, 1 and 2, which inhibit therapeutically important human CDKs. 1 and 2 exhibit a remarkable mechanism, forming a site-selective covalent bond to the CDK active site Lys. We explored the structure-activity relationship via semi- and total synthesis, generating an analog, 43, with improved kinase selectivity, bioavailability, and efficacy. This work highlights the power of TD3 to identify mechanistically and structurally novel molecules for the development of new medicines.
Assuntos
Quinases Ciclina-Dependentes , Descoberta de Drogas , Inibidores de Proteínas Quinases , Humanos , Relação Estrutura-Atividade , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Animais , Genômica/métodos , Modelos MolecularesRESUMO
From an initial lead 1, a structure-based design approach led to identification of a novel, high-affinity iminohydantoin BACE1 inhibitor that lowers CNS-derived Aß following oral administration to rats. Herein we report SAR development in the S3 and F' subsites of BACE1 for this series, the synthetic approaches employed in this effort, and in vivo data for the optimized compound.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/antagonistas & inibidores , Anticonvulsivantes/síntese química , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Hidantoínas/síntese química , Administração Oral , Doença de Alzheimer/sangue , Doença de Alzheimer/líquido cefalorraquidiano , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Animais , Anticonvulsivantes/farmacologia , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Modelos Animais de Doenças , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Hidantoínas/farmacologia , Modelos Moleculares , Ligação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
Stereochemically and structurally complex cyclic dinucleotide-based stimulator of interferon genes (STING) agonists were designed and synthesized to access a previously unexplored chemical space. The assessment of biochemical affinity and cellular potency, along with computational, structural, and biophysical characterization, was applied to influence the design and optimization of novel STING agonists, resulting in the discovery of MK-1454 as a molecule with appropriate properties for clinical development. When administered intratumorally to immune-competent mice-bearing syngeneic tumors, MK-1454 exhibited robust tumor cytokine upregulation and effective antitumor activity. Tumor shrinkage in mouse models that are intrinsically resistant to single-agent therapy was further enhanced when treating the animals with MK-1454 in combination with a fully murinized antimouse PD-1 antibody, mDX400. These data support the development of STING agonists in combination with pembrolizumab (humanized anti-PD-1 antibody) for patients with tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 therapy.
Assuntos
Proteínas de Membrana , Neoplasias , Animais , Citocinas , Humanos , Imunoterapia/métodos , Interferons , Camundongos , Neoplasias/tratamento farmacológicoRESUMO
The action of arginase, a metalloenzyme responsible for the hydrolysis of arginine to urea and ornithine, is hypothesized to suppress immune-cell activity within the tumor microenvironment, and thus its inhibition may constitute a means by which to potentiate the efficacy of immunotherapeutics such as anti-PD-1 checkpoint inhibitors. Taking inspiration from reported enzyme-inhibitor cocrystal structures, we designed and synthesized novel inhibitors of human arginase possessing a fused 5,5-bicyclic ring system. The prototypical member of this class, 3, when dosed orally, successfully demonstrated serum arginase inhibition and concomitant arginine elevation in a syngeneic mouse carcinoma model, despite modest oral bioavailability. Structure-based design strategies to improve the bioavailability of this class, including scaffold modification, fluorination, and installation of active-transport recognition motifs were explored.
RESUMO
Pharmacological activation of the STING (stimulator of interferon genes)-controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-ß secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti-PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Membrana/metabolismo , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , HumanosRESUMO
Herein we describe structure-activity relationship (SAR) and metabolite identification (Met-ID) studies that provided insight into the origin of time-dependent inhibition (TDI) of cytochrome P450 3A4 (CYP3A4) by compound 1. Collectively, these efforts revealed that bioactivation of the fluoropyrimidine moiety of 1 led to reactive metabolite formation via oxidative defluorination and was responsible for the observed TDI. We discovered that substitution at both the 4- and 6-positions of the 5-fluoropyrimidine of 1 was necessary to ameliorate this TDI as exemplified by compound 19.
Assuntos
Inibidores do Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A/farmacologia , Citocromo P-450 CYP3A/metabolismo , Pirimidinas/química , Pirimidinas/farmacologia , Animais , Inibidores do Citocromo P-450 CYP3A/farmacocinética , Humanos , Cinética , Pirimidinas/farmacocinética , Ratos , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
ß-Amyloid (Aß) peptides are thought to be critically involved in the etiology of Alzheimer's disease (AD). The aspartyl protease ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) is required for the production of Aß, and BACE1 inhibition is thus an attractive target for the treatment of AD. We show that verubecestat (MK-8931) is a potent, selective, structurally unique BACE1 inhibitor that reduced plasma, cerebrospinal fluid (CSF), and brain concentrations of Aß40, Aß42, and sAPPß (a direct product of BACE1 enzymatic activity) after acute and chronic administration to rats and monkeys. Chronic treatment of rats and monkeys with verubecestat achieved exposures >40-fold higher than those being tested in clinical trials in AD patients yet did not elicit many of the adverse effects previously attributed to BACE inhibition, such as reduced nerve myelination, neurodegeneration, altered glucose homeostasis, or hepatotoxicity. Fur hypopigmentation was observed in rabbits and mice but not in monkeys. Single and multiple doses were generally well tolerated and produced reductions in Aß40, Aß42, and sAPPß in the CSF of both healthy human subjects and AD patients. The human data were fit to an amyloid pathway model that provided insight into the Aß pools affected by BACE1 inhibition and guided the choice of doses for subsequent clinical trials.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Sistema Nervoso Central/metabolismo , Óxidos S-Cíclicos/farmacologia , Tiadiazinas/farmacologia , Administração Oral , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Encéfalo/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Feminino , Glucose/metabolismo , Macaca fascicularis , Espectroscopia de Ressonância Magnética , Camundongos , Bainha de Mielina/química , Peptídeos/química , Coelhos , RatosRESUMO
We describe successful efforts to optimize the in vivo profile and address off-target liabilities of a series of BACE1 inhibitors represented by 6 that embodies the recently validated fused pyrrolidine iminopyrimidinone scaffold. Employing structure-based design, truncation of the cyanophenyl group of 6 that binds in the S3 pocket of BACE1 followed by modification of the thienyl group in S1 was pursued. Optimization of the pyrimidine substituent that binds in the S2'-S2â³ pocket of BACE1 remediated time-dependent CYP3A4 inhibition of earlier analogues in this series and imparted high BACE1 affinity. These efforts resulted in the discovery of difluorophenyl analogue 9 (MBi-4), which robustly lowered CSF and cortex Aß40 in both rats and cynomolgus monkeys following a single oral dose. Compound 9 represents a unique molecular shape among BACE inhibitors reported to potently lower central Aß in nonrodent preclinical species.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Desenho de Fármacos , Compostos Heterocíclicos/química , Iminas/química , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Animais , Córtex Cerebral/metabolismo , Inibidores Enzimáticos/farmacologia , Macaca fascicularis , Estrutura Molecular , Ratos , Relação Estrutura-AtividadeRESUMO
Verubecestat 3 (MK-8931), a diaryl amide-substituted 3-imino-1,2,4-thiadiazinane 1,1-dioxide derivative, is a high-affinity ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor currently undergoing Phase 3 clinical evaluation for the treatment of mild to moderate and prodromal Alzheimer's disease. Although not selective over the closely related aspartyl protease BACE2, verubecestat has high selectivity for BACE1 over other key aspartyl proteases, notably cathepsin D, and profoundly lowers CSF and brain Aß levels in rats and nonhuman primates and CSF Aß levels in humans. In this annotation, we describe the discovery of 3, including design, validation, and selected SAR around the novel iminothiadiazinane dioxide core as well as aspects of its preclinical and Phase 1 clinical characterization.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Óxidos S-Cíclicos/farmacologia , Descoberta de Drogas , Tiadiazinas/farmacologia , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Óxidos S-Cíclicos/síntese química , Óxidos S-Cíclicos/química , Cães , Relação Dose-Resposta a Droga , Humanos , Macaca fascicularis , Modelos Moleculares , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tiadiazinas/síntese química , Tiadiazinas/químicaRESUMO
In early 1999, beta-amyloid cleaving enzyme-1 (BACE-1) was identified as the protease responsible for the critical first step in the processing of beta-amyloid precursor protein that ultimately leads to the production of Abeta peptides in the brain. Accumulation of these peptides has been implicated in the pathology of Alzheimer's disease (AD). An inhibitor of BACE-1 would therefore have therapeutic potential to slow or halt the progression of this debilitating and ultimately fatal disease. This review provides a perspective on the recent developments in the design of BACE-1 inhibitors. An overview of early research is also included, with particular emphasis on a comprehensive survey of the patent literature.
Assuntos
Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/uso terapêutico , Desenho de Fármacos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Doença de Alzheimer/fisiopatologia , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases/química , Avaliação Pré-Clínica de Medicamentos/métodos , Indústria Farmacêutica/métodos , Endopeptidases , Humanos , Estrutura MolecularRESUMO
Inhibition of BACE1 to prevent brain Aß peptide formation is a potential disease-modifying approach to the treatment of Alzheimer's disease. Despite over a decade of drug discovery efforts, the identification of brain-penetrant BACE1 inhibitors that substantially lower CNS Aß levels following systemic administration remains challenging. In this report we describe structure-based optimization of a series of brain-penetrant BACE1 inhibitors derived from an iminopyrimidinone scaffold. Application of structure-based design in tandem with control of physicochemical properties culminated in the discovery of compound 16, which potently reduced cortex and CSF Aß40 levels when administered orally to rats.
RESUMO
On the basis of our observation that the biaryl substituent of iminopyrimidinone 7 must be in a pseudoaxial conformation to occupy the contiguous S1-S3 subsites of BACE1, we have designed a novel fused bicyclic iminopyrimidinone scaffold intended to favor this bioactive conformation. Strategic incorporation of a nitrogen atom in the new constrained ring allowed us to develop SAR around the S2' binding pocket and ultimately resulted in analogues with low nanomolar potency for BACE1. In particular, optimization of the prime side substituent led to major improvements in potency by displacement of two conserved water molecules from a region near S2'. Further optimization of the pharmacokinetic properties of this fused pyrrolidine series, in conjunction with facile access to a rat pharmacodynamic model, led to identification of compound 43, which is an orally active, brain penetrant inhibitor that reduces Aß(40) in the plasma, CSF, and cortex of rats in a dose-dependent manner.