RESUMO
CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.
Assuntos
Sistemas CRISPR-Cas , Endonucleases/metabolismo , Edição de Genes , Linhagem Celular Tumoral , Endonucleases/genética , Células Hep G2 , Humanos , Estrutura Molecular , Engenharia de ProteínasRESUMO
A compact and stable bicyclic bridged ketal was developed as a ligand for the asialoglycoprotein receptor (ASGPR). This compound showed excellent ligand efficiency, and the molecular details of binding were revealed by the first X-ray crystal structures of ligand-bound ASGPR. This analogue was used to make potent di- and trivalent binders of ASGPR. Extensive characterization of the function of these compounds showed rapid ASGPR-dependent cellular uptake in vitro and high levels of liver/plasma selectivity in vivo. Assessment of the biodistribution in rodents of a prototypical Alexa647-labeled trivalent conjugate showed selective hepatocyte targeting with no detectable distribution in nonparenchymal cells. This molecule also exhibited increased ASGPR-directed hepatocellular uptake and prolonged retention compared to a similar GalNAc derived trimer conjugate. Selective release in the liver of a passively permeable small-molecule cargo was achieved by retro-Diels-Alder cleavage of an oxanorbornadiene linkage, presumably upon encountering intracellular thiol. Therefore, the multicomponent construct described here represents a highly efficient delivery vehicle to hepatocytes.
Assuntos
Receptor de Asialoglicoproteína/metabolismo , Compostos Bicíclicos com Pontes/química , Hepatócitos/metabolismo , Cetonas/química , Fígado/metabolismo , Polímeros/química , Compostos Bicíclicos com Pontes/metabolismo , Cristalografia por Raios X , Portadores de Fármacos/química , Humanos , Cetonas/metabolismo , Fígado/citologia , Modelos Moleculares , Estrutura Molecular , Polímeros/metabolismoRESUMO
A series of small-molecule YEATS4 binders have been discovered as part of an ongoing research effort to generate high-quality probe molecules for emerging and/or challenging epigenetic targets. Analogues such as 4d and 4e demonstrate excellent potency and selectivity for YEATS4 binding versus YEATS1,2,3 and exhibit good physical properties and in vitro safety profiles. A new X-ray crystal structure confirms direct binding of this chemical series to YEATS4 at the lysine acetylation recognition site of the YEATS domain. Multiple analogues engage YEATS4 with nanomolar potency in a whole-cell nanoluciferase bioluminescent resonance energy transfer assay. Rodent pharmacokinetic studies demonstrate the competency of several analogues as in vivo-capable binders.
Assuntos
Regulação da Expressão Gênica , Processamento de Proteína Pós-Traducional , Domínios Proteicos , Acetilação , Epigênese GenéticaRESUMO
Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (ß6 Glu â Val) on the ß-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 (23). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23, a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.
Assuntos
Anemia Falciforme/tratamento farmacológico , Hemoglobina A/efeitos dos fármacos , Hemoglobina Falciforme/efeitos dos fármacos , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Animais , Eritrócitos/metabolismo , Camundongos , Oxigênio/metabolismo , Quinolinas/químicaRESUMO
Modifications to the sugar portion of C-aryl glycoside sodium glucose transporter 2 (SGLT2) inhibitors were explored, including systematic deletion and modification of each of the glycoside hydroxyl groups. Based on results showing activity to be quite tolerant of structural change at the C-5 position, a series of novel C-5 spiro analogues was prepared. Some of these analogues exhibit low nanomolar potency versus SGLT2 and promote urinary glucose excretion (UGE) in rats. However, due to sub-optimal pharmacokinetic parameters (in particular half-life), predicted human doses did not meet criteria for further advancement.
Assuntos
Glicosídeos/química , Hipoglicemiantes/química , Inibidores do Transportador 2 de Sódio-Glicose , Compostos de Espiro/química , Animais , Ciclização , Glicosídeos/síntese química , Glicosídeos/farmacocinética , Humanos , Hipoglicemiantes/síntese química , Hipoglicemiantes/farmacocinética , Masculino , Microssomos Hepáticos/metabolismo , Ratos , Ratos Sprague-Dawley , Transportador 2 de Glucose-Sódio/metabolismoRESUMO
In the title compound, C(19)H(21)ClOS(2), the dithiane ring adopts a chair conformation. The dihedral angle between the benzene rings is 87.88â (4)°. In the crystal, inversion dimmers linked by pairs of C-Hâ¯O inter-actions occur.
RESUMO
Increased fructose consumption and its subsequent metabolism have been implicated in metabolic disorders such as nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH) and insulin resistance. Ketohexokinase (KHK) converts fructose to fructose-1-phosphate (F1P) in the first step of the metabolic cascade. Herein we report the discovery of a first-in-class KHK inhibitor, PF-06835919 (8), currently in phase 2 clinical trials. The discovery of 8 was built upon our originally reported, fragment-derived lead 1 and the recognition of an alternative, rotated binding mode upon changing the ribose-pocket binding moiety from a pyrrolidinyl to an azetidinyl ring system. This new binding mode enabled efficient exploration of the vector directed at the Arg-108 residue, leading to the identification of highly potent 3-azabicyclo[3.1.0]hexane acetic acid-based KHK inhibitors by combined use of parallel medicinal chemistry and structure-based drug design.
Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Frutoquinases/antagonistas & inibidores , Frutoquinases/metabolismo , Frutose/efeitos adversos , Doenças Metabólicas/enzimologia , Animais , Cristalografia por Raios X , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Frutose/administração & dosagem , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Resistência à Insulina/fisiologia , Masculino , Doenças Metabólicas/induzido quimicamente , Doenças Metabólicas/tratamento farmacológico , Estrutura Secundária de Proteína , Ratos , Ratos WistarRESUMO
Preclinical and clinical data suggest that acetyl-CoA carboxylase (ACC) inhibitors have the potential to rebalance disordered lipid metabolism, leading to improvements in nonalcoholic steatohepatitis (NASH). Consistent with these observations, first-in-human clinical trials with our ACC inhibitor PF-05175157 led to robust reduction of de novo lipogenesis (DNL), albeit with concomitant reductions in platelet count, which were attributed to the inhibition of fatty acid synthesis within bone marrow. Herein, we describe the design, synthesis, and evaluation of carboxylic acid-based ACC inhibitors with organic anion transporting polypeptide (OATP) substrate properties, which facilitated selective distribution of the compounds at the therapeutic site of action (liver) relative to the periphery. These efforts led to the discovery of clinical candidate PF-05221304 (12), which selectively inhibits liver DNL in animals, while demonstrating considerable safety margins against platelet reduction in a nonhuman primate model.
Assuntos
Acetil-CoA Carboxilase/antagonistas & inibidores , Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/farmacologia , Fígado/efeitos dos fármacos , Acetil-CoA Carboxilase/metabolismo , Animais , Inibidores Enzimáticos/uso terapêutico , Humanos , Lipogênese , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Especificidade por SubstratoRESUMO
C-X-C chemokine receptor type 7 (CXCR7) is involved in cardiac and immune pathophysiology. We report the discovery of a novel 1,4-diazepine CXCR7 modulator, demonstrating for the first time the role of pharmacological CXCR7 intervention in cardiac repair. Structure-activity-relationship (SAR) studies demonstrated that a net reduction in lipophilicity (log D) and an incorporation of saturated ring systems yielded compounds with good CXCR7 potencies and improvements in oxidative metabolic stability in human-liver microsomes (HLM). Tethering an ethylene amide further improved the selectivity profile (e.g., for compound 18, CXCR7 Ki = 13 nM, adrenergic α 1a Kb > 10â¯000 nM, and adrenergic ß 2 Kb > 10â¯000 nM). The subcutaneous administration of 18 in mice led to a statistically significant increase in circulating concentrations of plasma stromal-cell-derived factor 1α (SDF-1α) of approximately 2-fold. Chronic dosing of compound 18 in a mouse model of isoproterenol-induced cardiac injury further resulted in a statistically significant reduction of cardiac fibrosis.
Assuntos
Acetamidas/uso terapêutico , Azepinas/uso terapêutico , Cardiotônicos/uso terapêutico , Fibrose/tratamento farmacológico , Cardiopatias/tratamento farmacológico , Receptores CXCR/metabolismo , Acetamidas/síntese química , Acetamidas/química , Acetamidas/farmacologia , Animais , Azepinas/síntese química , Azepinas/química , Azepinas/farmacologia , Cardiotônicos/síntese química , Cardiotônicos/química , Cardiotônicos/farmacologia , Cães , Fibrose/induzido quimicamente , Cardiopatias/induzido quimicamente , Humanos , Interações Hidrofóbicas e Hidrofílicas , Isoproterenol , Células Madin Darby de Rim Canino , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The optimization of a new class of small molecule PCSK9 mRNA translation inhibitors is described. The potency, physicochemical properties, and off-target pharmacology associated with the hit compound (1) were improved by changes to two regions of the molecule. The last step in the synthesis of the congested amide center was enabled by three different routes. Subtle structural changes yielded significant changes in pharmacology and off-target margins. These efforts led to the identification of 7l and 7n with overall profiles suitable for in vivo evaluation. In a 14-day toxicology study, 7l demonstrated an improved safety profile vs lead 7f. We hypothesize that the improved safety profile is related to diminished binding of 7l to nontranslating ribosomes and an apparent improvement in transcript selectivity due to the lower strength of 7l stalling of off-target proteins.
Assuntos
Inibidores de PCSK9 , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Animais , Desenho de Fármacos , Masculino , Inibidores de Proteases/efeitos adversos , Inibidores de Proteases/metabolismo , Ratos , Ratos Sprague-Dawley , Segurança , Relação Estrutura-AtividadeRESUMO
Optimization of the pharmacokinetic (PK) properties of a series of activators of adenosine monophosphate-activated protein kinase (AMPK) is described. Derivatives of the previously described 5-aryl-indole-3-carboxylic acid clinical candidate (1) were examined with the goal of reducing glucuronidation rate and minimizing renal excretion. Compounds 10 (PF-06679142) and 14 (PF-06685249) exhibited robust activation of AMPK in rat kidneys as well as desirable oral absorption, low plasma clearance, and negligible renal clearance in preclinical species. A correlation of in vivo renal clearance in rats with in vitro uptake by human and rat renal organic anion transporters (human OAT/rat Oat) was identified. Variation of polar functional groups was critical to mitigate active renal clearance mediated by the Oat3 transporter. Modification of either the 6-chloroindole core to a 4,6-difluoroindole or the 5-phenyl substituent to a substituted 5-(3-pyridyl) group provided improved metabolic stability while minimizing propensity for active transport by OAT3.
Assuntos
Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Ativadores de Enzimas/síntese química , Ativadores de Enzimas/farmacologia , Indóis/síntese química , Indóis/farmacologia , Animais , Ativação Enzimática/efeitos dos fármacos , Ativadores de Enzimas/farmacocinética , Humanos , Indóis/farmacocinética , Absorção Intestinal , Rim/efeitos dos fármacos , Rim/enzimologia , Masculino , Modelos Moleculares , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
Increased fructose consumption and its subsequent metabolism have been implicated in hepatic steatosis, dyslipidemia, obesity, and insulin resistance in humans. Since ketohexokinase (KHK) is the principal enzyme responsible for fructose metabolism, identification of a selective KHK inhibitor may help to further elucidate the effect of KHK inhibition on these metabolic disorders. Until now, studies on KHK inhibition with small molecules have been limited due to the lack of viable in vivo pharmacological tools. Herein we report the discovery of 12, a selective KHK inhibitor with potency and properties suitable for evaluating KHK inhibition in rat models. Key structural features interacting with KHK were discovered through fragment-based screening and subsequent optimization using structure-based drug design, and parallel medicinal chemistry led to the identification of pyridine 12.
Assuntos
Desenho de Fármacos , Frutoquinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Cristalografia por Raios X , Frutoquinases/química , Frutoquinases/metabolismo , Humanos , Masculino , Simulação de Acoplamento Molecular , Piridinas/química , Piridinas/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Compound 4 (PF-04971729) belongs to a new class of potent and selective sodium-dependent glucose cotransporter 2 inhibitors incorporating a unique dioxa-bicyclo[3.2.1]octane (bridged ketal) ring system. In this paper we present the design, synthesis, preclinical evaluation, and human dose predictions related to 4. This compound demonstrated robust urinary glucose excretion in rats and an excellent preclinical safety profile. It is currently in phase 2 clinical trials and is being evaluated for the treatment of type 2 diabetes.