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1.
J Pharmacol Exp Ther ; 361(2): 229-244, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28193636

RESUMEN

Reversible janus associated kinase (JAK) inhibitors such as tofacitinib and decernotinib block cytokine signaling and are efficacious in treating autoimmune diseases. However, therapeutic doses are limited due to inhibition of other JAK/signal transducer and activator of transcription pathways associated with hematopoiesis, lipid biogenesis, infection, and immune responses. A selective JAK3 inhibitor may have a better therapeutic index; however, until recently, no compounds have been described that maintain JAK3 selectivity in cells, as well as against the kinome, with good physicochemical properties to test the JAK3 hypothesis in vivo. To quantify the biochemical basis for JAK isozyme selectivity, we determined that the apparent Km value for each JAK isozyme ranged from 31.8 to 2.9 µM for JAK1 and JAK3, respectively. To confirm compound activity in cells, we developed a novel enzyme complementation assay that read activity of single JAK isozymes in a cellular context. Reversible JAK3 inhibitors cannot achieve sufficient selectivity against other isozymes in the cellular context due to inherent differences in enzyme ATP Km values. Therefore, we developed irreversible JAK3 compounds that are potent and highly selective in vitro in cells and against the kinome. Compound 2, a potent inhibitor of JAK3 (0.15 nM) was 4300-fold selective for JAK3 over JAK1 in enzyme assays, 67-fold [interleukin (IL)-2 versus IL-6] or 140-fold [IL-2 versus erythropoietin or granulocyte-macrophage colony-stimulating factor (GMCSF)] selective in cellular reporter assays and >35-fold selective in human peripheral blood mononuclear cell assays (IL-7 versus IL-6 or GMCSF). In vivo, selective JAK3 inhibition was sufficient to block the development of inflammation in a rat model of rheumatoid arthritis, while sparing hematopoiesis.


Asunto(s)
Enfermedades Autoinmunes , Janus Quinasa 1 , Janus Quinasa 3 , Piperidinas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Animales , Artritis Experimental/tratamiento farmacológico , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/metabolismo , Relación Dosis-Respuesta a Droga , Monitoreo de Drogas/métodos , Humanos , Isoenzimas , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/química , Janus Quinasa 1/metabolismo , Janus Quinasa 3/antagonistas & inhibidores , Janus Quinasa 3/química , Janus Quinasa 3/metabolismo , Monitorización Inmunológica/métodos , Inhibidores de Proteínas Quinasas/farmacología , Ratas
2.
J Med Chem ; 67(18): 16807-16819, 2024 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-39231262

RESUMEN

Inhibition of leucine-rich repeat kinase 2 is a genetically supported mechanism for the treatment of Parkinson's disease. We previously disclosed the discovery of an indazole series lead that demonstrated both safety and translational risks. The safety risks were hypothesized to be of unknown origin, so structural diversity in subsequent chemical matter was prioritized. The translational risks were identified due to a low brain Kpu,u in nonhuman primate studies, which raised concern over the use of an established peripheral biomarker as a surrogate for central target engagement. Given these challenges, the team sought to leverage structure- and property-based drug design and expanded efflux transporter profiling to identify structurally distinct leads with enhanced CNS drug-likeness. Herein, we describe the discovery of a "reinvented" indazole series with improved physicochemical properties and efflux transporter profiles while maintaining excellent potency and off-target kinase selectivity, which resulted in advanced lead, compound 23.


Asunto(s)
Indazoles , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Inhibidores de Proteínas Quinasas , Indazoles/farmacología , Indazoles/química , Indazoles/síntesis química , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/antagonistas & inhibidores , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Animales , Relación Estructura-Actividad , Descubrimiento de Drogas , Ratas , Estructura Molecular
3.
J Med Chem ; 66(21): 14912-14927, 2023 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-37861679

RESUMEN

Genetic mutation of the leucine-rich repeat kinase 2 (LRRK2) protein has been associated with Parkinson's disease (PD), a disabling and progressive neurodegenerative disorder that is devoid of efficacious disease-modifying therapies. Herein, we describe the invention of an amidoisoquinoline (IQ)-derived LRRK2 inhibitor lead chemical series. Knowledge-, structure-, and property-based drug design in concert with rigorous application of in silico calculations and presynthesis predictions enabled the prioritization of molecules with favorable CNS "drug-like" physicochemical properties. This resulted in the discovery of compound 8, which was profiled extensively before human ether-a-go-go (hERG) ion channel inhibition halted its progression. Strategic reduction of lipophilicity and basicity resulted in attenuation of hERG ion channel inhibition while maintaining a favorable CNS efflux transporter profile. Further structure- and property-based optimizations resulted in the discovery of preclinical candidate MK-1468. This exquisitely selective LRRK2 inhibitor has a projected human dose of 48 mg BID and a preclinical safety profile that supported advancement toward GLP toxicology studies.


Asunto(s)
Enfermedad de Parkinson , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/química , Encéfalo/metabolismo , Mutación , Canales Iónicos/metabolismo
4.
J Med Chem ; 65(7): 5675-5689, 2022 04 14.
Artículo en Inglés | MEDLINE | ID: mdl-35332774

RESUMEN

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.


Asunto(s)
Proteínas de la Membrana , Neoplasias , Animales , Citocinas , Humanos , Inmunoterapia/métodos , Interferones , Ratones , Neoplasias/tratamiento farmacológico
5.
Mol Cancer Ther ; 21(2): 282-293, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34815361

RESUMEN

The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2'3'-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I IFN production. This promotes systemic antigen-specific CD8+ T-cell priming that eventually provides potent antitumor activity. To exploit this mechanism, we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher in vitro potency than cGAMP. Following intratumoral administration of MSA-1 to a panel of syngeneic mouse tumors on immune-competent mice, cytokine upregulation and its exposure were detected in plasma, other tissues, injected tumors, and noninjected tumors. This was accompanied by effective antitumor activity. Mechanistic studies in immune-deficient mice suggested that antitumor activity of intratumorally dosed STING agonists is in part due to necrosis and/or innate immune responses such as TNF-α activity, but development of a robust adaptive antitumor immunity is necessary for complete tumor elimination. Combination with PD-1 blockade in anti-PD-1-resistant murine models showed that MSA-1 may synergize with checkpoint inhibitors but can also provide superior tumor control as a single agent. We show for the first time that potent cyclic dinucleotides can promote a rapid and stronger induction of the same genes eventually regulated by PD-1 blockade. This may have contributed to the relatively early tumor control observed with MSA-1. Taken together, these data strongly support the development of STING agonists as therapy for patients with aggressive tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 treatment by enhancing the anti-PD-1 immune profile.


Asunto(s)
Inmunidad Innata/inmunología , Inmunoterapia/métodos , Interferones/metabolismo , Neoplasias/inmunología , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones
6.
J Med Chem ; 65(24): 16801-16817, 2022 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-36475697

RESUMEN

Inhibition of leucine-rich repeat kinase 2 (LRRK2) kinase activity represents a genetically supported, chemically tractable, and potentially disease-modifying mechanism to treat Parkinson's disease. Herein, we describe the optimization of a novel series of potent, selective, central nervous system (CNS)-penetrant 1-heteroaryl-1H-indazole type I (ATP competitive) LRRK2 inhibitors. Type I ATP-competitive kinase physicochemical properties were integrated with CNS drug-like properties through a combination of structure-based drug design and parallel medicinal chemistry enabled by sp3-sp2 cross-coupling technologies. This resulted in the discovery of a unique sp3-rich spirocarbonitrile motif that imparted extraordinary potency, pharmacokinetics, and favorable CNS drug-like properties. The lead compound, 25, demonstrated exceptional on-target potency in human peripheral blood mononuclear cells, excellent off-target kinase selectivity, and good brain exposure in rat, culminating in a low projected human dose and a pre-clinical safety profile that warranted advancement toward pre-clinical candidate enabling studies.


Asunto(s)
Enfermedad de Parkinson , Ratas , Humanos , Animales , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Enfermedad de Parkinson/tratamiento farmacológico , Indazoles/farmacología , Indazoles/uso terapéutico , Leucocitos Mononucleares/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/química , Encéfalo/metabolismo , Adenosina Trifosfato
7.
J Med Chem ; 65(1): 838-856, 2022 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-34967623

RESUMEN

The leucine-rich repeat kinase 2 (LRRK2) protein has been genetically and functionally linked to Parkinson's disease (PD), a disabling and progressive neurodegenerative disorder whose current therapies are limited in scope and efficacy. In this report, we describe a rigorous hit-to-lead optimization campaign supported by structural enablement, which culminated in the discovery of brain-penetrant, candidate-quality molecules as represented by compounds 22 and 24. These compounds exhibit remarkable selectivity against the kinome and offer good oral bioavailability and low projected human doses. Furthermore, they showcase the implementation of stereochemical design elements that serve to enable a potency- and selectivity-enhancing increase in polarity and hydrogen bond donor (HBD) count while maintaining a central nervous system-friendly profile typified by low levels of transporter-mediated efflux and encouraging brain penetration in preclinical models.


Asunto(s)
Antiparkinsonianos/síntesis química , Antiparkinsonianos/farmacología , Encéfalo/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/antagonistas & inhibidores , Quinazolinas/síntesis química , Quinazolinas/farmacología , Antiparkinsonianos/farmacocinética , Disponibilidad Biológica , Diseño de Fármacos , Humanos , Modelos Moleculares , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacocinética , Relación Estructura-Actividad
8.
ACS Med Chem Lett ; 12(3): 459-466, 2021 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-33738073

RESUMEN

Hematopoietic progenitor kinase (HPK1), a negative regulator of TCR-mediated T-cell activation, has been recognized as a novel antitumor immunotherapy target. Structural optimization of kinase inhibitor 4 through a systematic two-dimensional diversity screen of pyrazolopyridines led to the identification of potent and selective compounds. Crystallographic studies with HPK1 revealed a favorable water-mediated interaction with Asp155 and a salt bridge to Asp101 with optimized heterocyclic solvent fronts that were critical for enhanced potency and selectivity. Computational studies of model systems revealed differences in torsional profiles that allowed for these beneficial protein-ligand interactions. Further optimization of molecular properties led to identification of potent and selective reverse indazole inhibitor 36 that inhibited phosphorylation of adaptor protein SLP76 in human PBMC and exhibited low clearance with notable bioavailability in in vivo rat studies.

9.
SLAS Discov ; 26(1): 88-99, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32844715

RESUMEN

Hematopoietic progenitor kinase 1 (HPK1), also referred to as mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1), is a serine/threonine kinase that negatively regulates T-cell signaling by phosphorylating Ser376 of Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76), a critical mediator of T-cell receptor activation. HPK1 loss of function mouse models demonstrated enhanced immune cell activation and beneficial antitumor activity. To enable discovery and functional characterization of high-affinity small-molecule HPK1 inhibitors, we have established high-throughput biochemical, cell-based, and novel pharmacodynamic (PD) assays. Kinase activity-based time-resolved fluorescence energy transfer (TR-FRET) assays were established as the primary biochemical approach to screen for potent inhibitors and assess selectivity against members of MAP4K and other closely related kinases. A proximal target engagement (TE) assay quantifying pSLP-76 levels as a readout and a distal assay measuring IL-2 secretion as a functional response were established using human peripheral blood mononuclear cells (PBMCs) from two healthy donors. Significant correlations between biochemical and cellular assays as well as excellent correlation between the two donors for the cellular assays were observed. pSLP-76 levels were further used as a PD marker in the preclinical murine model. This effort required the development of a novel ultrasensitive single-molecule array (SiMoA) assay to monitor pSLP-76 changes in mouse spleen.


Asunto(s)
Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Animales , Línea Celular , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Ratones , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo
10.
ACS Med Chem Lett ; 12(4): 653-661, 2021 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-33859804

RESUMEN

Hematopoietic progenitor kinase 1 (HPK1), a serine/threonine kinase, is a negative immune regulator of T cell receptor (TCR) and B cell signaling that is primarily expressed in hematopoietic cells. Accordingly, it has been reported that HPK1 loss-of-function in HPK1 kinase-dead syngeneic mouse models shows enhanced T cell signaling and cytokine production as well as tumor growth inhibition in vivo, supporting its value as an immunotherapeutic target. Herein, we present the structurally enabled discovery of novel, potent, and selective diaminopyrimidine carboxamide HPK1 inhibitors. The key discovery of a carboxamide moiety was essential for enhanced enzyme inhibitory potency and kinome selectivity as well as sustained elevation of cellular IL-2 production across a titration range in human peripheral blood mononuclear cells. The elucidation of structure-activity relationships using various pendant amino ring systems allowed for the identification of several small molecule type-I inhibitors with promising in vitro profiles.

11.
Bioorg Med Chem Lett ; 20(10): 3158-60, 2010 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-20399652

RESUMEN

We have identified macrocyclic inhibitors of the aspartic protease BACE, implicated in the etiology of Alzheimer's disease. An X-ray structure of screening hit 1 in the BACE active site revealed a hairpin conformation suggesting that constrained macrocyclic derivatives may also bind there. Several of the analogs we prepared were >100x more potent than 1, such as 7 (5 nM K(i)).


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Compuestos Macrocíclicos/química , Inhibidores de Proteasas/química , Quinazolinas/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Sitios de Unión , Simulación por Computador , Humanos , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/farmacología , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Quinazolinas/síntesis química , Quinazolinas/farmacología , Relación Estructura-Actividad
12.
Science ; 369(6506)2020 08 21.
Artículo en Inglés | MEDLINE | ID: mdl-32820094

RESUMEN

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.


Asunto(s)
Antineoplásicos/farmacología , Proteínas de la Membrana/metabolismo , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Humanos
13.
ACS Med Chem Lett ; 9(12): 1193-1198, 2018 Dec 13.
Artículo en Inglés | MEDLINE | ID: mdl-30613325

RESUMEN

We report herein the design and synthesis of a series of orally active, liver-targeted hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitors for the treatment of anemia. In order to mitigate the concerns for potential systemic side effects, we pursued liver-targeted HIF-PHD inhibitors relying on uptake via organic anion transporting polypeptides (OATPs). Starting from a systemic HIF-PHD inhibitor (1), medicinal chemistry efforts directed toward reducing permeability and, at the same time, maintaining oral absorption led to the synthesis of an array of structurally diverse hydroxypyridone analogues. Compound 28a was chosen for further profiling, because of its excellent in vitro profile and liver selectivity. This compound significantly increased hemoglobin levels in rats, following chronic QD oral administration, and displayed selectivity over systemic effects.

14.
Nat Commun ; 6: 8833, 2015 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-26640126

RESUMEN

RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors.


Asunto(s)
Interleucina-17/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/química , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Sitio Alostérico , Animales , Diferenciación Celular , Humanos , Interleucina-17/química , Ligandos , Ratones , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Estructura Terciaria de Proteína , Células Th17/química , Células Th17/metabolismo
15.
J Biomol Screen ; 8(1): 65-71, 2003 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-12854999

RESUMEN

Target validation is one of rate-limiting steps in the modern drug discovery. The authors developed a strategy of combining adenovirus-mediated gene transfer for efficient target functionality validation, both in vivo and in vitro, with baculovirus expression to produce sufficient quantities of protein for high-throughput screening (HTS). The incorporation of green fluorescent protein (GFP) in the adenovirus vectors accelerates recombinant adenovirus plaque purification, whereas the use of epitope and affinity tags facilitates the identification and purification of recombinant protein. In this generalized scheme, the flexible modular design of viral vectors facilitates the transition between target validation and HTS. In the example presented, functional target validation in vivo was achieved by overexpressing the target gene in cell-based models and in the mouse cortex following adenovirus-mediated gene delivery. In this context, target overexpression resulted in the accumulation of a disease-related biomarker both in vitro and in vivo. A baculovirus-based expressional system was then generated to produce enough target protein for HTS. Thus, the use of these viral expression systems represents a generalized method for rapid target functionality validation and HTS assay development, which could be applied to numerous target candidates being elucidated in gene discovery programs.


Asunto(s)
Adenoviridae , Vectores Genéticos , Proteínas Recombinantes/análisis , Biología Computacional
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