RESUMO
The Hippo signaling pathway acts as a brake on regeneration in many tissues. This cascade of kinases culminates in the phosphorylation of the transcriptional cofactors Yap and Taz, whose concentration in the nucleus consequently remains low. Various types of cellular signals can reduce phosphorylation, however, resulting in the accumulation of Yap and Taz in the nucleus and subsequently in mitosis. We earlier identified a small molecule, TRULI, that blocks the final kinases in the pathway, Lats1 and Lats2, and thus elicits proliferation of several cell types that are ordinarily postmitotic and aids regeneration in mammals. In the present study, we present the results of chemical modification of the original compound and demonstrate that a derivative, TDI-011536, is an effective blocker of Lats kinases in vitro at nanomolar concentrations. The compound fosters extensive proliferation in retinal organoids derived from human induced pluripotent stem cells. Intraperitoneal administration of the substance to mice suppresses Yap phosphorylation for several hours and induces transcriptional activation of Yap target genes in the heart, liver, and skin. Moreover, the compound initiates the proliferation of cardiomyocytes in adult mice following cardiac cryolesions. After further chemical refinement, related compounds might prove useful in protective and regenerative therapies.
Assuntos
Inibidores de Proteínas Quinases , Proteínas Serina-Treonina Quinases , Regeneração , Animais , Proliferação de Células/efeitos dos fármacos , Coração/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas , Regeneração Hepática/efeitos dos fármacos , Regeneração Hepática/genética , Regeneração Hepática/fisiologia , Camundongos , Organoides/fisiologia , Fosforilação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Regeneração/efeitos dos fármacos , Regeneração/genética , Retina/fisiologia , Fenômenos Fisiológicos da Pele/efeitos dos fármacos , Fenômenos Fisiológicos da Pele/genética , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Proteínas de Sinalização YAP/metabolismoRESUMO
Tuberculosis remains a leading cause of death from a single infection worldwide. Drug resistance to existing and even new antimycobacterials calls for research into novel targets and unexplored mechanisms of action. Recently we reported on the development of tight-binding inhibitors of Mycobacterium tuberculosis (Mtb) lipoamide dehydrogenase (Lpd), which selectively inhibit the bacterial but not the human enzyme based on a differential modality of inhibitor interaction with these targets. Here we report on the striking improvement in inhibitor residence time on the Mtb enzyme associated with scaffold progression from an indazole to 2-cyanoindole. Cryo-EM of Lpd with the bound 2-cyanoindole inhibitor 19 confirmed displacement of the buried water molecule deep in the binding channel with a cyano group. The ensuing hours-long improvement in on-target residence time is associated with enhanced antibacterial activity in axenic culture and in primary mouse macrophages. Resistance to 2-cyanoindole inhibitors involves mutations within the inhibitor binding site that have little effect on inhibitor affinity but change the modality of inhibitor-target interaction, resulting in fast dissociation from Lpd. These findings underscore that on-target residence time is a major determinant of antibacterial activity and in vivo efficacy.
RESUMO
Free energy perturbation is a computational technique that can be used to predict how small changes to an inhibitor structure will affect the binding free energy to its target. In this paper, we describe the utility of free energy perturbation with FEP+ in the hit-to-lead stage of a drug discovery project targeting soluble adenyl cyclase. The project was structurally enabled by X-ray crystallography throughout. We employed free energy perturbation to first scaffold hop to a preferable chemotype and then optimize the binding affinity to sub-nanomolar levels while retaining druglike properties. The results illustrate that effective use of free energy perturbation can enable a drug discovery campaign to progress rapidly from hit to lead, facilitating proof-of-concept studies that enable target validation.
Assuntos
Adenilil Ciclases , Descoberta de Drogas , Termodinâmica , EntropiaRESUMO
Eleven-nineteen leukemia (ENL) is an epigenetic reader protein that drives oncogenic transcriptional programs in acute myeloid leukemia (AML). AML is one of the deadliest hematopoietic malignancies, with an overall 5-year survival rate of 27%. The epigenetic reader activity of ENL is mediated by its YEATS domain that binds to acetyl and crotonyl marks on histone tails and colocalizes with promoters of actively transcribed genes that are essential for leukemia. Prior to the discovery of TDI-11055, existing inhibitors of ENL YEATS showed in vitro potency, but had not shown efficacy in in vivo animal models. During the course of the medicinal chemistry campaign described here, we identified ENL YEATS inhibitor TDI-11055 that has an improved pharmacokinetic profile and is appropriate for in vivo evaluation of the ENL YEATS inhibition mechanism in AML.
RESUMO
With increasing reports of resistance to artemisinins and artemisinin-combination therapies, targeting the Plasmodium proteasome is a promising strategy for antimalarial development. We recently reported a highly selective Plasmodium falciparum proteasome inhibitor with anti-malarial activity in the humanized mouse model. To balance the permeability of the series of macrocycles with other drug-like properties, we conducted further structure-activity relationship studies on a biphenyl ether-tethered macrocyclic scaffold. Extensive SAR studies around the P1, P3, and P5 groups and peptide backbone identified compound TDI-8414. TDI-8414 showed nanomolar antiparasitic activity, no toxicity to HepG2 cells, high selectivity against the Plasmodium proteasome over the human constitutive proteasome and immunoproteasome, improved solubility and PAMPA permeability, and enhanced metabolic stability in microsomes and plasma of both humans and mice.
Assuntos
Antimaláricos , Plasmodium , Humanos , Animais , Camundongos , Antimaláricos/farmacologia , Antimaláricos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Relação Estrutura-Atividade , Plasmodium falciparum/metabolismo , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/químicaRESUMO
Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer disease (AD) and likely contributes to neuropathology through various pathways. Here we report that the intracellular trafficking of apoE4 is impaired in Neuro-2a cells and primary neurons, as shown by measuring fluorescence recovery after photobleaching. In Neuro-2a cells, more apoE4 than apoE3 molecules remained immobilized in the endoplasmic reticulum (ER) and the Golgi apparatus, and the lateral motility of apoE4 was significantly lower in the Golgi apparatus (but not in the ER) than that of apoE3. Likewise, the immobile fraction was larger, and the lateral motility was lower for apoE4 than apoE3 in mouse primary hippocampal neurons. ApoE4 with the R61T mutation, which abolishes apoE4 domain interaction, was less immobilized, and its lateral motility was comparable with that of apoE3. The trafficking impairment of apoE4 was also rescued by disrupting domain interaction with the small-molecule structure correctors GIND25 and PH002. PH002 also rescued apoE4-induced impairments of neurite outgrowth in Neuro-2a cells and dendritic spine development in primary neurons. ApoE4 did not affect trafficking of amyloid precursor protein, another AD-related protein, through the secretory pathway. Thus, domain interaction renders more newly synthesized apoE4 molecules immobile and slows their trafficking along the secretory pathway. Correcting the pathological structure of apoE4 by disrupting domain interaction is a potential therapeutic approach to treat or prevent AD related to apoE4.
Assuntos
Apolipoproteína E4/metabolismo , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Complexo de Golgi/metabolismo , Hipocampo/citologia , Humanos , Camundongos , Modelos Biológicos , Mutação , Neurônios/metabolismoRESUMO
HCV NS3/4a protease inhibitors are proven therapeutic agents against chronic hepatitis C virus infection, with boceprevir and telaprevir having recently received regulatory approval as add-on therapy to pegylated interferon/ribavirin for patients harboring genotype 1 infections. Overcoming antiviral resistance, broad genotype coverage, and a convenient dosing regimen are important attributes for future agents to be used in combinations without interferon. In this communication, we report the preclinical profile of MK-5172, a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor currently in clinical development. The compound demonstrates subnanomolar activity against a broad enzyme panel encompassing major hepatitis C virus (HCV) genotypes as well as variants resistant to earlier protease inhibitors. In replicon selections, MK-5172 exerted high selective pressure, which yielded few resistant colonies. In both rat and dog, MK-5172 demonstrates good plasma and liver exposures, with 24-h liver levels suggestive of once-daily dosing. When administered to HCV-infected chimpanzees harboring chronic gt1a or gt1b infections, MK-5172 suppressed viral load between 4 to 5 logs at a dose of 1 mg/kg of body weight twice daily (b.i.d.) for 7 days. Based on its preclinical profile, MK-5172 is anticipated to be broadly active against multiple HCV genotypes and clinically important resistance variants and highly suited for incorporation into newer all-oral regimens.
Assuntos
Hepacivirus/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Quinoxalinas/farmacologia , Quinoxalinas/farmacocinética , Proteínas não Estruturais Virais/antagonistas & inibidores , Amidas , Animais , Antivirais/farmacologia , Carbamatos , Ciclopropanos , Cães , Farmacorresistência Viral , Genótipo , Hepacivirus/enzimologia , Hepacivirus/genética , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/virologia , Fígado/efeitos dos fármacos , Pan troglodytes , Quinoxalinas/metabolismo , Ratos , Sulfonamidas , Carga Viral/efeitos dos fármacosRESUMO
Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 4 (mGluR4) have been proposed as a novel therapeutic approach for the treatment of Parkinson's disease. However, evaluation of this proposal has been limited by the availability of appropriate pharmacological tools to interrogate the target. In this study, we describe the properties of a novel mGluR4 PAM. 5-Methyl-N-(4-methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine (ADX88178) enhances glutamate-mediated activation of human and rat mGluR4 with EC(50) values of 4 and 9 nM, respectively. The compound is highly selective for mGluR4 with minimal activities at other mGluRs. Oral administration of ADX88178 in rats is associated with high bioavailability and results in cerebrospinal fluid exposure of >50-fold the in vitro EC(50) value. ADX88178 reverses haloperidol-induced catalepsy in rats at 3 and 10 mg/kg. It is noteworthy that this compound alone has no impact on forelimb akinesia resulting from a bilateral 6-hydroxydopamine lesion in rats. However, coadministration of a low dose of L-DOPA (6 mg/kg) enabled a robust, dose-dependent reversal of the forelimb akinesia deficit. ADX88178 also increased the effects of quinpirole in lesioned rats and enhanced the effects of L-DOPA in MitoPark mice. It is noteworthy that the enhancement of the actions of L-DOPA was not associated with an exacerbation of L-DOPA-induced dyskinesias in rats. ADX88178 is a novel, potent, and selective mGluR4 PAM that is a valuable tool for exploring the therapeutic potential of mGluR4 modulation. The use of this novel tool molecule supports the proposal that activation of mGluR4 may be therapeutically useful in Parkinson's disease.
Assuntos
Modelos Animais de Doenças , Agonistas de Aminoácidos Excitatórios/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Receptores de Glutamato Metabotrópico/fisiologia , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Agonistas de Aminoácidos Excitatórios/farmacologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doença de Parkinson/fisiopatologia , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/biossínteseRESUMO
A series of macrocyclic compounds containing a cyclic constraint in the P2-P4 linker region have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K, A156T, A156V, and D168V mutant activity while maintaining high rat liver exposure. The effect of the constraint is most dramatic against gt 1b A156 mutants where ~20-fold improvements in potency are achieved by introduction of a variety of ring systems into the P2-P4 linker.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Hepacivirus/enzimologia , Compostos Macrocíclicos/química , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Sítios de Ligação , Proteínas de Transporte/metabolismo , Domínio Catalítico , Ciclização , Genótipo , Meia-Vida , Hepacivirus/genética , Peptídeos e Proteínas de Sinalização Intracelular , Cinética , Fígado/metabolismo , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/farmacocinética , Simulação de Acoplamento Molecular , Mutação , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacocinética , Ratos , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismoRESUMO
A series of macrocyclic compounds containing 2-substituted-quinoline moieties have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K mutant activity while maintaining the high rat liver exposure. Cyclization of the 2-substituted quinoline substituent led to a series of tricyclic P2 compounds which also display superb gt3a potency.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Hepacivirus/enzimologia , Compostos Macrocíclicos/química , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Proteínas de Transporte/metabolismo , Ciclização , Genótipo , Meia-Vida , Hepacivirus/genética , Peptídeos e Proteínas de Sinalização Intracelular , Cinética , Fígado/metabolismo , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/farmacocinética , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacocinética , Quinolinas/química , Ratos , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismoRESUMO
Soluble adenylyl cyclase (sAC: ADCY10) is an enzyme involved in intracellular signaling. Inhibition of sAC has potential therapeutic utility in a number of areas. For example, sAC is integral to successful male fertility: sAC activation is required for sperm motility and ability to undergo the acrosome reaction, two processes central to oocyte fertilization. Pharmacologic evaluation of existing sAC inhibitors for utility as on-demand, nonhormonal male contraceptives suggested that both high intrinsic potency, fast on and slow dissociation rates are essential design elements for successful male contraceptive applications. During the course of the medicinal chemistry campaign described here, we identified sAC inhibitors that fulfill these criteria and are suitable for in vivo evaluation of diverse sAC pharmacology.
Assuntos
Adenilil Ciclases , Motilidade dos Espermatozoides , Animais , Masculino , Adenilil Ciclases/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Oócitos/metabolismo , Transdução de Sinais/fisiologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Anticoncepcionais Masculinos/química , Anticoncepcionais Masculinos/farmacologiaRESUMO
Aberrant gene-silencing through dysregulation of polycomb protein activity has emerged as an important oncogenic mechanism in cancer, implicating polycomb proteins as important therapeutic targets. Recently, an inhibitor targeting EZH2, the methyltransferase component of PRC2, received U.S. Food and Drug Administration approval following promising clinical responses in cancer patients. However, the current array of EZH2 inhibitors have poor brain penetrance, limiting their use in patients with central nervous system malignancies, a number of which have been shown to be sensitive to EZH2 inhibition. To address this need, we have identified a chemical strategy, based on computational modeling of pyridone-containing EZH2 inhibitor scaffolds, to minimize P-glycoprotein activity, and here we report the first brain-penetrant EZH2 inhibitor, TDI-6118 (compound 5). Additionally, in the course of our attempts to optimize this compound, we discovered TDI-11904 (compound 21), a novel, highly potent, and peripherally active EZH2 inhibitor based on a 7 member ring structure.
RESUMO
The chromatin reader eleven-nineteen leukemia (ENL) has been identified as a critical dependency in acute myeloid leukemia (AML), but its therapeutic potential remains unclear. We describe a potent and orally bioavailable small-molecule inhibitor of ENL, TDI-11055, which displaces ENL from chromatin by blocking its YEATS domain interaction with acylated histones. Cell lines and primary patient samples carrying MLL rearrangements or NPM1 mutations are responsive to TDI-11055. A CRISPR-Cas9-mediated mutagenesis screen uncovers an ENL mutation that confers resistance to TDI-11055, validating the compound's on-target activity. TDI-11055 treatment rapidly decreases chromatin occupancy of ENL-associated complexes and impairs transcription elongation, leading to suppression of key oncogenic gene expression programs and induction of differentiation. In vivo treatment with TDI-11055 blocks disease progression in cell line- and patient-derived xenograft models of MLL-rearranged and NPM1-mutated AML. Our results establish ENL displacement from chromatin as a promising epigenetic therapy for molecularly defined AML subsets and support the clinical translation of this approach. SIGNIFICANCE: AML is a poor-prognosis disease for which new therapeutic approaches are desperately needed. We developed an orally bioavailable inhibitor of ENL, demonstrated its potent efficacy in MLL-rearranged and NPM1-mutated AML, and determined its mechanisms of action. These biological and chemical insights will facilitate both basic research and clinical translation. This article is highlighted in the In This Issue feature, p. 2483.
Assuntos
Leucemia Mieloide Aguda , Lisina , Humanos , Leucemia Mieloide Aguda/genética , Histonas/metabolismo , Cromatina , Proteína de Leucina Linfoide-Mieloide/metabolismoRESUMO
Efforts to develop novel, interferon-sparing therapies for treatment of chronic hepatitis C (HCV) infection are contingent on the ability of combination therapies consisting of direct antiviral inhibitors to achieve a sustained virologic response. This work demonstrates a proof of concept that coadministration of the nucleoside analogue MK-0608 with the protease inhibitor MK-7009, both of which produced robust viral load declines as monotherapy, to an HCV-infected chimpanzee can achieve a cure of infection.
Assuntos
Antivirais/administração & dosagem , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Indóis/administração & dosagem , Pan troglodytes/virologia , Tubercidina/análogos & derivados , Carga Viral/efeitos dos fármacos , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Ciclopropanos , Relação Dose-Resposta a Droga , Esquema de Medicação , Quimioterapia Combinada , Hepacivirus/enzimologia , Hepacivirus/fisiologia , Hepatite C Crônica/virologia , Indóis/farmacologia , Indóis/uso terapêutico , Isoindóis , Lactamas Macrocíclicas , Leucina/análogos & derivados , Prolina/análogos & derivados , Inibidores de Proteases/administração & dosagem , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Sulfonamidas , Resultado do Tratamento , Tubercidina/administração & dosagem , Tubercidina/farmacologia , Tubercidina/uso terapêutico , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
Tuberculosis remains a leading cause of death from a single bacterial infection worldwide. Efforts to develop new treatment options call for expansion into an unexplored target space to expand the drug pipeline and bypass resistance to current antibiotics. Lipoamide dehydrogenase is a metabolic and antioxidant enzyme critical for mycobacterial growth and survival in mice. Sulfonamide analogs were previously identified as potent and selective inhibitors of mycobacterial lipoamide dehydrogenase in vitro but lacked activity against whole mycobacteria. Here we present the development of analogs with improved permeability, potency, and selectivity, which inhibit the growth of Mycobacterium tuberculosis in axenic culture on carbohydrates and within mouse primary macrophages. They increase intrabacterial pyruvate levels, supporting their on-target activity within mycobacteria. Distinct modalities of binding between the mycobacterial and human enzymes contribute to improved potency and hence selectivity through induced-fit tight binding interactions within the mycobacterial but not human enzyme, as indicated by kinetic analysis and crystallography.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Animais , Antibacterianos/uso terapêutico , Di-Hidrolipoamida Desidrogenase/metabolismo , Humanos , Cinética , Camundongos , Mycobacterium tuberculosis/metabolismo , Tuberculose/tratamento farmacológicoRESUMO
Soluble adenylyl cyclase (sAC) has gained attention as a potential therapeutic target given the role of this enzyme in intracellular signaling. We describe successful efforts to design improved sAC inhibitors amenable for in vivo interrogation of sAC inhibition to assess its potential therapeutic applications. This work culminated in the identification of TDI-10229 (12), which displays nanomolar inhibition of sAC in both biochemical and cellular assays and exhibits mouse pharmacokinetic properties sufficient to warrant its use as an in vivo tool compound.
RESUMO
The administration of hepatitis C virus (HCV) NS3/4A protease inhibitors to patients with chronic HCV infections has demonstrated that they have dramatic antiviral effects and that compounds acting via this mechanism are likely to form a key component of future anti-HCV therapy. We report here on the preclinical profile of MK-7009, an inhibitor of genotype 1a and 1b proteases at subnanomolar concentrations with modestly shifted potency against genotype 2a and 2b proteases at low nanomolar concentrations. Potent activity was also observed in a cell-based HCV replicon assay in the presence of added human serum (50%). In multiple species evaluated in preclinical studies, the MK-7009 concentrations in the liver were maintained at a significant multiple of the cell-based replicon 50% effective concentration over 12 to 24 h following the administration of moderate oral doses (5 to 10 mg per kg of body weight). MK-7009 also had excellent selectivity against both a range of human proteases and a broad panel of pharmacologically relevant ion channels, receptors, and enzymes. On the basis of this favorable profile, MK-7009 was selected for clinical development and is currently being evaluated in controlled clinical trials with both healthy volunteers and HCV-infected patients.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Indóis/farmacologia , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/farmacocinética , Área Sob a Curva , Linhagem Celular , Ciclopropanos , Cães , Genótipo , Meia-Vida , Hepacivirus/enzimologia , Hepacivirus/genética , Humanos , Indóis/farmacocinética , Interferon alfa-2 , Interferon-alfa/farmacologia , Isoindóis , Lactamas Macrocíclicas , Leucina/análogos & derivados , Macaca mulatta , Pan troglodytes , Prolina/análogos & derivados , Inibidores de Proteases/farmacocinética , Ratos , Proteínas Recombinantes , Replicon , Especificidade por Substrato , Sulfonamidas , Proteínas não Estruturais Virais/genéticaRESUMO
A series of triarylethanolamine inhibitors of the Kv1.5 potassium channel have been prepared and evaluated for their effects in vitro and in vivo. The structure-activity relationship (SAR) studies described herein led to the development of potent, selective and orally active inhibitors of Kv1.5.
Assuntos
Etanolaminas/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Etanolaminas/química , Humanos , Bloqueadores dos Canais de Potássio/química , Relação Estrutura-AtividadeRESUMO
HCV-NS3 protease is essential for viral replication and NS3 protease inhibitors have shown proof of concept in clinical trials. Novel P2-P4 macrocycle inhibitors of NS3/4A comprising a P1 C-terminal carboxylic acid have recently been disclosed. A series of analogs, in which the carboxylic residue is replaced by phosphorous acid functionalities were synthesized and found to be inhibitors of the NS3 protease. Among them the methylphosphinate analogue showed nanomolar level of enzyme inhibition and sub-micromolar potency in the replication assay.