RESUMEN
Autophagy is a catabolic process that was described to play a critical role in advanced stages of cancer, wherein it maintains tumor cell homeostasis and growth by supplying nutrients. Autophagy is also described to support alternative cellular trafficking pathways, providing a non-canonical autophagy-dependent inflammatory cytokine secretion mechanism. Therefore, autophagy inhibitors have high potential in the treatment of cancer and acute inflammation. In our study, we identified compound 1 as an inhibitor of the ATG12-ATG3 protein-protein interaction. We focused on the systematic modification of the original hit 1, a casein kinase 2 (CK2) inhibitor, to find potent disruptors of ATG12-ATG3 protein-protein interaction. A systematic modification of the hit structure led us to a wide plethora of compounds that maintain its ATG12-ATG3 inhibitory activity, which could act as a viable starting point to design new compounds with diverse therapeutic applications.
Asunto(s)
Proteínas Relacionadas con la Autofagia , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Humanos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/síntesis química , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/antagonistas & inhibidores , Unión Proteica , Estructura Molecular , Autofagia/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismoRESUMEN
Receptor-interacting protein kinases 2 and 3 (RIPK2 and RIPK3) are considered attractive therapeutic enzyme targets for the treatment of a multitude of inflammatory diseases and cancers. In this study, we developed three interrelated series of novel quinazoline-based derivatives to investigate the effects of extensive modifications of positions 6 and 7 of the central core on the inhibitory activity and the selectivity against these RIPKs. The design of the derivatives was inspired by analyses of available literary knowledge on both RIPK2 and RIPK3 in complex with known quinazoline or quinoline inhibitors. Enzymatic investigations for bioactivity of the prepared molecules against purified RIPKs (RIPK1-4) shed light on multiple potent and selective RIPK2 and dual RIPK2/3 inhibitors. Furthermore, evaluations in living cells against the RIPK2-NOD1/2-mediated signaling pathways, identified as the potential primary targets, demonstrated nanomolar inhibition for a majority of the compounds. In addition, we have demonstrated overall good stability of various lead inhibitors in both human and mouse microsomes and plasma. Several of these compounds also were evaluated for selectivity across 58 human kinases other than RIPKs, exhibiting outstanding specificity profiles. We have thus clearly demonstrated that tuning appropriate substitutions at positions 6 and 7 of the developed quinazoline derivatives may lead to interesting potency and specificities against RIPK2 and RIPK3. This knowledge might therefore be employed for the targeted preparation of new, highly potent and selective tools against these RIPKs, which could be of utility in biological and clinical research.
Asunto(s)
Microsomas , Quinazolinas , Humanos , Animales , Ratones , Quinazolinas/farmacología , Proteína Serina-Treonina Quinasa 2 de Interacción con ReceptorRESUMEN
FLT3 kinase is a potential drug target in acute myeloid leukemia (AML). Patients with FLT3 mutations typically have higher relapse rates and worse outcomes than patients without FLT3 mutations. In this study, we investigated the suitability of various heterocycles as central cores of FLT3 inhibitors, including thieno[3,2-d]pyrimidine, pyrazolo[1,5-a]pyrimidine, imidazo[4,5-b]pyridine, pyrido[4,3-d]pyrimidine, and imidazo[1,2-b]pyridazine. Our assays revealed a series of imidazo[1,2-b]pyridazines with high potency against FLT3. Compound 34f showed nanomolar inhibitory activity against recombinant FLT3-ITD and FLT3-D835Y (IC50 values 4 and 1 nM, respectively) as well as in the FLT3-ITD-positive AML cell lines MV4-11, MOLM-13, and MOLM-13 expressing the FLT3-ITD-D835Y mutant (GI50 values of 7, 9, and 4 nM, respectively). In contrast, FLT3-independent cell lines were much less sensitive. In vitro experiments confirmed suppression of FLT3 downstream signaling pathways. Finally, the treatment of MV4-11 xenograft-bearing mice with 34f at doses of 5 and 10 mg/kg markedly blocked tumor growth without any adverse effects.
Asunto(s)
Antineoplásicos , Leucemia Mieloide Aguda , Piridazinas , Humanos , Ratones , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridazinas/farmacología , Piridazinas/uso terapéutico , Leucemia Mieloide Aguda/patología , Pirimidinas/farmacología , Tirosina Quinasa 3 Similar a fms/genética , Mutación , ApoptosisRESUMEN
The nuclear constitutive androstane receptor (CAR, NR1I3) plays significant roles in many hepatic functions, such as fatty acid oxidation, biotransformation, liver regeneration, as well as clearance of steroid hormones, cholesterol, and bilirubin. CAR has been proposed as a hypothetical target receptor for metabolic or liver disease therapy. Currently known prototype high-affinity human CAR agonists such as CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) have limited selectivity, activating the pregnane X receptor (PXR) receptor, a related receptor of the NR1I subfamily. We have discovered several derivatives of 3-(1H-1,2,3-triazol-4-yl)imidazo[1,2-a]pyridine that directly activate human CAR in nanomolar concentrations. While compound 39 regulates CAR target genes in humanized CAR mice as well as human hepatocytes, it does not activate other nuclear receptors and is nontoxic in cellular and genotoxic assays as well as in rodent toxicity studies. Our findings concerning potent human CAR agonists with in vivo activity reinforce the role of CAR as a possible therapeutic target.
Asunto(s)
Receptor de Androstano Constitutivo , Receptores de Esteroides , Animales , Humanos , Ratones , Receptor de Androstano Constitutivo/agonistas , Receptor de Androstano Constitutivo/química , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Piridinas/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/agonistas , Receptores de Esteroides/químicaRESUMEN
A major structural retroviral protein, capsid protein (CA), is able to oligomerize into two different hexameric lattices, which makes this protein a key component for both the early and late stages of HIV-1 replication. During the late stage, the CA protein, as part of the Gag polyprotein precursor, facilitates protein-protein interactions that lead to the assembly of immature particles. Following protease activation and Gag polyprotein processing, CA also drives the assembly of the mature viral core. In the early stage of infection, the role of the CA protein is distinct. It controls the disassembly of the mature CA hexameric lattice i.e., uncoating, which is critical for the reverse transcription of the single-stranded RNA genome into double stranded DNA. These properties make CA a very attractive target for small molecule functioning as inhibitors of HIV-1 particle assembly and/or disassembly. Of these, inhibitors containing the PF74 scaffold have been extensively studied. In this study, we reported a series of modifications of the PF74 molecule and its characterization through a combination of biochemical and structural approaches. Our data supported the hypothesis that PF74 stabilizes the mature HIV-1 CA hexameric lattice. We identified derivatives with a higher in vitro stabilization activity in comparison to the original PF74 molecule.
Asunto(s)
VIH-1/efectos de los fármacos , Indoles/química , Indoles/farmacología , Virión/efectos de los fármacos , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Proteínas de la Cápside/antagonistas & inhibidores , Técnicas de Química Sintética , Diseño de Fármacos , Humanos , Indoles/síntesis química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Proteínas Recombinantes , Virión/ultraestructura , Ensamble de Virus/efectos de los fármacosRESUMEN
Shortly after entering the cell, HIV-1 copies its genomic RNA into double-stranded DNA in a process known as reverse transcription. This process starts inside a core consisting of an enclosed lattice of capsid proteins that protect the viral RNA from cytosolic sensors and degradation pathways. To accomplish reverse transcription and integrate cDNA into the host cell genome, the capsid shell needs to be disassembled, or uncoated. Premature or delayed uncoating attenuates reverse transcription and blocks HIV-1 infectivity. Small molecules that bind to the capsid lattice of the HIV-1 core and either destabilize or stabilize its structure could thus function as effective HIV-1 inhibitors. To screen for such compounds, we modified our recently developed FAITH assay to allow direct assessment of the stability of in vitro preassembled HIV-1 capsid-nucleocapsid (CANC) tubular particles. This new assay is a high-throughput fluorescence method based on measuring the amount of nucleic acid released from CANC complexes under disassembly conditions. The amount of disassembled CANC particles and released nucleic acid is proportional to the fluorescence signal, from which the relative percentage of CANC stability can be calculated. We consider our assay a potentially powerful tool for in vitro screening for compounds that alter HIV disassembly.