RESUMEN
Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.
Asunto(s)
Infecciones por VIH , VIH-1 , Alquinos , Benzoxazinas , Proteínas Adaptadoras de Señalización CARD/metabolismo , Ciclopropanos , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Infecciones por VIH/tratamiento farmacológico , VIH-1/metabolismo , Humanos , Inflamasomas/metabolismo , Leucocitos Mononucleares , Proteínas de Neoplasias/metabolismoRESUMEN
Acute kidney injury (AKI) is a life-threatening disease with no known curative or preventive therapies. Data from multiple animal models and human studies have linked dysregulation of bone morphogenetic protein (BMP) signaling to AKI. Small molecules that potentiate endogenous BMP signaling should have a beneficial effect in AKI. We performed a high-throughput phenotypic screen and identified a series of FK506 analogs that act as potent BMP potentiators by sequestering FKBP12 from BMP type I receptors. We further showed that calcineurin inhibition was not required for this activity. We identified a calcineurin-sparing FK506 analog oxtFK through late-stage functionalization and structure-guided design. OxtFK demonstrated an improved safety profile in vivo relative to FK506. OxtFK stimulated BMP signaling in vitro and in vivo and protected the kidneys in an AKI mouse model, making it a promising candidate for future development as a first-in-class therapeutic for diseases with dysregulated BMP signaling.
Asunto(s)
Lesión Renal Aguda/tratamiento farmacológico , Proteínas Morfogenéticas Óseas/metabolismo , Tacrolimus/farmacología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Ensayos Analíticos de Alto Rendimiento , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Fenotipo , Tacrolimus/análogos & derivados , Tacrolimus/químicaRESUMEN
Type 2 diabetes (T2D) is caused by loss of pancreatic ß-cell mass and failure of the remaining ß-cells to deliver sufficient insulin to meet demand. ß-Cell glucolipotoxicity (GLT), which refers to combined, deleterious effects of elevated glucose and fatty acid levels on ß-cell function and survival, contributes to T2D-associated ß-cell failure. Drugs and mechanisms that protect ß-cells from GLT stress could potentially improve metabolic control in patients with T2D. In a phenotypic screen seeking low-molecular-weight compounds that protected ß-cells from GLT, we identified compound A that selectively blocked GLT-induced apoptosis in rat insulinoma cells. Compound A and its optimized analogs also improved viability and function in primary rat and human islets under GLT. We discovered that compound A analogs decreased GLT-induced cytosolic calcium influx in islet cells, and all measured ß-cell-protective effects correlated with this activity. Further studies revealed that the active compound from this series largely reversed GLT-induced global transcriptional changes. Our results suggest that taming cytosolic calcium overload in pancreatic islets can improve ß-cell survival and function under GLT stress and thus could be an effective strategy for T2D treatment.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Calcio/toxicidad , Glucolípidos/antagonistas & inhibidores , Glucolípidos/toxicidad , Células Secretoras de Insulina/efectos de los fármacos , Animales , Apoptosis , Línea Celular , Supervivencia Celular , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacología , Humanos , Estructura Molecular , Ratas , Ratas Sprague-Dawley , TranscriptomaRESUMEN
The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the ß-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.
Asunto(s)
Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Triazinas/farmacología , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Proteínas de la Membrana Bacteriana Externa/genética , Transporte Biológico/fisiología , Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , Evaluación Preclínica de Medicamentos , Farmacorresistencia Bacteriana/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Pruebas de Sensibilidad MicrobianaRESUMEN
In an attempt to identify novel therapeutics and mechanisms to differentially kill tumor cells using phenotypic screening, we identified N-benzyl indole carbinols (N-BICs), synthetic analogs of the natural product indole-3-carbinol (I3C). To understand the mode of action for the molecules we employed Cancer Cell Line Encyclopedia viability profiling and correlative informatics analysis to identify and ultimately confirm the phase II metabolic enzyme sulfotransferase 1A1 (SULT1A1) as the essential factor for compound selectivity. Further studies demonstrate that SULT1A1 activates the N-BICs by rendering the compounds strong electrophiles which can alkylate cellular proteins and thereby induce cell death. This study demonstrates that the selectivity profile for N-BICs is through conversion by SULT1A1 from an inactive prodrug to an active species that induces cell death and tumor suppression.
Asunto(s)
Arilsulfotransferasa/metabolismo , Compuestos de Bencilo/farmacología , Indoles/farmacología , Animales , Compuestos de Bencilo/farmacocinética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Células HCT116 , Humanos , Indoles/farmacocinética , Ratones , Ratones Desnudos , Distribución Aleatoria , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The power and scope of chemical synthesis offer considerable opportunities to broaden the lexicon of chemical tools that can be implemented for the study of complex biological systems. To investigate individual signaling proteins and pathways, chemical tools provide a powerful complement to existing genetic, chemical genetic and immunologic methods. In particular, understanding phosphorylation-mediated signaling in real time yields important information about the regulation of cellular function and insights into the origin of disease. Recent advances in the development of photolabile caged analogs of bioactive species and fluorescence-based sensors of protein kinase activities are useful for investigating protein phosphorylation and the roles of phosphoproteins. Photolabile caged analogs allow spatial and temporal control over the release of a compound, while fluorescence-based sensors allow the real-time visualization of kinase activity. Here, we discuss recent advances that have increased the specificity and availability of these tools.
Asunto(s)
Proteínas Quinasas/metabolismo , Transducción de Señal/fisiología , Animales , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/química , Humanos , Modelos Químicos , Sondas Moleculares/química , Estructura Molecular , Péptidos/química , Fosfoproteínas/química , Fosforilación , Fotoquímica , Proteínas Quinasas/química , Especificidad por SustratoRESUMEN
We present the chemical and biological synthesis of caged phosphoproteins using the in vitro nonsense codon suppression methodology. Specifically, phosphoamino acid analogues of serine, threonine, and tyrosine with a single photocleavable o-nitrophenylethyl caging group were synthesized as the amino acyl tRNA adducts for insertion into full-length proteins. For this purpose, a novel phosphitylating agent was developed. The successful incorporation of these bulky and charged amino acids into the alpha-subunit of the nicotinic acetyl choline receptor (nAChR) and the vasodilator-stimulated phosphoprotein (VASP) using an in vitro translation system is reported.
Asunto(s)
Fosfoproteínas/biosíntesis , Fosfoproteínas/síntesis química , Serina/análogos & derivados , Treonina/análogos & derivados , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfoproteínas/química , ARN de Transferencia Aminoácido-Específico/química , ARN de Transferencia Aminoácido-Específico/metabolismo , Serina/metabolismo , Treonina/metabolismoRESUMEN
A new 4-(N,N-dimethylamino) phthalimide-based environment-sensitive fluorescent building block for solid phase peptide synthesis, has been synthesized and incorporated into peptides. Peptides incorporating this residue show great potential for biological applications in sensing protein/protein interactions.
Asunto(s)
Aminoácidos/química , Fluorenos/química , Colorantes Fluorescentes/química , Péptidos/síntesis química , Estructura Molecular , Péptidos/química , Ftalimidas/síntesis química , Ftalimidas/químicaRESUMEN
Using classical genetics to study modular phosphopeptide-binding domains within a family of proteins that are functionally redundant is difficult when other members of the domain family compensate for the product of the knocked-out gene. Here we describe a chemical genetics approach that overcomes this limitation by using UV-activatable caged phosphopeptides. By incorporating a caged phosphoserine residue within a consensus motif, these reagents simultaneously and synchronously inactivate all phosphoserine/phosphothreonine-binding domain family members in a rapid and temporally regulated manner. We applied this approach to study the global function of 14-3-3 proteins in cell cycle control. Activation of the caged phosphopeptides by UV irradiation displaced endogenous proteins from 14-3-3-binding, causing premature cell cycle entry, release of G1 cells from interphase arrest and loss of the S-phase checkpoint after DNA damage, accompanied by high levels of cell death. This class of reagents will greatly facilitate molecular dissection of kinase-dependent signaling pathways when applied to other phosphopeptide-binding domains including SH2, Polo-box and tandem BRCT domains.
Asunto(s)
Proteínas 14-3-3/metabolismo , Fase G1 , Osteosarcoma/metabolismo , Osteosarcoma/patología , Fosfopéptidos/metabolismo , Fotoquímica/métodos , Fase S , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Humanos , Fosfopéptidos/efectos de la radiación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efectos de la radiación , Transducción de Señal , Relación Estructura-Actividad , Factores de Tiempo , Rayos UltravioletaRESUMEN
Three 1-(2-nitrophenyl)ethyl-caged phospho-amino acids have been synthesized for use in standard N(alpha)-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis (SPPS). The most common naturally occurring phospho-amino acids, serine, threonine, and tyrosine, were prepared as protected caged building blocks by modification with a unique phosphitylating reagent. In previous work, caged phospho-peptides were made using an interassembly approach (Rothman, D. M.; Vazquez, M. E.; Vogel, E. M.; Imperiali, B. Org. Lett. 2002, 4, 2865-2868). However, this technique is limited to creating peptides without oxidation sensitive residues C-terminal to the amino acid to be modified and the methodology involves synthetic manipulations on the solid phase that may limit the utilization of the methodology. Herein we report the facile synthesis of N-alpha-Fmoc-phospho(1-nitrophenylethyl-2-cyanoethyl)-L-serine 1, N-alpha-Fmoc-phospho(1-nitrophenylethyl-2-cyanoethyl)-L-threonine 2, and N-alpha-Fmoc-phospho(1-nitrophenylethyl-2-cyanoethyl)-L-tyrosine 3. These building blocks allow the synthesis of any caged phospho-peptide sequence using standard Fmoc-based SPPS procedures.
Asunto(s)
Aminoácidos/química , Péptidos/síntesis química , Ácidos Fosfóricos/química , Indicadores y Reactivos , Espectroscopía de Resonancia MagnéticaRESUMEN
[reaction: see text] An interassembly approach for the synthesis of peptides containing 1-(2-nitrophenyl)ethyl-caged phosphoserine, -threonine, and -tyrosine has been developed. Photochemical uncaging of these peptides releases the 2-nitrophenylethyl protecting group to afford the corresponding phosphopeptide. The peptides described herein are based on phosphorylation sites of kinases involved in cell movement or cell cycle regulation and demonstrate the versatility of the method and compatibility with the synthesis of polypeptides, including a variety of encoded amino acids.