RESUMEN
BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.
Asunto(s)
Proteína BRCA2/antagonistas & inhibidores , Recombinasa Rad51/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Proteína BRCA2/química , Proteína BRCA2/metabolismo , Muerte Celular/efectos de los fármacos , Cristalografía por Rayos X , Daño del ADN , Humanos , Modelos Moleculares , Conformación Molecular , Unión Proteica/efectos de los fármacos , Recombinasa Rad51/química , Recombinasa Rad51/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Células Tumorales CultivadasRESUMEN
DNA-encoded combinatorial synthesis provides efficient and dense coverage of chemical space around privileged molecular structures. The indole side chain of tryptophan plays a prominent role in key, or "hot spot", regions of protein-protein interactions. A DNA-encoded combinatorial peptoid library was designed based on the Ugi four-component reaction by employing tryptophan-mimetic indole side chains to probe the surface of target proteins. Several peptoids were synthesized on a chemically stable hexathymidine adapter oligonucleotide "hexT", encoded by DNA sequences, and substituted by azide-alkyne cycloaddition to yield a library of 8112 molecules. Selection experiments for the tumor-relevant proteins MDM2 and TEAD4 yielded MDM2 binders and a novel class of TEAD-YAP interaction inhibitors that perturbed the expression of a gene under the control of these Hippo pathway effectors.
Asunto(s)
ADN/metabolismo , Indoles/metabolismo , Peptidomiméticos , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Factores de Transcripción/metabolismo , Humanos , Unión ProteicaRESUMEN
Small-molecule modulation of protein-protein interactions (PPIs) is a very promising but also challenging area in drug discovery. The tumor suppressor protein p53 is one of the most frequently altered proteins in human cancers, making it an attractive target in oncology. 14-3-3 proteins have been shown to bind to and positively regulate p53 activity by protecting it from MDM2-dependent degradation or activating its DNA binding affinity. PPIs can be modulated by inhibiting or stabilizing specific interactions by small molecules. Whereas inhibition has been widely explored by the pharmaceutical industry and academia, the opposite strategy of stabilizing PPIs still remains relatively underexploited. This is rather interesting considering the number of natural compounds like rapamycin, forskolin and fusicoccin that exert their activity by stabilizing specific PPIs. In this review, we give an overview of 14-3-3 interactions with p53, explain isoform specific stabilization of the tumor suppressor protein, explore the approach of stabilizing the 14-3-3σ-p53 complex and summarize some promising small molecules inhibiting the p53-MDM2 protein-protein interaction.
Asunto(s)
Unión Proteica/fisiología , Proteína p53 Supresora de Tumor/metabolismo , HumanosRESUMEN
The focal adhesion kinase-growth factor receptor 2 (FAK-Grb2) protein-protein interaction is implicated in pathogenesis of stress-induced cardiac hypertrophy. The focal adhesion targeting (FAT) domain of FAK unfolds to form a structural intermediate that interacts with a multibinding hot spot in the SH2 domain of Grb2. Disruption of the Grb2-FAT interaction is a therapeutic strategy for prevention of pathological cardiac hypertrophy. A pharmacophore was generated on the basis of structural and electrostatic properties of FAT bound to FAK using the Forge tool (Cresset). This pharmacophore was used as a query for Blaze server (Cresset) to screen a selectively enriched chemical library of 4,32,508 small molecules. The compounds selected were further filtered by hierarchical flexible docking approach using AutoDock v4. From the favorably docked compounds, five were selected on the basis of good adsorption, distribution, metabolism, excretion, and toxicity (ADMET) properties using SwissADME, MedChem Designer v.3, and MOLINSPIRATION. Stability of the binding mode of the inhibitors was further confirmed by molecular dynamic simulation study with AMBER v15 for a simulation time of 50 ns in aqueous environment. PM2307 was identified as the best inhibitor in terms of pharmacophoric features, dock score, and in silico ADMET analysis. The calculated binding affinity of PM2307 was better than that of the FAT-Grb2 complex as well as a previously reported small molecule inhibitor. PM2307 is also a quinolyl derivative sharing a similar scaffold with ofloxacin drugs, asserting its drug-like properties. Thus, it was proposed as a lead compound for development of drugs for pathological cardiac hypertrophy.
Asunto(s)
Cardiomegalia/tratamiento farmacológico , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Proteína Adaptadora GRB2/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Cardiomegalia/metabolismo , Evaluación Preclínica de Medicamentos , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Proteína Adaptadora GRB2/metabolismo , Humanos , Ligandos , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Protein-protein interactions (PPIs) are principle biological processes that control normal cell growth, differentiation, and homeostasis but are also crucial in diseases such as malignancy, neuropathy, and infection. Despite the importance of PPIs in biology, this target class has been very challenging to convert to therapeutics. In the last decade, much progress has been made in the inhibition of PPIs involved in diseases, but many remain difficult such as RAS-effector interactions in cancers. We describe here a protocol for using Bioluminescence Resonance Energy Transfer 2 (BRET2)-based RAS biosensors to detect and characterize RAS PPI inhibition by macromolecules and small molecules. This method could be extended to any other small GTPases or any other PPIs of interest. © 2019 by John Wiley & Sons, Inc.
Asunto(s)
Transferencia de Energía , Mediciones Luminiscentes/métodos , Proteínas ras/análisis , Técnicas Biosensibles , Células HEK293 , Humanos , Immunoblotting , Ingeniería de ProteínasRESUMEN
The RAS family of proteins is amongst the most highly mutated in human cancers and has so far eluded drug therapy. Currently, much effort is being made to discover mutant RAS inhibitors and in vitro screening for RAS-binding drugs must be followed by cell-based assays. Here, we have developed a robust set of bioluminescence resonance energy transfer (BRET)-based RAS biosensors that enable monitoring of RAS-effector interaction inhibition in living cells. These include KRAS, HRAS and NRAS and a variety of different mutations that mirror those found in human cancers with the major RAS effectors such as CRAF, PI3K and RALGDS. We highlighted the utility of these RAS biosensors by showing a RAS-binding compound is a potent pan-RAS-effector interactions inhibitor in cells. The RAS biosensors represent a useful tool to investigate and characterize the potency of anti-RAS inhibitors in cells and more generally any RAS protein-protein interaction (PPI) in cells.
Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Técnicas Biosensibles/métodos , Mutación , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Transferencia de Energía , Células HEK293 , Humanos , Unión Proteica , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de SeñalRESUMEN
The actual strategy to improve current therapies in advanced prostate cancer involves targeting genes activated by androgen withdrawal, either to delay or prevent the emergence of the castration-refractory phenotype. However, these genes are often implicated in several physiological processes, and long-term inhibition of survival proteins might be accompanied with cytotoxic effects. To avoid this problem, an alternative therapeutic strategy relies on the identification and use of compounds that disrupt specific protein-protein interactions involved in androgen withdrawal. Specifically, the interaction of the chaperone protein Hsp27 with the initiation factor eIF4E leads to the protection of protein synthesis initiation process and enhances cell survival during cell stress induced by castration or chemotherapy. Thus, in this work we aimed at i) identifying the interaction site of the Hsp27/eIF4E complex and ii) interfere with the relevant protein/protein association mechanism involved in castration-resistant progression of prostate cancer. By a combination of experimental and modeling techniques, we proved that eIF4E interacts with the C-terminal part of Hsp27, preferentially when Hsp27 is phosphorylated. We also observed that the loss of this interaction increased cell chemo-and hormone-sensitivity. In order to find a potential inhibitor of Hsp27/eIF4E interaction, BRET assays in combination with molecular simulations identified the phenazine derivative 14 as the compound able to efficiently interfere with this protein/protein interaction, thereby inhibiting cell viability and increasing cell death in chemo- and castration-resistant prostate cancer models in vitro and in vivo.
RESUMEN
Interference with protein-protein interactions of interfaces larger than 1500 Ų by small drug-like molecules is notoriously difficult, particularly if targeting homodimers. The tRNA modifying enzyme Tgt is only functionally active as a homodimer. Thus, blocking Tgt dimerization is a promising strategy for drug therapy as this protein is key to the development of Shigellosis. Our goal was to identify hot-spot residues which, upon mutation, result in a predominantly monomeric state of Tgt. The detailed understanding of the spatial location and stability contribution of the individual interaction hot-spot residues and the plasticity of motifs involved in the interface formation is a crucial prerequisite for the rational identification of drug-like inhibitors addressing the respective dimerization interface. Using computational analyses, we identified hot-spot residues that contribute particularly to dimer stability: a cluster of hydrophobic and aromatic residues as well as several salt bridges. This in silico prediction led to the identification of a promising double mutant, which was validated experimentally. Native nano-ESI mass spectrometry showed that the dimerization of the suggested mutant is largely prevented resulting in a predominantly monomeric state. Crystal structure analysis and enzyme kinetics of the mutant variant further support the evidence for enhanced monomerization and provide first insights into the structural consequences of the dimer destabilization.