RESUMEN
Transcription factors are generally considered challenging, if not "undruggable", targets but they promise new therapeutic options due to their fundamental involvement in many diseases. In this study, we aim to assess the ligandability of the C-terminal Rel-homology domain of nuclear factor of activated T cells 1 (NFAT1), a TF implicated in T-cell regulation. Using a combination of experimental and computational approaches, we demonstrate that small molecule fragments can indeed bind to this protein domain. The newly identified binder is the first small molecule binder to NFAT1 validated with biophysical methods and an elucidated binding mode by X-ray crystallography. The reported eutomer/distomer pair provides a strong basis for potential exploration of higher potency binders on the path toward degrader or glue modalities.
Asunto(s)
Factores de Transcripción NFATC , Sitios de Unión , Cristalografía por Rayos X , Ligandos , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción NFATC/química , Unión Proteica , Dominios Proteicos , Relación Estructura-ActividadRESUMEN
HUWE1 is a universal quality-control E3 ligase that marks diverse client proteins for proteasomal degradation. Although the giant HECT enzyme is an essential component of the ubiquitin-proteasome system closely linked with severe human diseases, its molecular mechanism is little understood. Here, we present the crystal structure of Nematocida HUWE1, revealing how a single E3 enzyme has specificity for a multitude of unrelated substrates. The protein adopts a remarkable snake-like structure, where the C-terminal HECT domain heads an extended alpha-solenoid body that coils in on itself and houses various protein-protein interaction modules. Our integrative structural analysis shows that this ring structure is highly dynamic, enabling the flexible HECT domain to reach protein targets presented by the various acceptor sites. Together, our data demonstrate how HUWE1 is regulated by its unique structure, adapting a promiscuous E3 ligase to selectively target unassembled orphan proteins.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Microsporidios/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas Fúngicas , Insectos , Microsporidios/genética , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The 3 human RAS genes, KRAS, NRAS, and HRAS, encode 4 different RAS proteins which belong to the protein family of small GTPases that function as binary molecular switches involved in cell signaling. Activating mutations in RAS are among the most common oncogenic drivers in human cancers, with KRAS being the most frequently mutated oncogene. Although KRAS is an excellent drug discovery target for many cancers, and despite decades of research, no therapeutic agent directly targeting RAS has been clinically approved. Using structure-based drug design, we have discovered BI-2852 (1), a KRAS inhibitor that binds with nanomolar affinity to a pocket, thus far perceived to be "undruggable," between switch I and II on RAS; 1 is mechanistically distinct from covalent KRASG12C inhibitors because it binds to a different pocket present in both the active and inactive forms of KRAS. In doing so, it blocks all GEF, GAP, and effector interactions with KRAS, leading to inhibition of downstream signaling and an antiproliferative effect in the low micromolar range in KRAS mutant cells. These findings clearly demonstrate that this so-called switch I/II pocket is indeed druggable and provide the scientific community with a chemical probe that simultaneously targets the active and inactive forms of KRAS.
Asunto(s)
Descubrimiento de Drogas , Preparaciones Farmacéuticas/química , Proteínas Proto-Oncogénicas p21(ras)/química , Guanosina Trifosfato/metabolismo , Humanos , Modelos Moleculares , Nanopartículas/químicaRESUMEN
While CH-π interactions with target proteins are crucial determinants for the affinity of arguably every drug molecule, no method exists to directly measure the strength of individual CH-π interactions in drug-protein complexes. Herein, we present a fast and reliable methodology called PI (π interactions) by NMR, which can differentiate the strength of protein-ligand CH-π interactions in solution. By combining selective amino-acid side-chain labeling with 1 H-13 Câ NMR, we are able to identify specific protein protons of side-chains engaged in CH-π interactions with aromatic ring systems of a ligand, based solely on 1 H chemical-shift values of the interacting protein aromatic ring protons. The information encoded in the chemical shifts induced by such interactions serves as a proxy for the strength of each individual CH-π interaction. PI by NMR changes the paradigm by which chemists can optimize the potency of drug candidates: direct determination of individual π interactions rather than averaged measures of all interactions.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Proteínas/química , Humanos , Modelos MolecularesRESUMEN
Natural products have proven to be a rich source of molecular architectures for drugs. Here, an integrated approach to natural product screening is proposed, which uncovered eight new natural product scaffolds for KRAS-the most frequently mutated oncogenic driver in human cancers, which has remained thus far undrugged. The approach combines aspects of virtual screening, fragment-based screening, structure-activity relationships (SAR) by NMR, and structure-based drug discovery to overcome the limitations in traditional natural product approaches. By using our approach, a new "snugness of fit" scoring function and the first crystal-soaking system of the active form of KRASG12D , the protein-ligand X-ray structures of a tricyclic indolopyrrole fungal alkaloid and an indoloisoquinolinone have been successfully elucidated. The natural product KRAS hits discovered provide fruitful ground for the optimization of highly potent natural-product-based inhibitors of the active form of oncogenic RAS. This integrated approach for screening natural products also holds promise for other "undruggable" targets.
RESUMEN
The WWE domain is a relatively under-researched domain found in twelve human proteins and characterized by a conserved tryptophan-tryptophan-glutamate (WWE) sequence motif. Six of these WWE domain-containing proteins also contain domains with E3 ubiquitin ligase activity. The general recognition of poly-ADP-ribosylated substrates by WWE domains suggests a potential avenue for development of Proteolysis-Targeting Chimeras (PROTACs). Here, we present novel crystal structures of the HUWE1, TRIP12, and DTX1 WWE domains in complex with PAR building blocks and their analogs, thus enabling a comprehensive analysis of the PAR binding site structural diversity. Furthermore, we introduce a versatile toolbox of biophysical and biochemical assays for the discovery and characterization of novel WWE domain binders, including fluorescence polarization-based PAR binding and displacement assays, 15N-NMR-based binding affinity assays and 19F-NMR-based competition assays. Through these assays, we have characterized the binding of monomeric iso-ADP-ribose (iso-ADPr) and its nucleotide analogs with the aforementioned WWE proteins. Finally, we have utilized the assay toolbox to screen a small molecule fragment library leading to the successful discovery of novel ligands targeting the HUWE1 WWE domain.
Asunto(s)
Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/química , Humanos , Ligandos , Unión Proteica , Sitios de Unión , Dominios Proteicos , Modelos Moleculares , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Cristalografía por Rayos X , Descubrimiento de Drogas/métodosRESUMEN
Identifying promising chemical starting points for small molecule inhibitors of active, GTP-loaded KRAS "on" remains of great importance to clinical oncology and represents a significant challenge in medicinal chemistry. Here, we describe broadly applicable learnings from a KRAS hit finding campaign: While we initially identified KRAS inhibitors in a biochemical high-throughput screen, we later discovered that compound potencies were all but assay artifacts linked to metal salts interfering with KRAS AlphaScreen assay technology. The source of the apparent biochemical KRAS inhibition was ultimately traced to unavoidable palladium impurities from chemical synthesis. This discovery led to the development of a Metal Ion Interference Set (MIIS) for up-front assay development and testing. Profiling of the MIIS across 74 assays revealed a reduced interference liability of label-free biophysical assays and, as a result, provided general estimates for luminescence- and fluorescence-based assay susceptibility to metal salt interference.
Asunto(s)
Paladio , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Paladio/química , Ensayos Analíticos de Alto Rendimiento/métodos , Sales (Química)/químicaRESUMEN
Activating mutations in KRAS are the most frequent oncogenic alterations in cancer. The oncogenic hotspot position 12, located at the lip of the switch II pocket, offers a covalent attachment point for KRASG12C inhibitors. To date, KRASG12C inhibitors have been discovered by first covalently binding to the cysteine at position 12 and then optimizing pocket binding. We report on the discovery of the in vivo active KRASG12C inhibitor BI-0474 using a different approach, in which small molecules that bind reversibly to the switch II pocket were identified and then optimized for non-covalent binding using structure-based design. Finally, the Michael acceptor containing warhead was attached. Our approach offers not only an alternative approach to discovering KRASG12C inhibitors but also provides a starting point for the discovery of inhibitors against other oncogenic KRAS mutants.
Asunto(s)
Neoplasias , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Genes ras , Mutación , Neoplasias/genética , CisteínaRESUMEN
Son of Sevenless (SOS) is a guanine nucleotide exchange factor that activates the important cell signaling switch KRAS. SOS acts as a pacemaker for KRAS, the beating heart of cancer, by catalyzing the "beating" from the KRAS(off) to the KRAS(on) conformation. Activating mutations in SOS1 are common in Noonan syndrome and oncogenic alterations in KRAS drive 1 in seven human cancers. Promising clinical efficacy has been observed for selective KRASG12C inhibitors, but the vast majority of oncogenic KRAS alterations remain undrugged. The discovery of a druggable pocket on SOS1 has led to potent SOS1 inhibitors such as BI-3406. SOS1 inhibition leads to antiproliferative effects against all major KRAS mutants. The first SOS1 inhibitor has entered clinical trials for KRAS-mutated cancers. In this review, we provide an overview of SOS1 function, its association with cancer and RASopathies, known SOS1 activators and inhibitors, and a future perspective is provided.
Asunto(s)
Antineoplásicos/química , Proteínas Mutantes/química , Neoplasias/terapia , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína SOS1/antagonistas & inhibidores , Acetonitrilos/farmacología , Antineoplásicos/farmacología , Regulación de la Expresión Génica , Humanos , Mutación , Marcapaso Artificial , Piperazinas/farmacología , Conformación Proteica , Piridinas/farmacología , Pirimidinas/farmacología , Proteína SOS1/metabolismo , Transducción de Señal , Relación Estructura-ActividadRESUMEN
It has taken four decades of research to see the first major breakthrough for KRAS-driven cancers. In particular, the last decade has seen a paradigm shift with the discovery of druggable pockets on KRAS and clinical efficacy with covalent KRASG12C inhibitors, culminating in the first approval of sotorasib monotherapy as second-line treatment in KRASG12C-driven non-small-cell lung cancer. Nevertheless, 85% of all KRAS-mutated cancers still lack novel agents. In this review, we will outline the structure, function, and post-translational modifications of KRAS and highlight the various approaches being adopted to drug KRAS, ranging from selective to pan concepts. The range of molecular modalities being explored, including PROTACs and glues, will also be described. Finally, an outlook toward the next wave of KRAS drugs and the challenges of resistance will be given.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Mutación , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Aberrant WNT pathway activation, leading to nuclear accumulation of ß-catenin, is a key oncogenic driver event. Mutations in the tumor suppressor gene APC lead to impaired proteasomal degradation of ß-catenin and subsequent nuclear translocation. Restoring cellular degradation of ß-catenin represents a potential therapeutic strategy. Here, we report the fragment-based discovery of a small molecule binder to ß-catenin, including the structural elucidation of the binding mode by X-ray crystallography. The difficulty in drugging ß-catenin was confirmed as the primary screening campaigns identified only few and very weak hits. Iterative virtual and NMR screening techniques were required to discover a compound with sufficient potency to be able to obtain an X-ray co-crystal structure. The binding site is located between armadillo repeats two and three, adjacent to the BCL9 and TCF4 binding sites. Genetic studies show that it is unlikely to be useful for the development of protein-protein interaction inhibitors but structural information and established assays provide a solid basis for a prospective optimization towards ß-catenin proteolysis targeting chimeras (PROTACs) as alternative modality.
Asunto(s)
Bibliotecas de Moléculas Pequeñas/química , beta Catenina/antagonistas & inhibidores , Sitios de Unión , Cristalografía por Rayos X , Humanos , Simulación de Dinámica Molecular , Mapas de Interacción de Proteínas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , beta Catenina/metabolismoRESUMEN
KRAS, the most common oncogenic driver in human cancers, is controlled and signals primarily through protein-protein interactions (PPIs). The interaction between KRAS and SOS1, crucial for the activation of KRAS, is a typical, challenging PPI with a large contact surface area and high affinity. Here, we report that the addition of only one atom placed between Y884SOS1 and A73KRAS is sufficient to convert SOS1 activators into SOS1 inhibitors. We also disclose the discovery of BI-3406. Combination with the upstream EGFR inhibitor afatinib shows in vivo efficacy against KRASG13D mutant colorectal tumor cells, demonstrating the utility of BI-3406 to probe SOS1 biology. These findings challenge the dogma that large molecules are required to disrupt challenging PPIs. Instead, a "foot in the door" approach, whereby single atoms or small functional groups placed between key PPI interactions, can lead to potent inhibitors even for challenging PPIs such as SOS1-KRAS.
Asunto(s)
Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína SOS1/metabolismo , Afatinib/química , Afatinib/metabolismo , Afatinib/uso terapéutico , Regulación Alostérica/efectos de los fármacos , Sitios de Unión , Dominio Catalítico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Humanos , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Mapas de Interacción de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genética , Quinazolinas/química , Quinazolinas/metabolismo , Quinazolinas/farmacología , Quinazolinas/uso terapéutico , Proteína SOS1/agonistas , Proteína SOS1/antagonistas & inhibidores , Proteína SOS1/genéticaRESUMEN
KRAS is the most frequently mutated driver of pancreatic, colorectal, and non-small cell lung cancers. Direct KRAS blockade has proved challenging, and inhibition of a key downstream effector pathway, the RAF-MEK-ERK cascade, has shown limited success because of activation of feedback networks that keep the pathway in check. We hypothesized that inhibiting SOS1, a KRAS activator and important feedback node, represents an effective approach to treat KRAS-driven cancers. We report the discovery of a highly potent, selective, and orally bioavailable small-molecule SOS1 inhibitor, BI-3406, that binds to the catalytic domain of SOS1, thereby preventing the interaction with KRAS. BI-3406 reduces formation of GTP-loaded RAS and limits cellular proliferation of a broad range of KRAS-driven cancers. Importantly, BI-3406 attenuates feedback reactivation induced by MEK inhibitors and thereby enhances sensitivity of KRAS-dependent cancers to MEK inhibition. Combined SOS1 and MEK inhibition represents a novel and effective therapeutic concept to address KRAS-driven tumors. SIGNIFICANCE: To date, there are no effective targeted pan-KRAS therapies. In-depth characterization of BI-3406 activity and identification of MEK inhibitors as effective combination partners provide an attractive therapeutic concept for the majority of KRAS-mutant cancers, including those fueled by the most prevalent mutant KRAS oncoproteins, G12D, G12V, G12C, and G13D.See related commentary by Zhao et al., p. 17.This article is highlighted in the In This Issue feature, p. 1.
Asunto(s)
Neoplasias Pulmonares , Proteínas Proto-Oncogénicas p21(ras) , Línea Celular Tumoral , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Mutación , Nucleótidos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Protein degraders, also known as proteolysis targeting chimeras (PROTACs), are bifunctional small molecules that promote cellular degradation of a protein of interest (POI). PROTACs act as molecular mediators, bringing an E3 ligase and a POI into proximity, thus promoting ubiquitination and degradation of the targeted POI. Despite their great promise as next-generation pharmaceutical drugs, the development of new PROTACs is challenged by the complexity of the system, which involves binary and ternary interactions between components. Here, we demonstrate the strength of native mass spectrometry (nMS), a label-free technique, to provide novel insight into PROTAC-mediated protein interactions. We show that nMS can monitor the formation of ternary E3-PROTAC-POI complexes and detect various intermediate species in a single experiment. A unique benefit of the method is its ability to reveal preferentially formed E3-PROTAC-POI combinations in competition experiments with multiple substrate proteins, thereby positioning it as an ideal high-throughput screening strategy during the development of new PROTACs.
RESUMEN
Activating mutations in the three human RAS genes, KRAS, NRAS and HRAS, are among the most common oncogenic drivers in human cancers. Covalent KRASG12C inhibitors, which bind to the switch II pocket in the 'off state' of KRAS, represent the first direct KRAS drugs that entered human clinical trials. However, the remaining 85% of non-KRASG12C-driven cancers remain undrugged as do NRAS and HRAS and no drugs targeting the 'on state' have been discovered so far. The switch I/II pocket is a second pocket for which the nanomolar inhibitor BI-2852 has been discovered. Here, we elucidate inhibitor binding modes in KRAS, NRAS and HRAS on and off and discuss future strategies to drug all RAS isoforms with this one pocket.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Isoenzimas/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Proteínas ras/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/enzimología , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Phosphoglycerate dehydrogenase (PHGDH) is known to be the rate-limiting enzyme in the serine synthesis pathway in humans. It converts glycolysis-derived 3-phosphoglycerate to 3-phosphopyruvate in a co-factor-dependent oxidation reaction. Herein, we report the discovery of BI-4916, a prodrug of the co-factor nicotinamide adenine dinucleotide (NADH/NAD+)-competitive PHGDH inhibitor BI-4924, which has shown high selectivity against the majority of other dehydrogenase targets. Starting with a fragment-based screening, a subsequent hit optimization using structure-based drug design was conducted to deliver a single-digit nanomolar lead series and to improve potency by 6 orders of magnitude. To this end, an intracellular ester cleavage mechanism of the ester prodrug was utilized to achieve intracellular enrichment of the actual carboxylic acid based drug and thus overcome high cytosolic levels of the competitive cofactors NADH/NAD+.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Fosfoglicerato-Deshidrogenasa/antagonistas & inhibidores , Serina/antagonistas & inhibidores , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Indoles/síntesis química , Indoles/química , Modelos Moleculares , Estructura Molecular , Fosfoglicerato-Deshidrogenasa/metabolismo , Serina/biosíntesis , Relación Estructura-ActividadRESUMEN
Fragment-based drug design exploits initial screening of low molecular weight compounds and their concomitant affinity improvement. The multitude of possible chemical modifications highlights the necessity to obtain structural information about the binding mode of a fragment. Herein we describe a novel NMR methodology (LOGSY titration) that allows the determination of binding modes of low affinity binders in the protein-ligand interface and reveals suitable ligand positions for the addition of functional groups that either address or substitute protein-bound water, information of utmost importance for drug design. The particular benefit of the methodology and in contrast to conventional ligand-based methods is the independence of the molecular weight of the protein under study. The validity of the novel approach is demonstrated on two ligands interacting with bromodomain 1 of bromodomain containing protein 4, a prominent cancer target in pharmaceutical industry.
Asunto(s)
Diseño de Fármacos , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Agua/química , Sitios de Unión , Proteínas de Ciclo Celular , Humanos , Ligandos , Modelos Moleculares , Proteínas Nucleares/química , Unión Proteica , Volumetría , Factores de Transcripción/químicaRESUMEN
The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B cell lymphoma (DLBCL). Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We used a structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also causes rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and anti-proliferative effects than compounds that merely inhibit co-repressor interactions. This work establishes the BTB domain as a highly druggable structure, paving the way for the use of other members of this protein family as drug targets. The magnitude of effects elicited by this class of BCL6-degrading compounds exceeds that of our equipotent non-degrading inhibitors, suggesting opportunities for the development of BCL6-based lymphoma therapeutics.