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1.
J Med Chem ; 67(11): 8585-8608, 2024 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-38809766

RESUMEN

The von Hippel-Lindau (VHL) protein plays a pivotal role in regulating the hypoxic stress response and has been extensively studied and utilized in the targeted protein degradation field, particularly in the context of bivalent degraders. In this study, we present a comprehensive peptidomimetic structure-activity relationship (SAR) approach, combined with cellular NanoBRET target engagement assays to enhance the existing VHL ligands. Through systematic modifications of the molecule, we identified the 1,2,3-triazole group as an optimal substitute of the left-hand side amide bond that yields 10-fold higher binding activity. Moreover, incorporating conformationally constrained alterations on the methylthiazole benzylamine moiety led to the development of highly potent VHL ligands with picomolar binding affinity and significantly improved oral bioavailability. We anticipate that our optimized VHL ligand, GNE7599, will serve as a valuable tool compound for investigating the VHL pathway and advancing the field of targeted protein degradation.


Asunto(s)
Disponibilidad Biológica , Peptidomiméticos , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/química , Peptidomiméticos/química , Peptidomiméticos/farmacocinética , Peptidomiméticos/farmacología , Humanos , Ligandos , Relación Estructura-Actividad , Administración Oral , Animales
3.
Nat Biotechnol ; 40(5): 769-778, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-34992247

RESUMEN

Small molecules that stabilize inactive protein conformations are an underutilized strategy for drugging dynamic or otherwise intractable proteins. To facilitate the discovery and characterization of such inhibitors, we created a screening platform to identify conformation-locking antibodies for molecular probes (CLAMPs) that distinguish and induce rare protein conformational states. Applying the approach to KRAS, we discovered CLAMPs that recognize the open conformation of KRASG12C stabilized by covalent inhibitors. One CLAMP enables the visualization of KRASG12C covalent modification in vivo and can be used to investigate response heterogeneity to KRASG12C inhibitors in patient tumors. A second CLAMP enhances the affinity of weak ligands binding to the KRASG12C switch II region (SWII) by stabilizing a specific conformation of KRASG12C, thereby enabling the discovery of such ligands that could serve as leads for the development of drugs in a high-throughput screen. We show that combining the complementary properties of antibodies and small molecules facilitates the study and drugging of dynamic proteins.


Asunto(s)
Anticuerpos , Neoplasias , Proteínas Proto-Oncogénicas p21(ras) , Anticuerpos/química , Humanos , Ligandos , Mutación , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores
4.
Angew Chem Int Ed Engl ; 59(35): 14809-14817, 2020 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-32363632

RESUMEN

Fragment-based lead discovery has become a fundamental approach to identify ligands that efficiently interact with disease-relevant targets. Among the numerous screening techniques, fluorine-detected NMR has gained popularity owing to its high sensitivity, robustness, and ease of use. To effectively explore chemical space, a universal NMR experiment, a rationally designed fragment library, and a sample composition optimized for a maximal number of compounds and minimal measurement time are required. Here, we introduce a comprehensive method that enabled the efficient assembly of a high-quality and diverse library containing nearly 4000 fragments and screening for target-specific binders within days. At the core of the approach is a novel broadband relaxation-edited NMR experiment that covers the entire chemical shift range of drug-like 19 F motifs in a single measurement. Our approach facilitates the identification of diverse binders and the fast ligandability assessment of new targets.

5.
J Am Chem Soc ; 142(9): 4445-4455, 2020 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-32064871

RESUMEN

The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multi-drug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA, and compound 2 is an uncompetitive inhibitor targeting the LpxA/product complex. Compound 2 exhibited more favorable biological and physicochemical properties than compound 1 and was optimized using structural information to achieve improved antibacterial activity against wild-type E. coli. These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme/product complexes in drug discovery.


Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Pirazoles/farmacología , Aciltransferasas/metabolismo , Antibacterianos/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Imidazoles/metabolismo , Pruebas de Sensibilidad Microbiana , Unión Proteica , Pirazoles/metabolismo
7.
Elife ; 92020 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-31960795

RESUMEN

In pursuit of therapeutics for human polyomaviruses, we identified a peptide derived from the BK polyomavirus (BKV) minor structural proteins VP2/3 that is a potent inhibitor of BKV infection with no observable cellular toxicity. The thirteen-residue peptide binds to major structural protein VP1 with single-digit nanomolar affinity. Alanine-scanning of the peptide identified three key residues, substitution of each of which results in ~1000 fold loss of binding affinity with a concomitant reduction in antiviral activity. Structural studies demonstrate specific binding of the peptide to the pore of pentameric VP1. Cell-based assays demonstrate nanomolar inhibition (EC50) of BKV infection and suggest that the peptide acts early in the viral entry pathway. Homologous peptide exhibits similar binding to JC polyomavirus VP1 and inhibits infection with similar potency to BKV in a model cell line. Lastly, these studies validate targeting the VP1 pore as a novel strategy for the development of anti-polyomavirus agents.


Asunto(s)
Antivirales/metabolismo , Virus BK , Proteínas de la Cápside/metabolismo , Virus JC/efectos de los fármacos , Péptidos/metabolismo , Antivirales/química , Antivirales/farmacología , Virus BK/efectos de los fármacos , Virus BK/genética , Virus BK/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Células Cultivadas , Células HEK293 , Humanos , Péptidos/química , Péptidos/genética , Unión Proteica
8.
Nat Chem Biol ; 16(1): 15-23, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31819272

RESUMEN

The anticancer agent indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor, leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which indisulam mediates the DCAF15-RBM39 interaction, we solved the DCAF15-DDB1-DDA1-indisulam-RBM39(RRM2) complex structure to a resolution of 2.3 Å. DCAF15 has a distinct topology that embraces the RBM39(RRM2) domain largely via non-polar interactions, and indisulam binds between DCAF15 and RBM39(RRM2), coordinating additional interactions between the two proteins. Studies with RBM39 point mutants and indisulam analogs validated the structural model and defined the RBM39 α-helical degron motif. The degron is found only in RBM23 and RBM39, and only these proteins were detectably downregulated in indisulam-treated HCT116 cells. This work further explains how indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade additional targets.


Asunto(s)
Antineoplásicos/farmacología , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas de Unión al ARN/química , Sulfonamidas/farmacología , Secuencias de Aminoácidos , Calorimetría , Clonación Molecular , Fluorometría , Células HCT116 , Células HEK293 , Humanos , Procesamiento de Imagen Asistido por Computador , Péptidos y Proteínas de Señalización Intracelular/genética , Cinética , Proteínas Nucleares/metabolismo , Péptidos/química , Mutación Puntual , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteoma , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Ubiquitina-Proteína Ligasas/metabolismo
9.
Sci Rep ; 9(1): 18389, 2019 12 05.
Artículo en Inglés | MEDLINE | ID: mdl-31804511

RESUMEN

Transient affinity binding interactions are central to life, composing the fundamental elements of biological networks including cell signaling, cell metabolism and gene regulation. Assigning a defined reaction mechanism to affinity binding interactions is critical to our understanding of the associated structure-function relationship, a cornerstone of biophysical characterization. Transient kinetics are currently measured using low throughput methods such as nuclear magnetic resonance, or stop-flow spectrometry-based techniques, which are not practical in many settings. In contrast, label-free biosensors measure reaction kinetics through direct binding, and with higher throughout, impacting life sciences with thousands of publications each year. Here we have developed a methodology enabling label-free biosensors to measure transient kinetic interactions towards providing a higher throughput approach suitable for mechanistic understanding of these processes. The methodology relies on hydrodynamic dispersion modeling of a smooth analyte gradient under conditions that maintain the quasi-steady-state boundary layer assumption. A transient peptide-protein interaction of relevance to drug discovery was analyzed thermodynamically using transition state theory and numerical simulations validated the approach over a wide range of operating conditions. The data establishes the technical feasibility of this approach to transient kinetic analyses supporting further development towards higher throughput applications in life science.


Asunto(s)
Técnicas Biosensibles , Proteínas de Unión a Maltosa/química , Modelos Estadísticos , Péptidos/química , Simulación por Computador , Humanos , Cinética , Unión Proteica , Termodinámica
10.
Methods Enzymol ; 614: 1-36, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30611421

RESUMEN

Escherichia coli expression protocols for selective labeling of methyl groups in proteins have been essential in expanding the size range of targets that can be studied by biomolecular NMR. Based on the initial work achieving selective labeling of isoleucine, leucine, and valine residues, additional methods were developed over the past years which enabled the individual and/or simultaneous combinatorial labeling of all methyl containing amino acids. Together with the introduction of new methyl-optimized NMR experiments, this now allows the detailed characterization of protein-ligand interactions as well as mechanistic and dynamic processes of protein-protein complexes up to 1MDa in size. In this chapter, we provide a general introduction to selective labeling of proteins using E. coli-based expression systems, describe the considerations taken into account prior to the selective labeling of a protein, and include the protocols used to produce such proteins. An overview of applications using selectively labeled proteins with an emphasis on examples relevant to the drug discovery process is then presented.


Asunto(s)
Proteínas de Escherichia coli/química , Marcaje Isotópico/métodos , Leucina/química , Espectroscopía de Resonancia Magnética/métodos , Coloración y Etiquetado/métodos , Valina/química , Descubrimiento de Drogas , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Humanos , Leucina/metabolismo , Ligandos , Espectroscopía de Resonancia Magnética/instrumentación , Metilación , Simulación de Dinámica Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Valina/metabolismo
11.
J Med Chem ; 59(7): 3303-10, 2016 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-26964888

RESUMEN

NMR binding assays are routinely applied in hit finding and validation during early stages of drug discovery, particularly for fragment-based lead generation. To this end, compound libraries are screened by ligand-observed NMR experiments such as STD, T1ρ, and CPMG to identify molecules interacting with a target. The analysis of a high number of complex spectra is performed largely manually and therefore represents a limiting step in hit generation campaigns. Here we report a novel integrated computational procedure that processes and analyzes ligand-observed proton and fluorine NMR binding data in a fully automated fashion. A performance evaluation comparing automated and manual analysis results on (19)F- and (1)H-detected data sets shows that the program delivers robust, high-confidence hit lists in a fraction of the time needed for manual analysis and greatly facilitates visual inspection of the associated NMR spectra. These features enable considerably higher throughput, the assessment of larger libraries, and shorter turn-around times.


Asunto(s)
Diseño de Fármacos , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Espectroscopía de Resonancia Magnética/métodos , Bibliotecas de Moléculas Pequeñas/química , Bases de Datos de Compuestos Químicos , Radioisótopos de Flúor/química , Humanos , Ligandos , Unión Proteica
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