RESUMEN
The structural conservation across the AAA (ATPases associated with diverse cellular activities) protein family makes designing selective chemical inhibitors challenging. Here, we identify a triazolopyridine-based fragment that binds the AAA domain of human katanin, a microtubule-severing protein. We have developed a model for compound binding and designed ASPIR-1 (allele-specific, proximity-induced reactivity-based inhibitor-1), a cell-permeable compound that selectively inhibits katanin with an engineered cysteine mutation. Only in cells expressing mutant katanin does ASPIR-1 treatment increase the accumulation of CAMSAP2 at microtubule minus ends, confirming specific on-target cellular activity. Importantly, ASPIR-1 also selectively inhibits engineered cysteine mutants of human VPS4B and FIGL1-AAA proteins, involved in organelle dynamics and genome stability, respectively. Structural studies confirm our model for compound binding at the AAA ATPase site and the proximity-induced reactivity-based inhibition. Together, our findings suggest a chemical genetics approach to decipher AAA protein functions across essential cellular processes and to test hypotheses for developing therapeutics.
Asunto(s)
Proteínas AAA/genética , Katanina/genética , Proteínas Asociadas a Microtúbulos/genética , Piridinas/química , Proteínas AAA/antagonistas & inhibidores , Proteínas AAA/ultraestructura , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/ultraestructura , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/ultraestructura , Humanos , Katanina/ultraestructura , Proteínas Asociadas a Microtúbulos/ultraestructura , Microtúbulos/genética , Microtúbulos/ultraestructura , Conformación Proteica/efectos de los fármacos , Dominios Proteicos/genética , Piridinas/farmacología , Triazoles/químicaRESUMEN
Drug-like inhibitors are often designed by mimicking cofactor or substrate interactions with enzymes. However, as active sites are comprised of conserved residues, it is difficult to identify the critical interactions needed to design selective inhibitors. We are developing an approach, named RADD (resistance analysis during design), which involves engineering point mutations in the target to generate active alleles and testing compounds against them. Mutations that alter compound potency identify residues that make key interactions with the inhibitor and predict target-binding poses. Here, we apply this approach to analyze how diaminotriazole-based inhibitors bind spastin, a microtubule-severing AAA (ATPase associated with diverse cellular activities) protein. The distinct binding poses predicted for two similar inhibitors were confirmed by a series of X-ray structures. Importantly, our approach not only reveals how selective inhibition of the target can be achieved but also identifies resistance-conferring mutations at the early stages of the design process.
Asunto(s)
Ingeniería de Proteínas/métodos , Espastina/efectos de los fármacos , Espastina/genética , Proteínas AAA/genética , Adenosina Trifosfatasas/metabolismo , Amitrol (Herbicida)/química , Fenómenos Bioquímicos , Dominio Catalítico , Cristalografía por Rayos X/métodos , Diseño de Fármacos , Humanos , Microtúbulos/metabolismo , Modelos Moleculares , Mutación Puntual/genética , Espastina/antagonistas & inhibidores , Triazoles/química , Tubulina (Proteína)/químicaRESUMEN
The bump-hole approach is a powerful chemical biology strategy to specifically probe the functions of closely related proteins. However, for many protein families, such as the ATPases associated with diverse cellular activities (AAA), we lack structural data for inhibitor-protein complexes to design allele-specific chemical probes. Here we report the X-ray structure of a pyrazolylaminoquinazoline-based inhibitor bound to spastin, a microtubule-severing AAA protein, and characterize the residues involved in inhibitor binding. We show that an inhibitor analogue with a single-atom hydrogen-to-fluorine modification can selectively target a spastin allele with an engineered cysteine mutation in its active site. We also report an X-ray structure of the fluoro analogue bound to the spastin mutant. Furthermore, analyses of other mutant alleles suggest how the stereoelectronics of the fluorine-cysteine interaction, rather than sterics alone, contribute to the inhibitor-allele selectivity. This approach could be used to design allele-specific probes for studying cellular functions of spastin isoforms. Our data also suggest how tuning stereoelectronics can lead to specific inhibitor-allele pairs for the AAA superfamily.
Asunto(s)
Alelos , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Espastina/antagonistas & inhibidores , Espastina/genética , Animales , Dominio Catalítico , Humanos , Modelos MolecularesRESUMEN
Spastin is a microtubule-severing AAA (ATPases associated with diverse cellular activities) protein needed for cell division and intracellular vesicle transport. Currently, we lack chemical inhibitors to probe spastin function in such dynamic cellular processes. To design a chemical inhibitor of spastin, we tested selected heterocyclic scaffolds against wild-type protein and constructs with engineered mutations in the nucleotide-binding site that do not substantially disrupt ATPase activity. These data, along with computational docking, guided improvements in compound potency and selectivity and led to spastazoline, a pyrazolyl-pyrrolopyrimidine-based cell-permeable probe for spastin. These studies also identified spastazoline-resistance-conferring point mutations in spastin. Spastazoline, along with the matched inhibitor-sensitive and inhibitor-resistant cell lines we generated, were used in parallel experiments to dissect spastin-specific phenotypes in dividing cells. Together, our findings suggest how chemical probes for AAA proteins, along with inhibitor resistance-conferring mutations, can be designed and used to dissect dynamic cellular processes.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Compuestos Heterocíclicos/farmacología , Mutación , Espastina/antagonistas & inhibidores , Espastina/genética , Dominio Catalítico/efectos de los fármacos , Dominio Catalítico/genética , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/química , Modelos Moleculares , Estructura Molecular , Espastina/metabolismoRESUMEN
Cytoplasmic dyneins 1 and 2 are related members of the AAA+ superfamily (ATPases associated with diverse cellular activities) that function as the predominant minus-end-directed microtubule motors in eukaryotic cells. Dynein 1 controls mitotic spindle assembly, organelle movement, axonal transport, and other cytosolic, microtubule-guided processes, whereas dynein 2 mediates retrograde trafficking within motile and primary cilia. Small-molecule inhibitors are important tools for investigating motor protein-dependent mechanisms, and ciliobrevins were recently discovered as the first dynein-specific chemical antagonists. Here, we demonstrate that ciliobrevins directly target the heavy chains of both dynein isoforms and explore the structure-activity landscape of these inhibitors in vitro and in cells. In addition to identifying chemical motifs that are essential for dynein blockade, we have discovered analogs with increased potency and dynein 2 selectivity. These antagonists effectively disrupt Hedgehog signaling, intraflagellar transport, and ciliogenesis, making them useful probes of these and other cytoplasmic dynein 2-dependent cellular processes.
Asunto(s)
Dineínas Citoplasmáticas/antagonistas & inhibidores , Dineínas Citoplasmáticas/química , Animales , Proteínas Hedgehog/fisiología , Ratones , Estructura Molecular , Células 3T3 NIH , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Quinazolinonas/química , Quinazolinonas/farmacología , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
An efficient and simple two-step procedure for the formation of hydroxy-Freidinger lactams is presented. The methodology allows assembly of the cyclic threonine motif (cThr) in solution and on solid support during conventional peptide synthesis.
Asunto(s)
Dipéptidos/síntesis química , Lactamas/síntesis química , Treonina/química , Biomimética , Técnicas Químicas Combinatorias/métodos , Dipéptidos/química , Lactamas/química , Estructura Molecular , Treonina/síntesis químicaRESUMEN
Eradicating hedgehogs: The title molecule has been previously identified as a potent inhibitor of the Hedgehog signaling pathway, which gives embryonic cells information needed to develop properly. This molecule is shown to modulate Hedgehog target gene expression by depolymerizing microtubules, thus revealing dual roles of the cytoskeleton in pathway regulation (see figure).
Asunto(s)
Proteínas Hedgehog/metabolismo , Compuestos Heterocíclicos con 2 Anillos/farmacología , Microtúbulos/metabolismo , Tiazoles/farmacología , Animales , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/antagonistas & inhibidores , Compuestos Heterocíclicos con 2 Anillos/química , Ratones , Microtúbulos/efectos de los fármacos , Células 3T3 NIH , Piridinas/química , Transducción de Señal , Tiazoles/químicaRESUMEN
The number of peptide-based pharmaceuticals has increased enormously over recent years; some of these peptides are derived from natural sources, and many are large and complex. This review provides an overview of the current state of peptide synthesis methodology, focusing mainly on the synthesis of natural peptides and their analogs.