RESUMEN
Serine/threonine protein kinase ULK3 is implicated in a variety of cellular processes, including autophagy, cell division, and execution of the Sonic hedgehog pathway. However, very little about how its biological activity could be controlled is known. This study focuses on unraveling biochemical insights into the mechanism of inhibition and activation of ULK3. We identify novel phosphorylation sites in ULK3 and show that autophosphorylation has no impact on the kinase activity of the protein. We further demonstrate that phosphorylation of two residues in the kinase domain of ULK3 by an as yet unidentified kinase may completely abolishes its catalytic activity. We show that a low-molecular weight inhibitor SU6668, designed as an ATP competitive inhibitor for tyrosine kinases, binds in the ATP pocket of ULK3 yet inhibits ULK3 kinase activity in a partially ATP noncompetitive manner. Finally, we demonstrate that the ULK3 kinase domain, annotated in silico, is not sufficient for its kinase activity, and additional amino acids in the 271-300 region are required.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Indoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Pirroles/farmacología , Secuencia de Aminoácidos , Dominio Catalítico , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Oxindoles , Fosforilación , Propionatos , Conformación Proteica , Proteínas Serina-Treonina Quinasas/genética , Homología de Secuencia , Transducción de SeñalRESUMEN
Treatments against leishmaniasis are limited and the development of new molecules is crucial. One class of developmental drug that has shown activity against the parasite Leishmania are thiophene derivatives. Here we synthetized thirty-eight novel thiophene compounds and characterized their activity and potential for resistance against L. infantum. Half of the molecules had an EC50 in the low micromolar range, the piperidine derivatives being more potent than the tetramethylpyran derivatives. Resistance was challenging to select for, and resistant cells could only be raised against one (GC1-19) of the four most active compounds. Using chemogenomic screens we show that a gene conversion event at the ABCG2 locus as well as the overexpression of a tryparedoxin peroxidase are responsible for a weak but significant resistance to the GC1-19 drug candidate. Together, our results suggest that thiophene is a scaffold of interest for further drug development against leishmaniasis.
Asunto(s)
Antiprotozoarios , Leishmania infantum , Leishmaniasis Visceral , Leishmaniasis , Parásitos , Animales , Leishmania infantum/genética , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Tiofenos/farmacología , Tiofenos/uso terapéutico , Leishmaniasis/tratamiento farmacológico , Leishmaniasis Visceral/tratamiento farmacológicoRESUMEN
Various conjugation techniques are used to affect the intracellular delivery of bioactive small molecules. However, the ability to track changes in the phenotype when applying these tools remains poorly studied. We addressed this issue by having prepared a focused library of heterobivalent constructs based on Rho kinase inhibitor HA-100. By comparing the induction of the phenotype of interest, cell viability and cellular uptake, we demonstrate that various conjugates indeed lead to divergent cellular outcomes.