Identification of small molecules that disrupt signaling between ABL and its positive regulator RIN1

Area of Science:

• Oncology
• Biochemistry
• Pharmacology

Background:

• Constitutively active BCR-ABL kinase fusions drive hematopoietic neoplasias like chronic myelogenous leukemia (CML).
• Existing kinase inhibitors face challenges with drug resistance and relapse, necessitating novel therapeutic strategies.
• Targeting positive regulatory interactions offers an alternative to direct catalytic inhibition for BCR-ABL1.

Purpose of the Study:

• To develop and implement a high-throughput screening (HTS) assay to identify compounds disrupting the BCR-ABL1 kinase stimulator RIN1 interaction.
• To discover novel small molecules that inhibit RIN1-dependent ABL signaling pathways.
• To identify compounds that reduce cellular BCR-ABL1 kinase activity through a novel mechanism of action.

Main Methods:

• Development of a time-resolved Förster resonance energy transfer (TR-FRET) assay to detect disruption of ABL-RIN1 binding.
• High-throughput screening of two small molecule libraries comprising 444,743 compounds.
• Counter-screening and reanalysis of hits to prioritize compounds with IC50 < 10 μM and validation in K562 cells for decreased MAPK1/3 phosphorylation.

Main Results:

• A TR-FRET assay successfully identified compounds disrupting the ABL kinase-RIN1 interaction.
• 508 confirmed hits were prioritized, with five compounds demonstrating efficacy in reducing MAPK1/3 phosphorylation in K562 cells.
• One thiadiazole and four related acyl piperidine amides were identified as inhibitors of RIN1-dependent ABL signaling.

Conclusions:

• Novel compounds were identified that inhibit BCR-ABL1 kinase activity by blocking the positive regulatory RIN1 interaction, not direct catalytic inhibition.
• These findings offer a new avenue for drug discovery targeting CML and potentially overcoming existing resistance mechanisms.
• The identified compounds represent promising leads for developing next-generation therapies for BCR-ABL1-driven leukemias.