Biology subjects

Vasta, J. D.

Publications and source records attributed to Vasta, J. D..

3 recordsLinked to original sources

bioRxiv2023

Development of Cell Permeable NanoBRET Probes for the Measurement of PLK1 Target Engagement in Live Cells

PLK1 is a protein kinase that regulates mitosis and is both an important oncology drug target and a potential anti-target of drugs for the DNA damage response pathway or anti-infective host kinases. To expand the range of live cell NanoBRET target engagement assays to include PLK1 we developed an energy transfer probe based on the anilino-tetrahydropteridine chemotype found in several selective PLK inhibitors. Probe 11 was used to configure NanoBRET target engagement assays for PLK1, PLK2, and PLK3 and measure the potency of several known PLK inhibitors. In cell target engagement for PLK1 was in good agreement with the reported cellular potency for inhibition of cell proliferation. Probe 11 enabled investigation of the promiscuity of adavosertib, which had been described as a dual PLK1/WEE1 inhibitor in biochemical assays. Live cell target engagement analysis of adavosertib by NanoBRET demonstrated PLK activity at micromolar concentrations but only selective engagement of WEE1 at clinically relevant doses. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/529946v3_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@914466org.highwire.dtl.DTLVardef@1219b65org.highwire.dtl.DTLVardef@1613ff6org.highwire.dtl.DTLVardef@1ae69a1_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Small Molecule Modulation of the Archetypal UbiB protein COQ8

Small molecule tools have enabled mechanistic investigations and therapeutic targeting of the protein kinase-like (PKL) superfamily. However, such tools are still lacking for many PKL members, including the highly conserved and disease-related UbiB family. Here, we sought to develop and characterize inhibitor and activator molecules for the archetypal UbiB member, COQ8, whose function is essential for coenzyme Q (CoQ) biosynthesis. Guided by crystallography, activity assays, and cellular CoQ measurements, we repurposed the 4-anilinoquinoline scaffold to selectively inhibit human COQ8A in cells. Second, using 1H-13C HMQC NMR and hydrogen-deuterium exchange mass spectrometry, we reveal that the CoQ precursor mimetic, 2-propylphenol (2-PP), modulates the quintessential UbiB KxGQ domain to increase COQ8A nucleotide affinity and ATPase activity. Our newfound chemical tools promise to lend new mechanistic insights into the activities of these widespread and understudied proteins and to offer potential therapeutic strategies for human diseases connected to their dysfunction.

biochemistry↗

bioRxiv2021

KRAS is vulnerable to reversible switch-II pocket engagement in cells

Current small molecule inhibitors of KRAS(G12C) bind irreversibly in the switch-II pocket, exploiting the strong nucleophilicity of the acquired cysteine as well as the preponderance of the GDP-bound form of this mutant. Nevertheless, many oncogenic KRAS mutants lack these two features, and it remains unknown whether targeting the switch-II pocket is a practical therapeutic approach for KRAS mutants beyond G12C. Here we use NMR spectroscopy and a novel cellular KRAS engagement assay to address this question by examining a collection of SII-P ligands from the literature and from our own laboratory. We show that the switch-II pockets of many GTP hydrolysis-deficient KRAS hotspot (G12, G13, Q61) mutants are accessible using non-covalent ligands, and that this accessibility is not necessarily coupled to the GDP state of KRAS. The results we describe here emphasize the switch-II pocket as a privileged drug binding site on KRAS and unveil new therapeutic opportunities in RAS-driven cancer.

cancer biology↗