Biology subjects

Robers, M. B.

Publications and source records attributed to Robers, M. B..

7 recordsLinked to original sources

bioRxiv2025

Cellular Selectivity Analyses Reveal Distinguishing Profiles for Type II Kinase Inhibitors

A pivotal part of kinase chemical probe and drug development is assessment of the selectivity of a putative lead compound. While there is no consensus around the panel size or the type of assay(s) that are most appropriate, there is concurrence that gauging the number of on- and off-targets of a kinase inhibitor is essential. As pharmacology takes place in cells, we have compared profiling results for ten kinase inhibitors generated using the cell-free assays to those obtained when a panel of cellular target engagement NanoBRET assays is used to assess selectivity in intact cells. This is the first systematic comparison of these two approaches across a broad kinase panel. Comparison of the data sets demonstrates divergent results that can influence chemical probe prioritization. We identify unanticipated kinase interactions in cells for type II kinase inhibitors that are not observed in biochemical, cell-free systems. Furthermore, we characterize TPKI-39 as a DDR1, DDR2, and FLT1 chemical probe based on its in-cell selectivity profile. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=149 HEIGHT=200 SRC="FIGDIR/small/681452v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@15abd7borg.highwire.dtl.DTLVardef@4dfbdcorg.highwire.dtl.DTLVardef@1d25bf6org.highwire.dtl.DTLVardef@1939228_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Development of SYK NanoBRET Cellular Target Engagement Assays for Gain-of-Function Variants

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that is activated by phosphorylation events downstream of FcR, B-cell and T-cell receptors, integrins, and C-type lectin receptors. When the tandem Src homology 2 (SH2) domains of SYK bind to phosphorylated immunoreceptor tyrosine-based activation motifs (pITAMs) contained within these immunoreceptors, or when SYK is phosphorylated in interdomain regions A and B, SYK is activated. SYK gain-of-function (GoF) variants were previously identified in six patients that had higher levels of phosphorylated SYK and phosphorylated downstream proteins JNK and ERK. Furthermore, the increased SYK activation resulted in the clinical manifestation of immune dysregulation, organ inflammation, and a predisposition for lymphoma. The knowledge that the SYK GoF variants have enhanced activity was leveraged to develop a SYK NanoBRET cellular target engagement assay in intact live cells with constructs for the SYK GoF variants. Herein, we developed a potent SYK-targeted NanoBRET tracer using a SYK donated chemical probe, MRL-SYKi, that enabled a NanoBRET cellular target engagement assay for SYK GoF variants, SYK(S550Y), SYK(S550F), and SYK(P342T). We determined that ATP-competitive SYK inhibitors bind potently to these SYK variants in intact live cells. Additionally, we demonstrated that MRL-SYKi can effectively reduce the catalytic activity of SYK variants, and the phosphorylation levels of SYK(S550Y) in an epithelial cell line (SW480) stably expressing SYK(S550Y).

cell biology↗

bioRxiv2024

Functional characterization of pathway inhibitors for the ubiquitin-proteasome system (UPS) as tool compounds for CRBN and VHL-mediated targeted protein degradation

Small molecule degraders such as PROteolysis TArgeting Chimeras (PROTACs) or molecular glues are new modalities for drug development and important tools for target validation. Both modalities recruit an E3 ubiquitin ligase to a protein of interest (POI) either via two independent, but linked ligands (PROTACs) or through binding of a small molecule that alters the E3 binding surface to recruit a neo-substrate (molecular glues). If optimized appropriately, both modalities result in the degradation of the POI. Due to the complexity of the induced multistep degradation process, controls for degrader evaluation are critical and they are commonly used in the literature. However, comparative studies and evaluation of cellular potencies of these control compounds and their appropriate uses have not been published so far. Additionally, the high diversity of mechanisms requires diverse small molecule controls to ensure appropriate inhibition of the investigated system while keeping potential cellular toxicity and unintended effects on cellular pathways as low as possible. Here, we scrutinized a diverse set of ubiquitin pathway inhibitors and evaluated their potency and utility within the CRBN and VHL mediated POI degradation pathway. We used the HiBiT system to measure the levels of target rescue after treatment with control compounds. Additionally, cell health was investigated using a multiplex high content assay. This assay panel allows us to determine non-toxic effective concentrations for control experiments and to perform rescue experiments in the absence of cellular toxicity, which has a profound effect on target degradation by ubiquitin-dependent and -independent pathways.

biochemistry↗

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↗

bioRxiv2021

Mutation in Abl kinase with altered drug binding kinetics indicates a novel mechanism of imatinib resistance

Protein kinase inhibitors are potent anti-cancer therapeutics (1). For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for Chronic Myeloid Leukemia (CML) by 80% (2, 3), but 22-41% of patients acquire resistance to imatinib (4). About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain (5), in which more than a hundred different mutations have been identified (6-8). Some mutations are located near the imatinib binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug binding site (9) and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies (10). Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease-relevant or resistance-causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than two-fold compared to Abl wild type. Surprisingly, one-third of mutations in Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified a clinical Abl mutation that binds imatinib with wild type-like affinity but dissociates from imatinib three times faster. Given the relevance of residence time for drug efficacy (11-14), mutations that alter binding kinetics could cause resistance in the non-equilibrium environment of the body where drug export and clearance play critical roles. SignificanceWe performed the first in cell screen of imatinib binding against a library of Abl kinase mutants derived from patients with imatinib-resistant CML. The majority of mutations readily bind imatinib, posing the question of how these mutations cause resistance in patients. We identified a kinetic mutant that binds imatinib with wild type affinity but dissociates considerably faster from the mutant kinase. Using NMR and molecular dynamics, we found that this mutation increases the conformational dynamics of the mutant protein, linking conformational dynamics of the protein to drug dissociation. The results underline the importance of drug dissociation kinetics for drug efficacy and propose a novel kinetic resistance mechanism that may be targetable by altering drug treatment schedules.

biochemistry↗