Conversion of a Single Polypharmacological Agent into Selective Bivalent Inhibitors of Intracellular Kinase Activity

Loss-of-function studies are valuable for elucidating kinase function and the validation of new drug targets. While genetic techniques, such as RNAi and genetic knockouts, are highly specific and easy to implement, in many cases post-translational perturbation of kinase activity, specifically pharmacological inhibition, is preferable. However, due to the high degree of structural similarity between kinase active sites and the large size of the kinome, identification of pharmacological agents that are sufficiently selective to probe the function of a specific kinase of interest is challenging, and there is currently no systematic method for accomplishing this goal. Here, we present a modular chemical genetic strategy that uses antibody mimetics as highly selective targeting components of bivalent kinase inhibitors. We demonstrate that it is possible to confer high kinase selectivity to a promiscuous ATP-competitive inhibitor by tethering it to an antibody mimetic fused to the self-labeling protein SNAPtag. With this approach, a potent bivalent inhibitor of the tyrosine kinase Abl was generated. Profiling in complex cell lysates, with competition-based quantitative chemical proteomics, revealed that this bivalent inhibitor possesses greatly enhanced selectivity for its target, BCR-Abl, in K562 cells. Importantly, we show that both components of the bivalent inhibitor can be assembled in K562 cells to block the ability of BCR-Abl to phosphorylate a direct cellular substrate. Finally, we demonstrate the generality of using antibody mimetics as components of bivalent inhibitors by generating a reagent that is selective for the activated state of the serine/threonine kinase ERK2

Discovery of Potent Small-Molecule Inhibitors of WDR5-MYC Interaction

WD repeat domain 5 (WDR5) is a member of the WD40-repeat protein family that plays a critical role in multiple processes. It is also a prominent target for pharmacological inhibition in diseases such as cancer, aging, and neurodegenerative disorders. Interactions between WDR5 and various partners are essential for sustaining its function. Most drug discovery efforts center on the WIN (WDR5 interaction motif) site of WDR5 that is responsible for the recruitment of WDR5 to chromatin. Here, we describe the discovery of novel WDR5 inhibitors for the other WBM (WDR5 binding motif) pocket on this scaffold protein, to disrupt WDR5 interaction with its binding partner MYC by high-throughput biochemical screening, subsequent molecule optimization, and biological assessment. These new WDR5 inhibitors provide useful probes for future investigations of WDR5 and an avenue for targeting WDR5 as a therapeutic strategy

Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

<div><p>Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. <i>In vivo</i> experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.</p></div

Englerin A affects proliferation of a subset of cancer cell lines across many cell lineages while englerin B is inactive.

<p>(<b>A</b>) Scatterplot of englerin A cell line profiling experiment. (<b>B</b>) Scatterplot of englerin B cell line profiling experiment. Each point represents effect of englerin A or B on growth of a single tumor cell line. Y-axis indicates maximal effect on growth and X-axis indicates potency. Tumor cell line lineage is indicated by color and the legend is in the figure. Englerin A sensitive (circles), englerin A refractory (squares) and englerin A intermediate (diamonds) cell line calls are indicated.</p

Englerin A selectivity against TRP family and non-TRP family ion channels.

<p>Englerin A selectivity against TRP family and non-TRP family ion channels.</p

TRPC4 expression is necessary and sufficient for cell proliferation effects of englerin A.

<p><b>(A</b>) Effect of TRPC4 siRNA knockdown on viability of A-498 cells in the presence of englerin A. An siRNA targeting luciferase was used as a control (mean+/- S.E.M.) Percent reduction of the TRPC4 mRNA levels are indicated in the legend, KD stands for knockdown. TRPC4 mRNA levels were normalized to peptidyl prolyl isomerase A (PPIA) mRNA levels. (<b>B</b>) Effect of TRPC4 siRNA knockdown on viability of A-673 cells in the presence of englerin A (mean +/- S.E.M.) (<b>C</b>) Effect of overexpression of TRPC4 by transient transfection on viability of HEK293T cells in the presence of englerin A. TRPC4 expression vector concentrations are indicated by different shapes (<b>D</b>) Effect of TRPC4 expression on cell viability in the presence of an englerin A in HEK293T cells engineered to express TRPC4 under control of a Doxycycline (Dox) regulated promoter (mean +/- standard deviation). 100 ng/ml Dox (black circles), 0 ng/ml Dox (open circles). (<b>E</b>) Western blot visualizing the levels of TRPC4 in the presence or absence of 100 ng/ml Dox. (<b>F</b>) Effect of PKCtheta inhibitor compound 27 on response to englerin A in A-498 cells. (<b>G</b>) Effect of PKCtheta inhibitor compound 27 on response to englerin A in A-673 cells.</p

Effects of englerin A and englerin B on mean arterial pressure in anesthetized rats.

<p>(<b>A</b>) Mean arterial blood pressure of anesthetized rats dosed with englerin A (mean +/- S.E.M.). (<b>B</b>) Mean arterial blood pressure of anesthetized rats dosed with englerin B (mean +/- S.E.M.).</p

Overexpression of TRPC5 can also confer sensitivity to englerin A.

<p>(<b>A</b>) Expression of TRPC4 and TRPC5 in the cell lines from the CLiP experiment. Each circle represents a single cell line and expression levels were measured by microarray (TRPC4 probe 220818_s_at, TRPC5 probe 220552_at). The circle representing the DMS-79 cell line is indicated. (<b>B</b>) The effect of englerin A on cell viability in HEK293T cells transiently transfected with a vector expressing TRPC5 (mean +/- standard deviation).</p

Englerin A agonizes the TRPC4/C5 ion channels and channel activation is needed for cell growth inhibition.

<p>(<b>A</b>) Calcium flux stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation): TRPC5 (closed diamonds), TRPC4beta (closed squares), TRPC4 (closed circles), TRPC6 (open squares), mock transfected cells (open circles). (<b>B</b>) Membrane depolarization stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation), markers as above. (<b>C</b>) TRPC4 current evoked by stimulation of 5 μM Englerin A, saline, or 5 μM Englerin A + 10 μM ML204 in 293T cells with Doxycyline-induced TRPC4. Currents were elicited by 200 ms voltage ramps from -100 to +100 mV, applied every 10 s from holding potential of 0 mV. (<b>D</b>) Summary of englerin A, englerin-B and ML-204 activity on membrane currents (mean +/- S.E.M.) (<b>E</b>) A-673 cell viability in the presence or absence of 50 nM englerin A and/or 50 μM ML204, a TRPC4/C5 channel blocker (mean +/- standard deviation).</p

Sensitivity to englerin A induced growth inhibition correlates with expression of TRPC4.

<p>(<b>A</b>) Volcano plot of gene expression features. X- axis indicates fold change of feature in sensitive versus refractory cell lines and Y-axis indicates statistical significance. (<b>B</b>) Waterfall plot showing TRPC4 expression levels (affymetrix microarray units) in englerin A sensitive (red) and refractory (blue) tumor cell lines.</p