B. Gayani K. Perera

Divergent allosteric control of the IRE1α endoribonuclease using kinase inhibitors

Under endoplasmic reticulum (ER) stress, unfolded proteins accumulate in the ER to activate the ER transmembrane kinase/endoribonuclease (RNase)—IRE1α. IRE1α oligomerizes, autophosphorylates, and initiates splicing of XBP1 mRNA, thus triggering the unfolded protein response (UPR). Here we show that IRE1α’s kinase-controlled RNase can be regulated in two distinct modes with kinase inhibitors: one class of ligands occupy IRE1α’s kinase ATP-binding site to activate RNase-mediated XBP1 mRNA splicing even without upstream ER stress, while a second class can inhibit the RNase through the same ATP-binding site, even under ER stress. Thus, alternative kinase conformations stabilized by distinct classes of ATP-competitive inhibitors can cause allosteric switching of IRE1α’s RNase—either on or off. As dysregulation of the UPR has been implicated in a variety of cell degenerative and neoplastic disorders, small molecule control over IRE1α should advance efforts to understand the UPR’s role in pathophysiology and to develop drugs for ER stress-related diseases.

Development of Toxoplasma gondii Calcium-Dependent Protein Kinase 1 (TgCDPK1) Inhibitors with Potent Anti-Toxoplasma Activity

Toxoplasmosis is a disease of prominent health concern that is caused by the protozoan parasite Toxoplasma gondii. Proliferation of T. gondii is dependent on its ability to invade host cells, which is mediated in part by calcium-dependent protein kinase 1 (CDPK1). We have developed ATP competitive inhibitors of TgCDPK1 that block invasion of parasites into host cells, preventing their proliferation. The presence of a unique glycine gatekeeper residue in TgCDPK1 permits selective inhibition of the parasite enzyme over human kinases. These potent TgCDPK1 inhibitors do not inhibit the growth of human cell lines and represent promising candidates as toxoplasmosis therapeutics.

Multiple Determinants for Selective Inhibition of Apicomplexan Calcium-Dependent Protein Kinase CDPK1.

Diseases caused by the apicomplexan protozoans Toxoplasma gondii and Cryptosporidium parvum are a major health concern. The life cycle of these parasites is regulated by a family of calcium-dependent protein kinases (CDPKs) that have no direct homologues in the human host. Fortuitously, CDPK1 from both parasites contains a rare glycine gatekeeper residue adjacent to the ATP-binding pocket. This has allowed creation of a series of C3-substituted pyrazolopyrimidine compounds that are potent inhibitors selective for CDPK1 over a panel of human kinases. Here we demonstrate that selectivity is further enhanced by modification of the scaffold at the C1 position. The explanation for this unexpected result is provided by crystal structures of the inhibitors bound to CDPK1 and the human kinase c-SRC. Furthermore, the insight gained from these studies was applied to transform an alternative ATP-competitive scaffold lacking potency and selectivity for CDPK1 into a low nanomolar inhibitor of this enzyme with no activity against SRC.

Affinity-Based Probes Based on Type II Kinase Inhibitors

Protein kinases are key components of most mammalian signal transduction networks and are therapeutically relevant drug targets. Efforts to study protein kinase function would benefit from new technologies that are able to profile kinases in complex proteomes. Here, we describe active site-directed probes for profiling kinases in whole cell extracts and live cells. These probes contain general ligands that stabilize a specific inactive conformation of the ATP-binding sites of protein kinases, as well as trifluoromethylphenyl diazirine and alkyne moieties that allow covalent modification and enrichment of kinases, respectively. A diverse group of serine/threonine and tyrosine kinases were identified as specific targets of these probes in whole cell extracts. In addition, a number of kinase targets were selectively labeled in live cells. Our chemical proteomics approach should be valuable for interrogating protein kinase active sites in physiologically relevant environments.

A chemical genetic method for generating bivalent inhibitors of protein kinases.

We report a new chemical genetic method for creating bivalent ligands of protein kinases. The kinase inhibitors that are generated with this methodology consist of two components: (1) a synthetic, small molecule that targets the ATP-binding cleft and (2) a peptidic ligand that enhances selectivity between kinases by targeting a secondary binding domain. A key feature of these bivalent inhibitors is that they are assembled on a protein scaffold with a chemoselective protein labeling technique. The utility of this methodology is demonstrated through the generation of a panel of protein-small molecule conjugates that simultaneously target the SH1 and SH3 domains of the closely related tyrosine kinases Src and Abl. The assembled bivalent ligands are significantly more potent inhibitors of Src and Abl than either modular component alone. Importantly, these protein-small molecule conjugates show a high degree of selectivity for their intended kinase target.

Targeting diverse signaling interaction sites allows the rapid generation of bivalent kinase inhibitors.

The identification of potent and selective modulators of protein kinase function remains a challenge, and new strategies are needed for generating these useful ligands. Here, we describe the generation of bivalent inhibitors of three unrelated protein kinases: the CAMK family kinase Pim1, the mitogen-activated protein kinase (MAPK) p38α, and the receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR). These bivalent inhibitors consist of an ATP-competitive inhibitor that is covalently tethered to an engineered form of the self-labeling protein O(6)-alkylguanine-DNA alkyltransferase (SNAP-tag). In each example, SNAP-tag is fused to a peptide ligand that binds to a signaling interaction site of the kinase being targeted. These interactions increase the overall selectivity and potency of the bivalent inhibitors that were generated. The ability to exploit disparate binding sites in diverse kinases points to the generality of the method described. Finally, we demonstrate that ATP-competitive inhibitors that are conjugated to the bio-orthogonal tag O(4)-benzyl-2-chloro-6-aminopyrimidine (CLP) are cell-permeable. The selective labeling of SNAP-tag with CLP conjugates allows the rapid assembly of bivalent inhibitors in living cells.

Conformation-Selective Inhibitors Reveal Differences in the Activation and Phosphate-Binding Loops of the Tyrosine Kinases Abl and Src

Over the past decade, an increasingly diverse array of potent and selective inhibitors that target the ATP-binding sites of protein kinases have been developed. Many of these inhibitors, like the clinically approved drug imatinib (Gleevec), stabilize a specific catalytically inactive ATP-binding site conformation of their kinases targets. Imatinib is notable in that it is highly selective for its kinase target, Abl, over other closely related tyrosine kinases, such as Src. In addition, imatinib is highly sensitive to the phosphorylation state of Abls activation loop, which is believed to be a general characteristic of all inhibitors that stabilize a similar inactive ATP-binding site conformation. In this report, we perform a systematic analysis of a diverse series of ATP-competitive inhibitors that stabilize a similar inactive ATP-binding site conformation as imatinib with the tyrosine kinases Src and Abl. In contrast to imatinib, many of these inhibitors have very similar potencies against Src and Abl. Furthermore, only a subset of this class of inhibitors is sensitive to the phosphorylation state of the activation loop of these kinases. In attempting to explain this observation, we have uncovered an unexpected correlation between Abls activation loop and another flexible active site feature, called the phosphate-binding loop (p-loop). These studies shed light on how imatinib is able to obtain its high target selectivity and reveal how the conformational preference of flexible active site regions can vary between closely related kinases.

Rapid profiling of protein kinase inhibitors by quantitative proteomics

The ability to determine structure-activity relationships (SAR) and identify cellular targets from cell lysates and tissues is of great utility for kinase inhibitor drug discovery. We describe a streamlined mass spectrometry-based chemoproteomics workflow to examine the SAR and target profiles of a small library of kinase inhibitors that consists of the drug dasatinib and a panel of general type II inhibitors. By combining a simplified affinity enrichment and on-bead protein digestion workflow with quantitative proteomics, we achieved sensitive and specific enrichment of target kinases using our small molecule probes. We applied the affinity matrices in competition experiments with soluble probes in HeLa cell lysates using less than 1 mg of protein per experiment. Each pull-down experiment was analyzed in a single nano LC-MS run. Stringent selection criteria for target identification were applied to deduce 28 protein targets for dasatinib and 31 protein targets for our general type II kinase inhibitor in HeLa cell lysate. Additional kinase and protein targets were identified with the general type II inhibitor analogs, with small structural changes leading to divergent target profiles. We observed surprisingly high sequence coverage on some proteins, enabling further analyses of phosphorylation sites for several target kinases without additional sample processing. Our rapid workflow profiled cellular targets for six small molecules within a week, demonstrating that an unbiased proteomics screen of cellular targets yields valuable SAR information and may be incorporated at an early stage in kinase inhibitor development.

Label Transfer Reagents to Probe p38 MAPK Binding Partners

Protein kinases are essential enzymes for cellular signaling, and are often regulated by participation in protein complexes. The mitogen‐activated protein kinase (MAPK) p38 is involved in multiple pathways, and its regulation depends on its interactions with other signaling proteins. However, the identification of p38‐interacting proteins is challenging. For this reason, we have developed label transfer reagents (LTRs) that allow labeling of p38 signaling complexes. These LTRs leverage the potency and selectivity of known p38 inhibitors to place a photo‐crosslinker and tag in the vicinity of p38 and its binding partners. Upon UV irradiation, proteins that are in close proximity to p38 are covalently crosslinked, and labeled proteins are detected and/or purified with an orthogonal chemical handle. Here we demonstrate that p38‐selective LTRs selectively label a diversity of p38 binding partners, including substrates, activators, and inactivators. Furthermore, these LTRs can be used in immunoprecipitations to provide low‐resolution structural information on p38‐containing complexes.