The BRAF kinase is mutated, typically V600E, to induce an active oncogenic state in a large fraction of melanoma, thyroid, hairy cell leukemia, and to a lesser extent, a wide spectrum of other cancers1,2. BRAFV600E phosphorylates and activates the kinases MEK1 and MEK2, which in turn phosphorylate and activate the kinases ERK1 and ERK2, stimulating the MAPK pathway to promote cancer3. Targeting MEK1/2 is proving to be an important therapeutic strategy, as a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma4, which is increased when co-administered with a BRAFV600E inhibitor5. In this regard, we previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu-MEK1 interaction6. We now show that genetic loss of the high affinity Cu transporter Ctr1 or mutations in MEK1 that disrupt Cu binding reduced BRAFV600E-driven signaling and tumorigenesis. Conversely, a MEK1-MEK5 chimera that phosphorylates ERK1/2 independent of Cu or an active ERK2 restored tumor growth to cells lacking Ctr1. Importantly, Cu chelators used in the treatment of Wilson disease7 reduced tumor growth of both BRAFV600E-transformed cells and cells resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat BRAFV600E mutation-positive cancers.

Background: BTB-Kelch proteins, including KLHL11, are proposed to bind Cul3 through a “3-box” motif to form E3 ubiquitin ligases.Results: We solved crystal structures of the KLHL11-Cul3 complex and four Kelch domains.Conclusion: The 3-box forms a hydrophobic groove that binds a specific N-terminal extension of Cul3.Significance: Dimeric BTB-Kelch proteins bind two Cul3 molecules and support a two-site model for substrate recognition.

Background: Mutations in the ALK2 kinase cause extraskeletal bone formation.Results: We solved the structure of ALK2 in complex with the inhibitor FKBP12.Conclusion: Disease mutations break critical interactions that stabilize the inactive ALK2-FKBP12 complex leading to kinase activation.Significance: We offer an explanation for the effects of mutation and a structural template for the design of small molecule inhibitors.

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Drugs that function through covalent bond formation represent a considerable fraction of our repository of effective medicines but safety concerns and the complexity of developing covalent inhibitors has rendered covalent targeting a less attractive strategy for rational drug design. The recent approval of four covalent kinase inhibitors and the development of highly potent covalent kinase probes with exceptional selectivity has raised significant interest in industry and academic research and validated the concept of covalent kinase targeting for clinical applications. The abundance of cysteines at diverse positions in and around the kinase active site suggests that a large fraction of kinases can be targeted by covalent inhibitors. Herein, we review recent developments of this rapidly growing area in kinase drug development and highlight the unique opportunities and challenges of this strategy.

Growth factor signaling pathways are tightly regulated by phosphorylation and include many important kinase targets of interest for drug discovery. Small molecule inhibitors of the bone morphogenetic protein (BMP) receptor kinase ALK2 (ACVR1) are needed urgently to treat the progressively debilitating musculoskeletal disease fibrodysplasia ossificans progressiva (FOP). Dorsomorphin analogues, first identified in zebrafish, remain the only BMP inhibitor chemotype reported to date. By screening an assay panel of 250 recombinant human kinases we identified a highly selective 2-aminopyridine-based inhibitor K02288 with in vitro activity against ALK2 at low nanomolar concentrations similar to the current lead compound LDN-193189. K02288 specifically inhibited the BMP-induced Smad pathway without affecting TGF-β signaling and induced dorsalization of zebrafish embryos. Comparison of the crystal structures of ALK2 with K02288 and LDN-193189 revealed additional contacts in the K02288 complex affording improved shape complementarity and identified the exposed phenol group for further optimization of pharmacokinetics. The discovery of a new chemical series provides an independent pharmacological tool to investigate BMP signaling and offers multiple opportunities for pre-clinical development.

Activation of the ERK pathway is a hallmark of cancer and targeting of upstream signalling partners led to the development of approved drugs. Recently SCH772984 has been shown to be a selective and potent ERK1/2 inhibitor. Here we report the structural mechanism for its remarkable selectivity. In ERK1/2, SCH772984 induced a so far unknown binding pocket that accommodated the piperazine-phenyl-pyrimidine decoration. This novel binding pocket was created by an inactive conformation of the phosphate binding loop and an outward tilt of helix αC. In contrast, structure determination of SCH772984 with the off-target haspin and JNK1 revealed canonical but two distinct type-I binding modes. Intriguingly, the novel binding mode with ERK1/2 was associated with slow binding kinetics in vitro as well as in cell based assay systems. The described binding mode of SCH772984 with ERK1/2 enables the design of a new type of specific kinase inhibitors with prolonged on-target activity.

Elevated production of the matrix glycosaminoglycan hyaluronan is strongly implicated in epithelial tumor progression. Inhibition of synthesis of the hyaluronan precursor UDP-glucuronic acid (UDP-GlcUA) therefore presents an emerging target for cancer therapy. Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes, in two NAD+-dependent steps without release of intermediate aldehyde, the biosynthetic oxidation of UDP-glucose (UDP-Glc) to UDP-GlcUA. Here, we present a structural characterization of the hUGDH reaction coordinate using crystal structures of the apoenzyme and ternary complexes of the enzyme bound with UDP-Glc/NADH and UDP-GlcUA/NAD+. The quaternary structure of hUGDH is a disc-shaped trimer of homodimers whose subunits consist of two discrete α/β domains with the active site located in the interdomain cleft. Ternary complex formation is accompanied by rigid-body and restrained movement of the N-terminal NAD+ binding domain, sequestering substrate and coenzyme in their reactive positions through interdomain closure. By alternating between conformations in and out of the active site during domain motion, Tyr14, Glu161, and Glu165 participate in control of coenzyme binding and release during 2-fold oxidation. The proposed mechanism of hUGDH involves formation and breakdown of thiohemiacetal and thioester intermediates whereby Cys276 functions as the catalytic nucleophile. Stopped-flow kinetic data capture the essential deprotonation of Cys276 in the course of the first oxidation step, allowing the thiolate side chain to act as a trap of the incipient aldehyde. Because thiohemiacetal intermediate accumulates at steady state under physiological reaction conditions, hUGDH inhibition might best explore ligand binding to the NAD+ binding domain.