Highlights d KI-ARv-03 reduces AR protein levels and AR-driven transcription d KI-ARv-03 is deduced to be a potent, ultraselective inhibitor of CDK9 d Optimization led to the orally bioavailable and selective CDK9 inhibitor KB-0742 d KB-0742 displays potent anti-tumor activity in cancer models in vitro and in vivo

The mammalian visual dim-light photoreceptor rhodopsin is considered a prototype G protein-coupled receptor. Here, we characterize the kinetics of its light-activation process. Milligram quantities of α,ε-(15)N-labeled tryptophan rhodopsin were produced in stably transfected HEK293 cells. Assignment of the chemical shifts of the indole signals was achieved by generating the single-point-tryptophan to phenylalanine mutants, and the kinetics of each of the five tryptophan residues were recorded. We find kinetic partitioning in rhodopsin decay, including three half-lives, that reveal two parallel processes subsequent to rhodopsin activation that are related to the photocycle. The meta II and meta III states emerge in parallel with a relative ratio of about 3:1. Transient formation of the meta III state was confirmed by flash photolysis experiments. From analysis of the site-resolved kinetic data we propose the involvement of the E2 -loop in the formation of the meta III state.

Highlights d Structure of the ULK4 ATP complex reveals a unique ATP binding mode d Disease-associated mutations modulate ATP binding and ULK4 stability d Loss of canonical motifs co-occurred in evolution with a specific activation loop d BioID suggests a role of ULK4 regulating centrosomal and cytoskeletal functions

Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules.Here, we describe the structural and dynamic properties of pseudokinase domains from the Wntbinding receptor tyrosine kinases (PTK7, ROR1, ROR2, RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family, and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity, and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed strikingly similar dynamic properties to IRK.Despite the inaccessible ATP site, screening identified ATP competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.

Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homologue of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson’s mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we were unable to confirm previous data suggesting that Rab7A phosphorylation is regulated by the PINK1/TBK1 axis. Altogether, these finding reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.