Leucine-rich repeat kinase 2 (LRRK2), a product of a causative gene for the autosomal-dominant form of familial Parkinson's disease (PARK8), harbors a Ras-like small GTP binding protein-like (ROC) domain besides the kinase domain, although the relationship between these two functional domains remains elusive. Here we show by thin-layer chromatographic analysis that LRRK2 stably binds GTP but lacks a GTPase activity in HEK293 and Neuro-2a cells. A ROC domain mutation that converts LRRK2 to a guanine nucleotide-free form (T1348N) abolishes the kinase activity of LRRK2 as well as its phosphate incorporation upon metabolic labeling. The phosphorylation of LRRK2 was inhibited by potential inhibitors for cyclic AMP-dependent protein kinase. These data suggest that binding of GTP to the ROC domain regulates the kinase activity of LRRK2 as well as its phosphorylation by other kinase(s).

We report the discovery of 7-oxo-2,4,5,7-tetrahydro-6 H-pyrazolo[3,4- c]pyridine derivatives as a novel class of receptor interacting protein 1 (RIP1) kinase inhibitors. On the basis of the overlay study between HTS hit 10 and GSK2982772 (6) in RIP1 kinase, we designed and synthesized a novel class of RIP1 kinase inhibitor 11 possessing moderate RIP1 kinase inhibitory activity and P-gp mediated efflux. The optimization of the core structure and the exploration of appropriate substituents utilizing SBDD approach led to the discovery of 22, a highly potent, orally available, and brain-penetrating RIP1 kinase inhibitor with excellent PK profiles. Compound 22 significantly suppressed necroptotic cell death both in mouse and human cells. Oral administration of 22 (10 mg/kg, bid) attenuated disease progression in the mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). Moreover, analysis of structure-kinetic relationship (SKR) for our novel chemical series was also discussed.

The small GTPase Ras family regulates a variety of cell functions including proliferation and differentiation. Here we have identified novel Ras members, human Di-Ras1 and Di-Ras2, belonging to a distinct branch of the GTPase family. Di-Ras1 and Di-Ras2 specifically expressed in heart and brain share 30 -40% overall identity with other members of Ras family, however, they have the following characteristic substitutions at highly conserved regions among the Ras family. 1) Thr-63 and Ser-65 in Di-Ras are substituted for Ala-59 and Gln-61 positions in Ha-Ras, respectively, that are known to be critical for GTP hydrolysis. 2) Within the effector domains, Di-Ras has Ile at a position corresponding to Asp-33 in Ha-Ras, which is important for its interaction with the downstream effector Raf. As predicted by these substitutions, Di-Ras has only a quite low level of GTPase activity and exists predominantly as a GTPbound form upon its expression in living cells. Moreover, Di-Ras fails to interact with the Ras-binding domain of Raf, resulting in no stimulation of mitogenactivated protein kinase. Interestingly, introduction of Di-Ras into HEK293T cells induces large cellular vacuolation. These findings raise the possibility that Di-Ras might regulate cell morphogenesis in a manner distinct from other members of Ras family.The small GTPase Ras family acts as a molecular switch that regulates a wide range of cell functions including proliferation and differentiation (1-3). Ras is activated in response to a variety of extracellular signals, resulting in stimulation of tyrosine kinases either directly or indirectly. The small GTPase, which cycles between active GTP-bound and inactive GDPbound states, is activated by guanine nucleotide exchange factors that enhance the exchange of bound GDP for GTP and is deactivated by GTPase-activating proteins that increase the intrinsic rate of hydrolysis of bound GTP. The GTP-bound form of Ras associates with several effector molecules, most notably members of the Raf family, the RalGDS family and phosphoinositide 3-kinase. Amino acid residues 32-40 of Ras are important for its interaction with effector molecules and designated as the "effector domain" (4). The biological consequences of their interactions depend greatly on the cell type and on the context of other signaling events.The members of Ras family, at least 13 at present, are characterized by extensive similarities in their effector domains (5). Besides three Ras proteins (Ha-Ras, Ki-Ras, and N-Ras), there are four Rap proteins (Rap1A, Rap1B, Rap2A, and Rap2B), two Ral proteins (RalA and RalB), R-Ras, TC21, R-Ras3/M-Ras, and Rheb in the Ras family. Although many Ras members can interact with the same effector molecules as the three Ras proteins, the physiological roles of most Ras-like GTPases are not fully understood.More recently, some members of the Ras family have begun to be analyzed. For example, Rap1, the closest relative of Ras, has attracted much attention because of the possibility that it functions independently or coor...

The small GTPase superfamily, which includes the Ras, Rho/Rac, Rab, Arf and Ran subfamilies, serves as a signal transducer to regulate cell proliferation and differentiation, actin cytoskeleton, membrane trafficking, and nuclear transport. Here, we identify novel GTPases (human Gie1 and Gie2) that form a distinct subfamily of the small GTPases in terms of their sequences and intracellular function. Gie stands for `novel GTPase indispensable for equal segregation of chromosomes', and this subfamily is conserved in multicellular organisms. Expression of dominant-negative Gie mutants in mammalian cells or knockdown of Gie transcripts using RNA interference in Drosophila S2 cells induced abnormal morphology in the chromosome segregation. Gie protein has ability to bind to tubulin and localizes with microtubules on the spindle mid-zone in late mitosis. Furthermore, overexpression of Gie mutants that lack putative effector domains but have tubulin-binding ability induced micronucleus formation. Thus, this is the first report showing that a small GTPase subfamily capable of associating with microtubules might be involved in chromosome segregation.

The proof of target engagement (TE) is a key element for evaluating potential investment in drug development. The cellular thermal shift assay (CETSA) is expected to facilitate direct measurement of intracellular TE at all stages of drug development. However, there have been no reports of applying this technology to comprehensive animal and clinical studies. This report demonstrates that CETSA can not only quantitatively evaluate the drug-TE in mouse peripheral blood, but also confirm TE in animal tissues exemplified by using the receptor interacting protein 1 kinase (RIPK1) lead compound we have developed. Our established semi-automated system allows evaluation of the structure-activity relationship using native RIPK1 in culture cell lines, and also enables estimation of drug occupancy ratio in mouse peripheral blood mononuclear cells. Moreover, optimized tissue homogenisation enables monitoring of the in vivo drug-TE in spleen and brain. Our results indicate that CETSA methodology will provide an efficient tool for preclinical and clinical drug development.