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Inhibitors of leucine-rich repeat kinase 2 (LRRK2) and mutants, such as G2019S, have potential utility in Parkinson's disease treatment. Fragment hit-derived pyrrolo[2,3-d]pyrimidines underwent optimization using X-ray structures of LRRK2 kinase domain surrogates, based on checkpoint kinase 1 (CHK1) and a CHK1 10-point mutant. (2R)-2-Methylpyrrolidin-1-yl derivative 18 (LRRK2 G2019S cKi 0.7 nM, LE 0.66) was identified, with increased potency consistent with an X-ray structure of 18/CHK1 10-pt. mutant showing the 2-methyl substituent proximal to Ala147 (Ala2016 in LRRK2). Further structure-guided elaboration of 18 gave the 2-[(1,3-dimethyl-1H-pyrazol-4-yl)amino] derivative 32. Optimization of 32 afforded diastereomeric oxolan-3-yl derivatives 44 and 45, which demonstrated a favorable in vitro PK profile, although they displayed species disconnects in the in vivo PK profile, and a propensity for P-gp- and/or BCRP-mediated efflux in a mouse model. Compounds 44 and 45 demonstrated high potency and exquisite selectivity for LRRK2 and utility as chemical probes for the study of LRRK2 inhibition.

Inhibitors of leucine-rich repeat kinase 2 (LRRK2) and mutants, such as G2019S, have potential utility in Parkinson's disease treatment. Fragment hit-derived pyrrolo[2,3

Mutations in leucine-rich repeat kinase 2 (LRRK2), such as G2019S, are associated with an increased risk of developing Parkinson’s disease. Surrogates for the LRRK2 kinase domain based on checkpoint kinase 1 (CHK1) mutants were designed, expressed in insect cells infected with baculovirus, purified, and crystallized. X-ray structures of the surrogates complexed with known LRRK2 inhibitors rationalized compound potency and selectivity. The CHK1 10-point mutant was preferred, following assessment of surrogate binding affinity with LRRK2 inhibitors. Fragment hit-derived arylpyrrolo[2,3-b]pyridine LRRK2 inhibitors underwent structure-guided optimization using this crystallographic surrogate. LRRK2-pSer935 HEK293 IC50 data for 22 were consistent with binding to Ala2016 in LRRK2 (equivalent to Ala147 in CHK1 10-point mutant structure). Compound 22 was shown to be potent, moderately selective, orally available, and brain-penetrant in wild-type mice, and confirmation of target engagement was demonstrated, with LR...

Genetic variation in the leucine-rich repeat kinase 2 (LRRK2) gene is associated with risk of familial and sporadic Parkinson’s disease (PD). To support clinical development of LRRK2 inhibitors as disease-modifying treatment in PD biomarkers for kinase activity, target engagement and kinase inhibition are prerequisite tools. In a combined proteomics and phosphoproteomics study on human peripheral mononuclear blood cells (PBMCs) treated with the LRRK2 inhibitor Lu AF58786 a number of putative biomarkers were identified. Among the phospho-site hits were known LRRK2 sites as well as two phospho-sites on human Rab10 and Rab12. LRRK2 dependent phosphorylation of human Rab10 and human Rab12 at positions Thr73 and Ser106, respectively, was confirmed in HEK293 and, more importantly, Rab10-pThr73 inhibition was validated in immune stimulated human PBMCs using two distinct LRRK2 inhibitors. In addition, in non-stimulated human PBMCs acute inhibition of LRRK2 with two distinct LRRK2 inhibitor compounds reduced Rab10-Thr73 phosphorylation in a concentration-dependent manner with apparent IC50’s equivalent to IC50’s on LRRK2-pSer935. The identification of Rab10 phosphorylated at Thr73 as a LRRK2 inhibition marker in human PBMCs strongly support inclusion of assays quantifying Rab10-pThr73 levels in upcoming clinical trials evaluating LRRK2 kinase inhibition as a disease-modifying treatment principle in PD.

We describe our work to establish structure- and fragment-based drug discovery to identify small molecules that inhibit the anti-apoptotic activity of the proteins Mcl-1 and Bcl-2. This identified hit series of compounds, some of which were subsequently optimized to clinical candidates in trials for treating various cancers. Many protein constructs were designed to identify protein with suitable properties for different biophysical assays and structural methods. Fragment screening using ligand-observed NMR experiments identified several series of compounds for each protein. The series were assessed for their potential for subsequent optimization using 1H and 15N heteronuclear single-quantum correlation NMR, surface plasmon resonance, and isothermal titration calorimetry measurements to characterize and validate binding. Crystal structures could not be determined for the early hits, so NMR methods were developed to provide models of compound binding to guide compound optimization. For Mcl-1, a benzodioxane/benzoxazine series was optimized to a Kd of 40 μM before a thienopyrimidine hit series was identified which subsequently led to the lead series from which the clinical candidate S 64315 (MIK 665) was identified. For Bcl-2, the fragment-derived series were difficult to progress, and a compound derived from a published tetrahydroquinone compound was taken forward as the hit from which the clinical candidate (S 55746) was obtained. For both the proteins, the work to establish a portfolio of assays gave confidence for identification of compounds suitable for optimization.

Leucine-rich repeat kinase 2 (LRRK2) has attracted considerable interest as a therapeutic target for the treatment of Parkinson's disease. Compounds derived from a 2-aminopyridine screening hit were optimised using a LRRK2 homology model based on mixed lineage kinase 1 (MLK1), such that a 2-aminopyridine-based lead molecule 45, with in vivo activity, was identified.

Linkage and genome-wide association studies have identified a genetic risk locus for late-onset Parkinson's disease in chromosome 12, originally identified as PARK6. The causative gene was identified to code for a large multifunctional protein, LRRK2 (leucine-rich repeat kinase 2). The combined genetic and biochemical evidence supports a hypothesis in which the LRRK2 kinase function is causally involved in the pathogenesis of sporadic and familial forms of PD, and therefore that LRRK2 kinase inhibitors could be useful for treatment. Although LRRK2 has so far not been crystallised, the use of homology modelling and crystallographic surrogates has allowed the optimisation of chemical structures such that compounds of high selectivity with good brain penetration and appropriate pharmacokinetic properties are now available for understanding the biology of LRRK2 in vitro and in vivo. This chapter reviews LRRK2 biology, the structural biology of LRRK2 and gives an overview of inhibitors of LRRK2.

The anti-apoptotic function of the 70 kDa family of heat shock proteins and their role in cancer is well documented. Dual targeting of Hsc70 and Hsp70 with siRNA induces proteasome-dependent degradation of Hsp90 client proteins and extensive tumor specific apoptosis as well as the potentiation of tumor cell apoptosis following pharmacological Hsp90 inhibition.We have previously described the discovery and synthesis of novel adenosine-derived inhibitors of the 70 kDa family of heat shock proteins; the first inhibitors described to target the ATPase binding domain. The in vitro activity of VER-155008 was evaluated in HCT116, HT29, BT474 and MDA-MB-468 carcinoma cell lines. Cell proliferation, cell apoptosis and caspase 3/7 activity was determined for VER-155008 in the absence or presence of small molecule Hsp90 inhibitors.VER-155008 inhibited the proliferation of human breast and colon cancer cell lines with GI(50)s in the range 5.3-14.4 microM, and induced Hsp90 client protein degradation in both HCT116 and BT474 cells. As a single agent, VER-155008 induced caspase-3/7 dependent apoptosis in BT474 cells and non-caspase dependent cell death in HCT116 cells. VER-155008 potentiated the apoptotic potential of a small molecule Hsp90 inhibitor in HCT116 but not HT29 or MDA-MB-468 cells. In vivo, VER-155008 demonstrated rapid metabolism and clearance, along with tumor levels below the predicted pharmacologically active level.These data suggest that small molecule inhibitors of Hsc70/Hsp70 phenotypically mimic the cellular mode of action of a small molecule Hsp90 inhibitor and can potentiate the apoptotic potential of a small molecule Hsp90 inhibitor in certain cell lines. The factors determining whether or not cells apoptose in response to Hsp90 inhibition or the combination of Hsp90 plus Hsc70/Hsp70 inhibition remain to be determined.

A series of pyrazolo[3,4-d]pyrimidine, pyrrolo[2,3-d]pyrimidine and 6-arylpurine adenosine A(2A) antagonists is described. Many examples were highly selective against the human A(1) receptor sub-type and were active in an in vivo model of Parkinson's disease.

The (-)-(11R,2'S)-enantiomer of the antimalarial drug mefloquine has been found to be a reasonably potent and moderately selective adenosine A(2A) receptor antagonist. Further investigation of this compound has led to the discovery of a series of keto-aryl thieno[3,2-d]pyrimidine derivatives, which are potent and selective antagonists of the adenosine A(2A) receptor. These derivatives show selectivity against the A(1) receptor. Furthermore, some of these compounds have been shown to have in vivo activity in a commonly used model, suggesting the potential for the treatment of Parkinson's disease.

Virtual screening against a pCDK2/cyclin A crystal structure led to the identification of a potent and novel CDK2 inhibitor, which exhibited an unusual mode of interaction with the kinase binding motif. With the aid of X-ray crystallography and modelling, a medicinal chemistry strategy was implemented to probe the interactions seen in the crystal structure and to establish SAR. A fragment-based approach was also considered but a different, more conventional, binding mode was observed. Compound selectivity against GSK-3beta was improved using a rational design strategy, with crystallographic verification of the CDK2 binding mode.

Crystallographic and modelling data, in conjunction with a medicinal chemistry template-hopping approach, led to the identification of a series of novel and potent inhibitors of human cyclin-dependent kinase 2 (CDK2), with selectivity over glycogen synthase kinase-3beta (GSK-3beta). One example had a CDK2 IC(50) of 120 nM and showed selectivity over GSK-3beta of 167-fold.

The potent neuroexcitatory activity of kainoid amino acids in the mammalian CNS places new analogues in high demand as tools for neuropharmacological research. A range of 4-arylsulfanyl-substituted kainoids has been synthesised in a parallel fashion via mesylate displacement by a number of aromatic and heteroaromatic thiolates upon (2S,3S,4R)-1-benzoyl-3-tert-butoxycarbonylmethyl-4-methanesulfonyloxy pyrrolidine-2-carboxylic acid methyl ester 8, which is obtainable in eight steps from trans-4-hydroxy- l -proline 5.

78 kDa glucose-regulated protein (Grp78) is a heat shock protein (HSP) involved in protein folding that plays a role in cancer cell proliferation. Binding of adenosine-derived inhibitors to Grp78 was characterized by surface plasmon resonance and isothermal titration calorimetry. The most potent compounds were 13 (VER-155008) with K(D) = 80 nM and 14 with K(D) = 60 nM. X-ray crystal structures of Grp78 bound to ATP, ADPnP, and adenosine derivative 10 revealed differences in the binding site between Grp78 and homologous proteins.

Anabolic steroid abuse is now widespread both in sport and outside the competitive arena. Psychiatrists may be asked to give an opinion in cases of violent crime involving anabolic steroid abusers. Literature suggesting that anabolic steroids can cause aggressive behaviour, or precipitate mental disorder, is reviewed. The possibility that aggression or mental disorder supposedly due to anabolic steroid abuse might provide a defence in cases of violent crime is discussed.

Antagonists of the human A2A receptor have been reported to have potential therapeutic benefit in the alleviation of the symptoms associated with neurodegenerative movement disorders such as Parkinson’s disease. As part of our efforts to discover potent and selective antagonists of this receptor, we herein describe the detailed optimization and structure–activity relationships of a series of pyrimidine-4-carboxamides. These optimized derivatives display desirable physiochemical and pharmacokinetic profiles, which have led to promising oral activity in clinically relevant models of Parkinson’s disease.

The design and synthesis of novel adenosine-derived inhibitors of HSP70, guided by modeling and X-ray crystallographic structures of these compounds in complex with HSC70/BAG-1, is described. Examples exhibited submicromolar affinity for HSP70, were highly selective over HSP90, and some displayed potency against HCT116 cells. Exposure of compound 12 to HCT116 cells caused significant reduction in cellular levels of Raf-1 and Her2 at concentrations similar to that which caused cell growth arrest.

Transient treatment with small molecule CDK inhibitors is toxic to cancer cells and leads to depletion of anti-apoptotic proteins and Chk1, coupled with DNA damage and induction of apoptosis. Here we have examined, which of these phenomena are necessary for CDK inhibitors to have an anti-proliferative effect. We find that 24 hours treatment with either a primarily CDK2-specific, or a primarily CDK7/9-specific, antagonist eliminates proliferative potential even if apoptosis is blocked and the tendency of CDK inhibition to result in DNA damage is overcome by expression of recombinant Chk1. Loss of proliferative potential is correlated with irreversible suppression of biomarkers of cell cycle progression. CDK inhibitors dramatically reduced levels of the anti-apoptotic proteins, Mcl-1 and XIAP, but siRNA-mediated suppression of Mcl-1 and XIAP did not induce cell death in the osteosarcoma cells used in this study. Finally, we found that many literature CDK inhibitors do not effectively suppress the CDK/cyclin complexes responsible for cell cycle progression at the minimum doses required to block proliferation: some are only effective after a substantial delay and may act via inhibition of CDK7.

We describe herein the discovery and development of a series of 4-arylthieno(3,2-d)pyrimidines which are potent adeno- sine A2A receptor antagonists. These novel compounds show high degrees of selectivity against the human A1 ,A 2B and A3 receptor sub-types. Moreover, a number of these compounds show promising activity in vivo, suggesting potential utility in the treatment of Parkinson's disease. 2008 Elsevier Ltd. All rights reserved. Adenosine receptors comprise four distinct sub-types, designated A1 ,A 2A ,A 2B and A3. In the brain, adenosine A2A receptors are located primarily in the striatum, playing a key role in regulating movement. There is strong evidence that adenosine A2A receptor antagonists may provide a novel therapy for the treatment of Par- kinson's disease, with a lower risk of dyskinesias. 1

Database searching led to the identification of potent A 2A antagonists which do not contain the privileged furan moiety and which show selectivity over A 1 receptors. Simple substructure searching on a proprietary database identified compounds with activities in the low nM range. A targeted approach to the identification of non-furan containing compounds resulted in the identification of two novel series, with potency, selectivity and directional SAR from screening 113 compounds.

The protein structure guided design of a series of pyrazolo[1,5-a]pyrimidines with high potency for human cyclin-dependent kinase 2 (CDK2) is described. Some examples were shown to inhibit the growth of human colon tumour cells, were equipotent for CDK1 and were selective against GSK-3β and other kinases.

The role of the 70 kDa heat shock protein isoforms (Hsc70 and Hsp70) in cancer development and progression through their ability to inhibit apoptosis and via their role as Hsp90 co-chaperones has been well documented. Dual targeting of Hsc70 and Hsp70 with siRNA has previously been demonstrated to induce proteasome-dependent degradation of Hsp90 client proteins and extensive tumor specific apoptosis as well as the potentiation of tumor cell apoptosis following pharmacological Hsp90 inhibition. The design and synthesis of novel adenosine-derived inhibitors of Hsp70, guided by modelling and X-ray crystallographic structures of these compounds in complex with Hsc70/BAG-1, has been described.1 These were the first inhibitors described to target the ATPase binding domain of this family of chaperones. Many of these compounds exhibited submicromolar affinity for Hsp70, were highly selective over Hsp90, and displayed in vitro activity against a variety of human tumor cell lines. We further describe the in vitro mode of action of one of the most potent analogues, VER-155008 in HCT116, HT29, BT474 and MDA-MB-468 carcinoma cell lines. Cell proliferation, cell cycle, cell apoptosis and caspase 3/7 activity was determined for VER-155008 in the absence or presence of small molecule Hsp90 inhibitors. VER-155008 inhibited the proliferation of human breast and colon cancer cell lines with GI50s in the range 5.3 to 14.4 M, and induced Hsp90 client protein degradation in both HCT116 and BT474 cells. As a single agent, VER-155008 induced caspase-3/7 dependent apoptosis in BT474 cells and non-caspase dependent cell death in HCT116 and HT29 cells. VER-155008 potentiated the apoptotic potential of the small molecule Hsp90 inhibitors VER-821602 and 17-AAG in HCT116 but not HT29 or MDA-MB-468 cells. In vivo, VER-155008 demonstrated rapid metabolism and clearance, along with tumor levels below the predicted pharmacologically active level. These data suggest that as a single agent, small molecule inhibitors of Hsc70/Hsp70 phenotypically mimic the cellular mode of action of a small molecule Hsp90 inhibitor and can potentiate the apoptotic potential of a small molecule Hsp90 inhibitor in certain cell lines. The factors determining whether or not cells apoptose in response to Hsp90 inhibition or the combination of Hsp90 plus Hsc70/Hsp70 inhibition remain to be determined. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A212.

The 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl auxiliary is effective in directing bromopropargyloxy additions to the olefinic bonds of vinylogous esters/carbonates; in the presence of AIBN and 1-ethylpiperidinium hypophosphite, the adducts undergo highly stereoselective reductive radical cyclisations in which quaternary carbon stereogenic centres are generated.