Parkinson's disease is a relatively common neurological disorder with incidence increasing with age. Present treatments merely alleviate the symptoms and do not alter the course of the disease, thus identification of disease modifying therapies represents a significant unmet medical need. Mutations in the LRRK2 gene are risk-factors for developing PD and it has been hypothesized that the increased kinase activity of certain LRRK2 mutants are responsible for the damage of the dopaminergic neurons, thus LRRK2 inhibitors offer the potential to target an underlying cause of the disease. In this communication, we describe hit-to-lead medicinal chemistry program on a novel series of 5-azaindazoles. Compound 1, obtained from high-throughput screening was optimized to a highly potent, selective series of molecules with promising DMPK properties. Introduction of heterocycles at the 3-position were found to significantly increase the potency and kinase selectivity, whilst changes to the 4-chlorobenzyl group improved the physicochemical properties. Our series was licensed to a major pharmaceutical company for further development.

The efficiency of drug research and development has paradoxically declined over the last decades despite major scientific and technological advances, promoting new cost-effective strategies such as drug repositioning by systematic screening for new actions of known drugs. Here, we performed a screening for positive allosteric modulators (PAMs) at melanocortin (MC) receptors. The non-steroidal anti-inflammatory drug fenoprofen, but not the similar compound ibuprofen, presented PAM activity at MC3, MC4, and MC5 receptors. In a model of inflammatory arthritis, fenoprofen afforded potent inhibition while ibuprofen was nearly inactive. Fenoprofen presented anti-arthritic actions on cartilage integrity and synovitis, effects markedly attenuated in Mc3r-/- mice. Fenoprofen displayed pro-resolving properties promoting macrophage phagocytosis and efferocytosis, independently of cyclooxygenase inhibition. In conclusion, combining repositioning with advances in G-protein coupled receptor biology (allosterism) may lead to potential new therapeutics. In addition, MC3 PAMs emerged as a viable approach to the development of innovative therapeutics for joint diseases.

Imidazopyridazine compounds are potent, ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1) and of Plasmodium falciparum parasite growth in vitro. Here, we show that these compounds can be divided into two classes depending on the nature of the aromatic linker between the core and the R2 substituent group. Class 1 compounds have a pyrimidine linker and inhibit parasite growth at late schizogony, whereas class 2 compounds have a nonpyrimidine linker and inhibit growth in the trophozoite stage, indicating different modes of action for the two classes. The compounds also inhibited cyclic GMP (cGMP)-dependent protein kinase (PKG), and their potency against this enzyme was greatly reduced by substitution of the enzyme's gatekeeper residue at the ATP binding site. The effectiveness of the class 1 compounds against a parasite line expressing the modified PKG was also substantially reduced, suggesting that these compounds kill the parasite primarily through inhibition of PKG rather than CDPK1. HSP90 was identified as a binding partner of class 2 compounds, and a representative compound bound to the ATP binding site in the N-terminal domain of HSP90. Reducing the size of the gatekeeper residue of CDPK1 enabled inhibition of the enzyme by bumped kinase inhibitors; however, a parasite line expressing the modified enzyme showed no change in sensitivity to these compounds. Taken together, these findings suggest that CDPK1 may not be a suitable target for further inhibitor development and that the primary mechanism through which the imidazopyridazines kill parasites is by inhibition of PKG or HSP90.

ABSTRACT PfCDPK1 is a Plasmodium falciparum calcium-dependent protein kinase, which has been identified as a potential target for novel antimalarial chemotherapeutics. In order to further investigate the role of PfCDPK1, we established a high-throughput in vitro biochemical assay and used it to screen a library of over 35,000 small molecules. Five chemical series of inhibitors were initially identified from the screen, from which series 1 and 2 were selected for chemical optimization. Indicative of their mechanism of action, enzyme inhibition by these compounds was found to be sensitive to both the ATP concentration and substitution of the amino acid residue present at the “gatekeeper” position at the ATP-binding site of the enzyme. Medicinal chemistry efforts led to a series of PfCDPK1 inhibitors with 50% inhibitory concentrations (IC 50 s) below 10 nM against PfCDPK1 in a biochemical assay and 50% effective concentrations (EC 50 s) less than 100 nM for inhibition of parasite growth in vitro . Potent inhibition was combined with acceptable absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties and equipotent inhibition of Plasmodium vivax CDPK1. However, we were unable to correlate biochemical inhibition with parasite growth inhibition for this series overall. Inhibition of Plasmodium berghei CDPK1 correlated well with PfCDPK1 inhibition, enabling progression of a set of compounds to in vivo evaluation in the P. berghei rodent model for malaria. These chemical series have potential for further development as inhibitors of CDPK1.

A structure-guided design approach using a homology model of Plasmodium falciparum calcium-dependent protein kinase 1 (Pf CDPK1) was used to improve the potency of a series of imidazopyridazine inhibitors as potential antimalarial agents. This resulted in high affinity compounds with PfCDPK1 enzyme IC50 values less than 10 nM and in vitro P. falciparum antiparasite EC50 values down to 12 nM, although these compounds did not have suitable ADME properties to show in vivo efficacy in a mouse model. Structural modifications designed to address the ADME issues, in particular permeability, were initially accompanied by losses in antiparasite potency, but further optimization allowed a good balance in the compound profile to be achieved. Upon testing in vivo in a murine model of efficacy against malaria, high levels of compound exposure relative to their in vitro activities were achieved, and the modest efficacy that resulted raises questions about the level of effect that is achievable through the targeting of PfCDPK1.

Table 4 appeared incorrectly. The corrected table appears below. Table 4 Selected cyanophenyl pyrazoles

A variety of G-protein–coupled receptor (GPCR) screening technologies have successfully partnered a number of GPCRs with their cognate ligands. GPCR-mediated β-arrestin recruitment is now recognized as a distinct intracellular signaling pathway, and ligand-receptor interactions may show a bias toward β-arrestin over classical GPCR signaling pathways. We hypothesized that the failure to identify native ligands for the remaining orphan GPCRs may be a consequence of biased β-arrestin signaling. To investigate this, we assembled 10 500 candidate ligands and screened 82 GPCRs using PathHunter β-arrestin recruitment technology. High-quality screening assays were validated by the inclusion of liganded receptors and the detection and confirmation of these established ligand-receptor pairings. We describe a candidate endogenous orphan GPCR ligand and a number of novel surrogate ligands. However, for the majority of orphan receptors studied, measurement of β-arrestin recruitment did not lead to the identification of cognate ligands from our screening sets. β-Arrestin recruitment represents a robust GPCR screening technology, and ligand-biased signaling is emerging as a therapeutically exploitable feature of GPCR biology. The identification of cognate ligands for the orphan GPCRs and the extent to which receptors may exist to preferentially signal through β-arrestin in response to their native ligand remain to be determined.

The structural diversity and SAR in a series of imidazopyridazine inhibitors of Plasmodium falciparum calcium dependent protein kinase 1 (PfCDPK1) has been explored and extended. The opportunity to further improve key ADME parameters by means of lowering log D was identified, and this was achieved by replacement of a six-membered (hetero)aromatic linker with a pyrazole. A short SAR study has delivered key examples with useful in vitro activity and ADME profiles, good selectivity against a human kinase panel and improved levels of lipophilic ligand efficiency. These new analogues thus provide a credible additional route to further development of the series.

The orphan G protein coupled receptor GPR35 has emerged as a therapeutic target in a number of disease states including metabolic disorder, cardiovascular disease, inflammation and nociception. Efforts to characterize this receptor, however, have been complicated by marked differences in the pharmacology of GPR35 species orthologs. We have identified novel agonist compounds with which to dissect the pharmacology and function of GPR35. Selected compounds were chosen to illustrate the differences in ligand structure activity relationship between human, mouse and rat orthologs of GPR35. Sequence alignment and receptor homology modeling techniques guided site directed mutagenesis, which highlighted a number of residues involved in GPR35 ligand interaction and revealed differences between rodent and human orthologs. These findings will benefit future drug development programs and efforts to generate therapeutically relevant GPR35‐specific pharmacological compounds. This work was funded by a BBSRC CASE studentship with MRC Technology

Drugs targeting the orphan receptor GPR35 have potential therapeutic application in a number of disease areas, including inflammation, metabolic disorders, nociception, and cardiovascular disease. Currently available surrogate GPR35 agonists identified from pharmacologically relevant compound libraries have limited utility due to the likelihood of off-target effects in vitro and in vivo and the variable potency that such ligands exhibit across species. We sought to identify and characterize novel GPR35 agonists to facilitate studies aimed at defining the physiologic role of GPR35. PathHunter β-arrestin recruitment technology was validated as a human GPR35 screening assay, and a high-throughput screen of 100,000 diverse low molecular weight compounds was conducted. Confirmed GPR35 agonists from five distinct chemotypes were selected for detailed characterization using both β-arrestin recruitment and G protein-dependent assays and each of the human, mouse, and rat GPR35 orthologs. These studies identified 4-{(Z)-[(2Z)-2-(2-fluorobenzylidene)-4-oxo-1,3-thiazolidin-5-ylidene]methyl}benzoic acid (compound 1) as the highest potency full agonist of human GPR35 yet described. As with certain other GPR35 agonists, compound 1 was markedly selective for human GPR35, but displayed elements of signal bias between β-arrestin-2 and G protein-dependent assays. Compound 1 also displayed competitive behavior when assessed against the human GPR35 antagonist, ML-145 (2-hydroxy-4-[4-(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]butanoylamino]benzoic acid). Of the other chemotypes studied, compounds 2 and 3 were selective for the human receptor, but compounds 4 and 5 demonstrated similar activity at human, rat, and mouse GPR35 orthologs. Further characterization of these compounds and related analogs is likely to facilitate a better understanding of GPR35 in health and disease.

Variation in pharmacology and function of ligands at species orthologs can be a confounding feature in understanding the biology and role of poorly characterized receptors. Substantial selectivity in potency of a number of GPR35 agonists has previously been demonstrated between human and rat orthologs of this G protein-coupled receptor. Via a bioluminescence resonance energy transfer-based assay of induced interactions between GPR35 and β-arrestin-2, addition of the mouse ortholog to such studies indicated that, as for the rat ortholog, murine GPR35 displayed very low potency for pamoate, whereas potency for the reference GPR35 agonist zaprinast was intermediate between the rat and human orthologs. This pattern was replicated in receptor internalization and G protein activation assays. The effectiveness and mode of action of two recently reported GPR35 antagonists, methyl-5-[(tert-butylcarbamothioylhydrazinylidene)methyl]-1-(2,4-difluorophenyl)pyrazole-4-carboxylate (CID-2745687) and 2-hydroxy-4-[4-(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]butanoylamino)benzoic acid (ML-145), were investigated. Both CID-2745687 and ML-145 competitively inhibited the effects at human GPR35 of cromolyn disodium and zaprinast, two agonists that share an overlapping binding site. By contrast, although ML-145 also competitively antagonized the effects of pamoate, CID-2745687 acted in a noncompetitive fashion. Neither ML-145 nor CID-2745687 was able to effectively antagonize the agonist effects of either zaprinast or cromolyn disodium at either rodent ortholog of GPR35. These studies demonstrate that marked species selectivity of ligands at GPR35 is not restricted to agonists and considerable care is required to select appropriate ligands to explore the function of GPR35 in nonhuman cells and tissues.

A high-throughput screen against PknB, an essential serine-threonine protein kinase present in Mycobacterium tuberculosis (M. tuberculosis), allowed the identification of an aminoquinazoline inhibitor which was used as a starting point for SAR investigations. Although a significant improvement in enzyme affinity was achieved, the aminoquinazolines showed little or no cellular activity against M. tuberculosis. However, switching to an aminopyrimidine core scaffold and the introduction of a basic amine side chain afforded compounds with nanomolar enzyme binding affinity and micromolar minimum inhibitory concentrations against M. tuberculosis. Replacement of the pyrazole head group with pyridine then allowed equipotent compounds with improved selectivity against a human kinase panel to be obtained.

PknB is an essential serine/threonine kinase of Mycobacterium tuberculosis with possible roles in a number of signalling pathways involved in cell division and metabolism. We screened a library of >50,000 compounds for inhibitors of the in vitro phosphorylation of GarA (Rv1827) by PknB and identified a number of inhibitors. A program of synthetic medicinal chemistry was subsequently conducted around one class of inhibitors and was successful in generating ATP competitive inhibitors with potency in the nanomolar range. Compounds in this class showed cross-reactivity with the related M. tuberculosis kinase, PknF, but not with PknG in an in vitro autophosphorylation assay. These synthesised inhibitors were able to prevent the growth of M. tuberculosis in an Alamar blue assay and in an intracellular model of infection, but only in the micromolar range. We attempted to determine if cell wall permeability was an explanation for the discrepancy between the potent in vitro compared with relatively poor in vivo activity, but found no evidence that the activity of the inhibitors could be improved by weakening the cell wall. Despite a number of drug discovery efforts attempting to develop inhibitors against PknB, it is yet to be reported that any such inhibitors prevent mycobacterial growth at submicromolar concentrations.

Mycobacterium tuberculosis has an on-going impact on global public health and new therapeutics to treat tuberculosis are urgently required. The emergence of drug resistant tuberculosis poses a serious threat to the control of this pathogen, and the development of drugs that are active against the resistant strains is vital. A medium-throughput assay using the Alamar Blue reagent was set-up to identify novel inhibitors of M. tuberculosis from a library of known drugs, for which there has already been extensive research investigating their suitability and safety as human therapeutics. Of the 1514 compounds screened, 53 were demonstrated to possess inhibitory properties against M. tuberculosis at a concentration of 5microM or below. Of these, 17 were novel inhibitors while 36 were known tuberculosis drugs or had been previously described as possessing anti-tuberculosis activity. Five compounds were selected as those which represent the most promising starting points for new anti-tuberculosis agents. It was demonstrated that all five were active against intracellular M. tuberculosis in a macrophage model of infection. The anti-tuberculosis agents identified in this screen represent promising new scaffolds on which future drug development efforts can be focused.

The synthesis, anti-HIV-l and anti-herpesvirus activities of new rigid acyclonucleotide analogs are described. 9-[2-methylidene-3-(phosphonomethoxy)propyl]guanine 1a exhibits in vitro anti-HIV-1 activity similar to that of the antiviral agent 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA). Compound la is 9-fold less toxic to human T-lymphoid cells MT-4 than PMEA.