Inhibition of striatal dopamine release by the L-type calcium channel inhibitor isradipine co-varies with risk factors for Parkinson's.

Ca2+ entry into nigrostriatal dopamine (DA) neurons and axons via L-type voltage-gated Ca2+ channels (LTCCs) contributes, respectively, to pacemaker activity and DA release and has long been thought to contribute to vulnerability to degeneration in Parkinson's disease. LTCC function is greater in DA axons and neurons from substantia nigra pars compacta than from ventral tegmental area, but this is not explained by channel expression level. We tested the hypothesis that LTCC control of DA release is governed rather by local mechanisms, focussing on candidate biological factors known to operate differently between types of DA neurons and/or be associated with their differing vulnerability to parkinsonism, including biological sex, α-synuclein, DA transporters (DATs) and calbindin-D28k (Calb1). We detected evoked DA release ex vivo in mouse striatal slices using fast-scan cyclic voltammetry and assessed LTCC support of DA release by detecting the inhibition of DA release by the LTCC inhibitors isradipine or CP8. Using genetic knockouts or pharmacological manipulations, we identified that striatal LTCC support of DA release depended on multiple intersecting factors, in a regionally and sexually divergent manner. LTCC function was promoted by factors associated with Parkinsonian risk, including male sex, α-synuclein, DAT and a dorsolateral co-ordinate, but limited by factors associated with protection, that is, female sex, glucocerebrosidase activity, Calb1 and ventromedial co-ordinate. Together, these data show that LTCC function in DA axons and isradipine effect are locally governed and suggest they vary in a manner that in turn might impact on, or reflect, the cellular stress that leads to parkinsonian degeneration.

The BET inhibitor CPI203 promotes ex vivo expansion of cord blood long-term repopulating HSCs and megakaryocytes.

Although cytokine-mediated expansion of human hematopoietic stem cells (HSCs) can result in high yields of hematopoietic progenitor cells, this generally occurs at the expense of reduced bone marrow HSC repopulating ability, thereby limiting potential therapeutic applications. Because bromodomain-containing proteins (BCPs) have been demonstrated to regulate mouse HSC self-renewal and stemness, we screened small molecules targeting various BCPs as potential agents for ex vivo expansion of human HSCs. Of 10 compounds tested, only the bromodomain and extra-terminal motif inhibitor CPI203 enhanced the expansion of human cord blood HSCs without losing cell viability in vitro. The expanded cells also demonstrated improved engraftment and repopulation in serial transplantation assays. Transcriptomic and functional studies showed that the expansion of long-term repopulating HSCs was accompanied by synchronized expansion and maturation of megakaryocytes consistent with CPI203-mediated reprogramming of cord blood hematopoietic stem and progenitor cells. This approach may therefore prove beneficial for ex vivo gene editing, for enhanced platelet production, and for the improved usage of cord blood for transplantation research and therapy.

Structure-based development of new RAS-effector inhibitors from a combination of active and inactive RAS-binding compounds.

The RAS gene family is frequently mutated in human cancers, and the quest for compounds that bind to mutant RAS remains a major goal, as it also does for inhibitors of protein-protein interactions. We have refined crystallization conditions for KRAS169Q61H-yielding crystals suitable for soaking with compounds and exploited this to assess new RAS-binding compounds selected by screening a protein-protein interaction-focused compound library using surface plasmon resonance. Two compounds, referred to as PPIN-1 and PPIN-2, with related structures from 30 initial RAS binders showed binding to a pocket where compounds had been previously developed, including RAS effector protein-protein interaction inhibitors selected using an intracellular antibody fragment (called Abd compounds). Unlike the Abd series of RAS binders, PPIN-1 and PPIN-2 compounds were not competed by the inhibitory anti-RAS intracellular antibody fragment and did not show any RAS-effector inhibition properties. By fusing the common, anchoring part from the two new compounds with the inhibitory substituents of the Abd series, we have created a set of compounds that inhibit RAS-effector interactions with increased potency. These fused compounds add to the growing catalog of RAS protein-protein inhibitors and show that building a chemical series by crossing over two chemical series is a strategy to create RAS-binding small molecules.

Identification and Preliminary Structure-Activity Relationship Studies of 1,5-Dihydrobenzo[e][1,4]oxazepin-2(3H)-ones That Induce Differentiation of Acute Myeloid Leukemia Cells In Vitro.

Acute myeloid leukemia (AML) is the most aggressive type of blood cancer, and there is a continued need for new treatments that are well tolerated and improve long-term survival rates in patients. Induction of differentiation has emerged as a promising alternative to conventional cytotoxic chemotherapy, but known agents lack efficacy in genetically distinct patient populations. Previously, we established a phenotypic screen to identify small molecules that could stimulate differentiation in a range of AML cell lines. Utilising this strategy, a 1,5-dihydrobenzo[e][1,4]oxazepin-2(3H)-one hit compound was identified. Herein, we report the hit validation in vitro, structure-activity relationship (SAR) studies and the pharmacokinetic profiles for selected compounds.

Aminothiazolones as potent, selective and cell active inhibitors of the PIM kinase family.

We have previously reported the discovery of a series of rhodanine-based inhibitors of the PIM family of serine/threonine kinases. Here we described the optimisation of those compounds to improve their physicochemical and ADME properties as well as reducing their off-targets activities against other kinases. Through molecular modeling and systematic structure activity relationship (SAR) studies, advanced molecules with high inhibitory potency, reduced off-target activity and minimal efflux were identified as new pan-PIM inhibitors. One example of an early lead, OX01401, was found to inhibit PIMs with nanomolar potency (15 nM for PIM1), inhibit proliferation of two PIM-expressing leukaemic cancer cell lines, MV4-11 and K562, and to reduce intracellular phosphorylation of a PIM substrate in a concentration dependent manner.

Anthelmintic drug discovery: target identification, screening methods and the role of open science.

Helminths, including cestodes, nematodes and trematodes, are a huge global health burden, infecting hundreds of millions of people. In many cases, existing drugs such as benzimidazoles, diethylcarbamazine, ivermectin and praziquantel are insufficiently efficacious, contraindicated in some populations, or at risk of the development of resistance, thereby impeding progress towards World Health Organization goals to control or eliminate these neglected tropical diseases. However, there has been limited recent progress in developing new drugs for these diseases due to lack of commercial attractiveness, leading to the introduction of novel, more efficient models for drug innovation that attempt to reduce the cost of research and development. Open science aims to achieve this by encouraging collaboration and the sharing of data and resources between organisations. In this review we discuss how open science has been applied to anthelmintic drug discovery. Open resources, including genomic information from many parasites, are enabling the identification of targets for new antiparasitic agents. Phenotypic screening remains important, and there has been much progress in open-source systems for compound screening with parasites, including motility assays but also high content assays with more detailed investigation of helminth physiology. Distributed open science compound screening programs, such as the Medicines for Malaria Venture Pathogen Box, have been successful at facilitating screening in diverse assays against many different parasite pathogens and models. Of the compounds identified so far in these screens, tolfenpyrad, a repurposed insecticide, shows significant promise and there has been much progress in creating more potent and selective derivatives. This work exemplifies how open science approaches can catalyse drug discovery against neglected diseases.

A Genomic DNA Reporter Screen Identifies Squalene Synthase Inhibitors That Act Cooperatively with Statins to Upregulate the Low-Density Lipoprotein Receptor.

Hypercholesterolemia remains one of the leading risk factors for the development of cardiovascular disease. Many large double-blind studies have demonstrated that lowering low-density lipoprotein (LDL) cholesterol using a statin can reduce the risk of having a cardiovascular event by approximately 30%. However, despite the success of statins, some patient populations are unable to lower their LDL cholesterol to meet the targeted lipid levels, due to compliance or potency issues. This is especially true for patients with heterozygous familial hypercholesterolemia who may require additional upregulation of the low-density lipoprotein receptor (LDLR) to reduce LDL cholesterol levels below those achievable with maximal dosing of statins. Here we identify a series of small molecules from a genomic DNA reporter screen that upregulate the LDLR in mouse and human liver cell lines at nanomolar potencies (EC50 = 39 nM). Structure-activity relationship studies carried out on the lead compound, OX03771 [(E)-N,N-dimethyl-3-(4-styrylphenoxy)propan-1-amine], led to the identification of compound OX03050 [(E)-3-(4-styrylphenoxy)propan-1-ol], which had similar potency (EC50 = 26 nM) but a much-improved pharmacokinetic profile and showed in vivo efficacy. Compounds OX03050 and OX03771 were found to inhibit squalene synthase, the first committed step in cholesterol biosynthesis. These squalene synthase inhibitors were shown to act cooperatively with statins to increase LDLR expression in vitro. Overall, we demonstrated here a novel series of small molecules with the potential to be further developed to treat patients either alone or in combination with statins.

Thiazolidine derivatives as potent and selective inhibitors of the PIM kinase family.

The PIM family of serine/threonine kinases have become an attractive target for anti-cancer drug development, particularly for certain hematological malignancies. Here, we describe the discovery of a series of inhibitors of the PIM kinase family using a high throughput screening strategy. Through a combination of molecular modeling and optimization studies, the intrinsic potencies and molecular properties of this series of compounds was significantly improved. An excellent pan-PIM isoform inhibition profile was observed across the series, while optimized examples show good selectivity over other kinases. Two PIM-expressing leukemic cancer cell lines, MV4-11 and K562, were employed to evaluate the in vitro anti-proliferative effects of selected inhibitors. Encouraging activities were observed for many examples, with the best example (44) giving an IC50 of 0.75µM against the K562 cell line. These data provide a promising starting point for further development of this series as a new cancer therapy through PIM kinase inhibition.

Ligand-based virtual screening identifies a family of selective cannabinoid receptor 2 agonists.

The cannabinoid receptor 2 (CB2R) has been linked with the regulation of inflammation, and selective receptor activation has been proposed as a target for the treatment of a range of inflammatory diseases such as atherosclerosis and arthritis. In order to identify selective CB2R agonists with appropriate physicochemical and ADME properties for future evaluation in vivo, we first performed a ligand-based virtual screen. Subsequent medicinal chemistry optimisation studies led to the identification of a new class of selective CB2R agonists. Several examples showed high levels of activity (EC50<200 nM) and binding affinity (Ki<200 nM) for the CB2R, and no detectable activity at the CB1R. The most promising example, DIAS2, also showed favourable in vitro metabolic stability and absorption properties along with a clean selectivity profile when evaluated against a panel of GPCRs and kinases.

Development of Highly Potent, G-Protein Pathway Biased, Selective, and Orally Bioavailable GPR84 Agonists.

Orphan G-protein-coupled receptor 84 (GPR84) is a receptor that has been linked to cancer, inflammatory, and fibrotic diseases. We have reported DL-175 as a biased agonist at GPR84 which showed differential signaling via Gαi/cAMP and ß-arrestin, but which is rapidly metabolized. Herein, we describe an optimization of DL-175 through a systematic structure-activity relationship (SAR) analysis. This reveals that the replacement of the naphthalene group improved metabolic stability and the addition of a 5-hydroxy substituent to the pyridine N-oxide group, yielding compounds 68 (OX04528) and 69 (OX04529), enhanced the potency for cAMP signaling by 3 orders of magnitude to low picomolar values. Neither compound showed detectable effects on ß-arrestin recruitment up to 80 µM. Thus, the new GPR84 agonists 68 and 69 displayed excellent potency, high G-protein signaling bias, and an appropriate in vivo pharmacokinetic profile that will allow investigation of GPR84 biased agonist activity in vivo.

Kinetic insights into agonist-dependent signalling bias at the pro-inflammatory G-protein coupled receptor GPR84.

GPR84 is an orphan G-protein coupled receptor (GPCR) linked to inflammation. Strategies targeting GPR84 to prevent excessive inflammation in disease are hampered by a lack of understanding of its precise functional role. We have developed heterologous cell lines with low GPR84 expression levels that phenocopy the response of primary cells in a label-free cell electrical impedance (CEI) sensing system that measures cell morphology and adhesion. We then investigated the signalling profile and membrane localisation of GPR84 upon treatment with 6-OAU and DL-175, two agonists known to differentially influence immune cell function. When compared to 6-OAU, DL-175 was found to exhibit a delayed impedance response, a delayed and suppressed activation of Akt, which together correlated with an impaired ability to internalise GPR84 from the plasma membrane. The signalling differences were transient and occurred only at early time points in the low expressing cell lines, highlighting the importance of receptor number and kinetic readouts when evaluating signalling bias. Our findings open new ways to understand GPR84 signalling and evaluate the effect of newly developed agonists.

Phenotypic screening identifies a trisubstituted imidazo[1,2-a]pyridine series that induces differentiation in multiple AML cell lines.

Acute myeloid leukaemia (AML) is an aggressive type of leukaemia with low rates of long-term survival. While the current standard of care is based on cytotoxic chemotherapy, a promising emerging approach is differentiation therapy. However, most current differentiating agents target specific mutations and are effective only in certain patient subtypes. To identify agents which may be effective in wider population cohorts, we performed a phenotypic screen with the myeloid marker CD11b and identified a compound series that was able to differentiate AML cell lines in vitro regardless of their mutation status. Structure-activity relationship studies revealed that replacing the formamide and catechol methyl ether groups with sulfonamide and indazole respectively improved the in vitro metabolic profile of the series while maintaining the differentiation profile in multiple cell lines. This optimisation exercise enabled progression of a lead compound to in vivo efficacy testing. Our work supports the promise of phenotypic screening to identify novel small molecules that induce differentiation in a wide range of AML subtypes.

Osmium-free direct syn-dihydroxylation of alkenes.

Numerous synthetic protocols for producing syn-diols from the corresponding alkenes have been developed and published over recent years. It is the intent of the following tutorial review to present a concise summary of the main methods used to prepare syn-diol fragments directly from alkene precursors, and that do not make use of osmium oxo complexes as catalysts.

A cell-based screening method using an intracellular antibody for discovering small molecules targeting the translocation protein LMO2.

Intracellular antibodies are tools that can be used directly for target validation by interfering with properties like protein-protein interactions. An alternative use of intracellular antibodies in drug discovery is developing small-molecule surrogates using antibody-derived (Abd) technology. We previously used this strategy with an in vitro competitive surface plasmon resonance method that relied on high-affinity antibody fragments to obtain RAS-binding compounds. We now describe a novel implementation of the Abd method with a cell-based intracellular antibody-guided screening method that we have applied to the chromosomal translocation protein LMO2. We have identified a chemical series of anti-LMO2 Abd compounds that bind at the same LMO2 location as the inhibitory anti-LMO2 intracellular antibody combining site. Intracellular antibodies could therefore be used in cell-based screens to identify chemical surrogates of their binding sites and potentially be applied to any challenging proteins, such as transcription factors that have been considered undruggable.

Structural Requirements for Dihydrobenzoxazepinone Anthelmintics: Actions against Medically Important and Model Parasites: Trichuris muris, Brugia malayi, Heligmosomoides polygyrus, and Schistosoma mansoni.

Nine hundred million people are infected with the soil-transmitted helminths Ascaris lumbricoides (roundworm), hookworm, and Trichuris trichiura (whipworm). However, low single-dose cure rates of the benzimidazole drugs, the mainstay of preventative chemotherapy for whipworm, together with parasite drug resistance, mean that current approaches may not be able to eliminate morbidity from trichuriasis. We are seeking to develop new anthelmintic drugs specifically with activity against whipworm as a priority and previously identified a hit series of dihydrobenzoxazepinone (DHB) compounds that block motility of ex vivo Trichuris muris. Here, we report a systematic investigation of the structure-activity relationship of the anthelmintic activity of DHB compounds. We synthesized 47 analogues, which allowed us to define features of the molecules essential for anthelmintic action as well as broadening the chemotype by identification of dihydrobenzoquinolinones (DBQs) with anthelmintic activity. We investigated the activity of these compounds against other parasitic nematodes, identifying DHB compounds with activity against Brugia malayi and Heligmosomoides polygyrus. We also demonstrated activity of DHB compounds against the trematode Schistosoma mansoni, a parasite that causes schistosomiasis. These results demonstrate the potential of DHB and DBQ compounds for further development as broad-spectrum anthelmintics.

NMR waterLOGSY as An Assay in Drug Development Programmes for Detecting Protein-Ligand Interactions-NMR waterLOGSY.

In drug development programmes, multiple assays are needed for the determination of protein-compound interactions and evaluation of potential use in assays with protein-protein interactions. In this protocol we describe the waterLOGSY NMR method for confirming protein-ligand binding events.

Immunopolymer Lipid Nanoparticles for Delivery of Macromolecules to Antigen-Expressing Cells.

Macromolecules such as antibodies and antibody fragments have been reported to interfere with intracellular targets, but their use is limited to delivery systems where expression is achieved from vectors such as plasmids or viruses. We have developed PEGylated nanoparticles of poly-lactic acid (PLA), including the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), which are functionalized with monoclonal anti-CD7, anti-CD53, or anti-GPR56 antibodies for receptor-mediated endocytic delivery into T-cell leukemia cell lines. Incorporation of DOTAP as the lipid component of the PLA nanoparticles enhanced the release of the immuno-nanoparticles from the endosomes into the cytosol compared to nanoparticles made from PLA alone. Systemic delivery of these anti-CD7 immuno-nanoparticles into humanized CD7 transgenic mice resulted in localization in the spleen, enhanced uptake into CD7-expressing splenocytes, and release of low amounts of reporter mRNA for translation. These functionalized polymer lipid nanoparticles are the basis for elaboration and optimization for realizing their potential in therapeutic applications to carry specific macromolecules such as mRNAs for translation into therapeutic proteins that can target intracellular proteins which mediate disease.

20 Years an Orphan: Is GPR84 a Plausible Medium-Chain Fatty Acid-Sensing Receptor?

GPR84 is an inflammation-induced receptor highly expressed on immune cells, yet its endogenous ligand is still unknown. This makes any interpretation of its physiological activity in vivo difficult. However, experiments with potent synthetic agonists have highlighted what the receptor can do, namely, enhance proinflammatory signaling and macrophage effector functions such as phagocytosis. Developing drugs to block these effects has attracted interest from the scientific community with the aim of decreasing disease activity in inflammatory disorders or enhancing inflammation resolution. In this review, we critically reassess the widely held belief that the major role of GPR84 is that of being a medium-chain fatty acid (MCFA) receptor. While MCFAs have been shown to activate GPR84, it remains to be demonstrated that they are present in relevant tissues at appropriate concentrations. In contrast to four other "full-time" free fatty acid receptor subtypes, GPR84 is not expressed by enteroendocrine cells and has limited expression in the gastrointestinal tract. Across multiple tissues and cell types, the highest expression levels of GPR84 are observed hours after exposure to an inflammatory stimulus. These factors obscure the relationship between ligand and receptor in the human body and do not support the exclusive physiological pairing of MCFAs with GPR84. To maximize the chances of developing efficacious drugs for inflammatory diseases, we must advance our understanding of GPR84 and what it does in vivo.

A Biased Agonist at Immunometabolic Receptor GPR84 Causes Distinct Functional Effects in Macrophages.

GPR84 is an orphan G-protein-coupled receptor that is expressed on immune cells and implicated in several inflammatory diseases. The validation of GPR84 as a therapeutic target is hindered by the narrow range of available chemical tools and consequent poor understanding of GPR84 pathophysiology. Here we describe the discovery and characterization of DL-175, a potent, selective, and structurally novel GPR84 agonist and the first to display significantly biased signaling across GPR84-overexpressing cells, primary murine macrophages, and human U937 cells. By comparing DL-175 with reported GPR84 ligands, we show for the first time that biased GPR84 agonists have markedly different abilities to induce chemotaxis in human myeloid cells, while causing similar levels of phagocytosis enhancement. This work demonstrates that biased agonism at GPR84 enables the selective activation of functional responses in immune cells and delivers a high-quality chemical probe for further investigation.

Enantiomerically pure bicyclo[3.3.1]nona-2,6-diene as the sole source of enantioselectivity in BIPHEP-Rh asymmetric hydrogenation.

In situ resolution of the rapidly racemising diphosphine BIPHEP and its relatives with the cationic Rh complex of (S,S)-bicyclonona-2,6-diene permits the asymmetric hydrogenation of dehydroamino esters.