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 G/cAMP and β-arrestin, but which is rapidly metabolized. Herein, we describe an optimization of DL-175 through a systematic structure-activity relationship (SAR) analysis.

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.

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.

Identification and Preliminary Structure-Activity Relationship Studies of 1,5-Dihydrobenzo[][1,4]oxazepin-2(3)-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.

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.

Structural Requirements for Dihydrobenzoxazepinone Anthelmintics: Actions against Medically Important and Model Parasites: , , , and .

Nine hundred million people are infected with the soil-transmitted helminths (roundworm), hookworm, and (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 Here, we report a systematic investigation of the structure-activity relationship of the anthelmintic activity of DHB compounds.

Competitive SPR using an intracellular anti-LMO2 antibody identifies novel LMO2-interacting compounds.

The use of intracellular antibodies as templates to derive surrogate compounds is an important objective because intracellular antibodies can be employed initially for target validation in pre-clinical assays and subsequently employed in compound library screens. LMO2 is a T cell oncogenic protein activated in the majority of T cell acute leukaemias. We have used an inhibitory intracellular antibody fragment as a competitor in a small molecule library screen using competitive surface plasmon resonance (cSPR) to identify compounds that bind to LMO2.

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.

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.

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 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.

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.

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.

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.

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

The 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 KRAS-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).

Chemical Instability and Promiscuity of Arylmethylidenepyrazolinone-Based MDMX Inhibitors.

Targeting the protein-protein interaction between p53 and MDM2/MDMX (MDM4) represents an attractive anticancer strategy for the treatment of p53-competent tumors. Several selective and potent MDM2 inhibitors have been developed and entered the clinic; however, the repertoire of MDMX antagonists is still limited. The arylmethylidenepyrazolinone SJ-172550 has been reported as a selective MDMX antagonist; yet, uncertainties about its mechanism of action have raised doubts about its use as a chemical probe.

Small molecule inhibitors of RAS-effector protein interactions derived using an intracellular antibody fragment.

Targeting specific protein-protein interactions (PPIs) is an attractive concept for drug development, but hard to implement since intracellular antibodies do not penetrate cells and most small-molecule drugs are considered unsuitable for PPI inhibition. A potential solution to these problems is to select intracellular antibody fragments to block PPIs, use these antibody fragments for target validation in disease models and finally derive small molecules overlapping the antibody-binding site. Here, we explore this strategy using an anti-mutant RAS antibody fragment as a competitor in a small-molecule library screen for identifying RAS-binding compounds.

2,4-Diaminothieno[3,2-d]pyrimidines, a new class of anthelmintic with activity against adult and egg stages of whipworm.

The human whipworm Trichuris trichiura is a parasite that infects around 500 million people globally, with consequences including damage to physical growth and educational performance. Current drugs such as mebendazole have a notable lack of efficacy against whipworm, compared to other soil-transmitted helminths. Mass drug administration programs are therefore unlikely to achieve eradication and new treatments for trichuriasis are desperately needed.

BRET-based RAS biosensors that show a novel small molecule is an inhibitor of RAS-effector protein-protein interactions.

The RAS family of proteins is amongst the most highly mutated in human cancers and has so far eluded drug therapy. Currently, much effort is being made to discover mutant RAS inhibitors and in vitro screening for RAS-binding drugs must be followed by cell-based assays. Here, we have developed a robust set of bioluminescence resonance energy transfer (BRET)-based RAS biosensors that enable monitoring of RAS-effector interaction inhibition in living cells.

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.

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.

Dihydrobenz[e][1,4]oxazepin-2(3H)-ones, a new anthelmintic chemotype immobilising whipworm and reducing infectivity in vivo.

Trichuris trichiura is a human parasitic whipworm infecting around 500 million people globally, damaging the physical growth and educational performance of those infected. Current drug treatment options are limited and lack efficacy against the worm, preventing an eradication programme. It is therefore important to develop new treatments for trichuriasis.