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.

A tubulin binding molecule drives differentiation of acute myeloid leukemia cells.

Despite much progress in developing better drugs, many patients with acute myeloid leukemia (AML) still die within a year of diagnosis. This is partly because it is difficult to identify therapeutic targets that are effective across multiple AML subtypes. One common factor across AML subtypes is the presence of a block in differentiation. Overcoming this block should allow for the identification of therapies that are not dependent on a specific mutation for their efficacy. Here, we used a phenotypic screen to identify compounds that stimulate differentiation in genetically diverse AML cell lines. Lead compounds were shown to decrease tumor burden and to increase survival in vivo. Using multiple complementary target deconvolution approaches, these compounds were revealed to be anti-mitotic tubulin disruptors that cause differentiation by inducing a G2-M mitotic arrest. Together, these results reveal a function for tubulin disruptors in causing differentiation of AML cells.

Structure-activity relationships of 2-pyrimidinecarbohydrazides as utrophin modulators for the potential treatment of Duchenne muscular dystrophy.

A therapeutic approach that holds the potential to treat all Duchenne muscular dystrophy (DMD) patient populations is utrophin modulation. Ezutromid, a first generation utrophin modulator which was later found to act via antagonism of the arylhydrocarbon receptor, progressed to Phase 2 clinical trials. Although interim data showed target engagement and functional improvements, ezutromid ultimately failed to meet its clinical endpoints. We recently described the identification of a new class of hydrazide utrophin modulators which has a different mechanism of action to ezutromid. In this study we report our early optimisation studies on this hydrazide series. The new analogues had significantly improved potency in cell-based assays, increased sp3 character and reduced lipophilicity, which also improved their physicochemical properties. A representative new analogue combining these attributes increased utrophin protein in dystrophic mouse cells showing it can be used as a chemical tool to reveal new insights regarding utrophin upregulation as a strategy for DMD therapeutic intervention.

Pilot Study to Quantify Palladium Impurities in Lead-like Compounds Following Commonly Used Purification Techniques.

Palladium-catalyzed reactions are among the most commonly used procedures in organic synthesis. The products have a range of uses, including as intermediates in total synthesis and as screening compounds for drug discovery or agrochemical projects. Despite the known and potentially deleterious effects of low-level metal impurities in biological assays, the quantification of metal remaining in reaction products to verify the effective removal of the transition element is rarely reported. Using palladium as an exemplar, we describe a pilot study that for the first time quantifies residual metal levels in reaction products following increasingly rigorous purification protocols. Our results demonstrate that significant levels of residual palladium can remain in isolated reaction products following chromatographic purification, and only by using a subsequent metal scavenging step are they reliably reduced to a low level. Finally, we provide a set of simple guidelines that should minimize the potential for issues associated with residual palladium in reaction products.

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.

A Phenotypic Screen Identifies a Compound Series That Induces Differentiation of Acute Myeloid Leukemia Cells In Vitro and Shows Antitumor Effects In Vivo.

Induction of differentiation is a promising therapeutic strategy against acute myeloid leukemia. However, current differentiation therapies are effective only to specific patient populations. To identify novel differentiation agents with wider efficacy, we developed a phenotypic high-throughput screen with a range of genetically diverse cell lines. From the resulting hits, one chemical scaffold was optimized in terms of activity and physicochemical properties to yield OXS007417, a proof-of-concept tool compound, which was also able to decrease tumor volume in a murine in vivo xenograft model.

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.

Discovery and mechanism of action studies of 4,6-diphenylpyrimidine-2-carbohydrazides as utrophin modulators for the treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy is a fatal disease with no cure, caused by lack of the cytoskeletal protein dystrophin. Upregulation of utrophin, a dystrophin paralogue, offers a potential therapy independent of mutation type. The failure of first-in-class utrophin modulator ezutromid/SMT C1100 in Phase II clinical trials necessitates development of compounds with better efficacy, physicochemical and ADME properties and/or complementary mechanisms. We have discovered and performed a preliminary optimisation of a novel class of utrophin modulators using an improved phenotypic screen, where reporter expression is derived from the full genomic context of the utrophin promoter. We further demonstrate through target deconvolution studies, including expression analysis and chemical proteomics, that this compound series operates via a novel mechanism of action, distinct from that of ezutromid.

Decreasing HepG2 Cytotoxicity by Lowering the Lipophilicity of Benzo[d]oxazolephosphinate Ester Utrophin Modulators.

Utrophin modulation is a disease-modifying therapeutic strategy for Duchenne muscular dystrophy that would be applicable to all patient populations. To improve the suboptimal profile of ezutromid, the first-in-class clinical candidate, a second generation of utrophin modulators bearing a phosphinate ester moiety was developed. This modification significantly improved the physicochemical and ADME properties, but one of the main lead molecules was found to have dose-limiting hepatotoxicity. In this work we describe how less lipophilic analogues retained utrophin modulatory activity in a reporter gene assay, upregulated utrophin protein in dystrophic mouse muscle cells, but also had improved physicochemical and ADME properties. Notably, ClogP was found to directly correlate with pIC50 in HepG2 cells, hence leading to a potentially safer toxicological profiles in this series. Compound 21 showed a balanced profile (H2K EC50: 4.17 µM, solubility: 477 µM, mouse hepatocyte T 1/2 > 240 min) and increased utrophin protein 1.6-fold in a Western blot assay.

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.

Synthesis of SMT022357 enantiomers and in vivo evaluation in a Duchenne muscular dystrophy mouse model.

Following on from ezutromid, the first-in-class benzoxazole utrophin modulator that progressed to Phase 2 clinical trials for the treatment of Duchenne muscular dystrophy, a new chemotype was designed to optimise its physicochemical and ADME profile. Herein we report the synthesis of SMT022357, a second generation utrophin modulator preclinical candidate, and an asymmetric synthesis of its constituent enantiomers. The pharmacological properties of both enantiomers were evaluated in vitro and in vivo. No significant difference in the activity or efficacy was observed between the two enantiomers; activity was found to be comparable to the racemic mixture.

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.

2-Arylbenzo[d]oxazole Phosphinate Esters as Second-Generation Modulators of Utrophin for the Treatment of Duchenne Muscular Dystrophy.

Utrophin modulation is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD), which should be applicable to all patient populations. Following on from ezutromid, the first-generation utrophin modulator, we describe the development of a second generation of utrophin modulators, based on the bioisosteric replacement of the sulfone group with a phosphinate ester and substitution of the metabolically labile naphthalene with a haloaryl substituent. The improved physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties, further reflected in the enhanced pharmacokinetic profile of the most advanced compounds, 30 and 27, led to significantly better in vivo exposure compared to ezutromid and alleviation of the dystrophic phenotype in mdx mice. While 30 was found to have dose-limiting hepatotoxicity, 27 and its enantiomers exhibited limited off-target effects, resulting in a safe profile and highlighting their potential utility as next-generation utrophin modulators suitable for progression toward a future DMD therapy.

From diagnosis to therapy in Duchenne muscular dystrophy.

Genetic approaches for the diagnosis and treatment of inherited muscle diseases have advanced rapidly in recent years. Many of the advances have occurred in the treatment of Duchenne muscular dystrophy (DMD), a muscle wasting disease where affected boys are typically wheelchair bound by age 12 years and generally die in their twenties from respiratory failure or cardiomyopathy. Dystrophin is a 421 kD protein which links F-actin to the extracellular matrix via the dystrophin-associated protein complex (DAPC) at the muscle membrane. In the absence of dystrophin, the DAPC is lost, making the muscle membrane more susceptible to contraction-induced injury. The identification of the gene causing DMD in 1986 resulted in improved diagnosis of the disease and the identification of hotspots for mutation. There is currently no effective treatment. However, there are several promising genetic therapeutic approaches at the preclinical stage or in clinical trials including read-through of stop codons, exon skipping, delivery of dystrophin minigenes and the modulation of expression of the dystrophin related protein, utrophin. In spite of significant progress, the problem of targeting all muscles, including diaphragm and heart at sufficiently high levels, remains a challenge. Any therapy also needs to consider the immune response and some treatments are mutation specific and therefore limited to a subgroup of patients. This short review provides a summary of the current status of DMD therapy with a particular focus on those genetic strategies that have been taken to the clinic.

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.

Chemical Proteomics and Phenotypic Profiling Identifies the Aryl Hydrocarbon Receptor as a Molecular Target of the Utrophin Modulator Ezutromid.

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease arising from mutations in the dystrophin gene. Upregulation of utrophin to compensate for the missing dystrophin offers a potential therapy independent of patient genotype. The first-in-class utrophin modulator ezutromid/SMT C1100 was developed from a phenotypic screen through to a Phase 2 clinical trial. Promising efficacy and evidence of target engagement was observed in DMD patients after 24 weeks of treatment, however trial endpoints were not met after 48 weeks. The objective of this study was to understand the mechanism of action of ezutromid which could explain the lack of sustained efficacy and help development of new generations of utrophin modulators. Using chemical proteomics and phenotypic profiling we show that the aryl hydrocarbon receptor (AhR) is a target of ezutromid. Several lines of evidence demonstrate that ezutromid binds AhR with an apparent KD of 50 nm and behaves as an AhR antagonist. Furthermore, other reported AhR antagonists also upregulate utrophin, showing that this pathway, which is currently being explored in other clinical applications including oncology and rheumatoid arthritis, could also be exploited in future DMD therapies.

Characterisation of utrophin modulator SMT C1100 as a non-competitive inhibitor of firefly luciferase.

Firefly luciferase (FLuc) is a powerful tool for molecular and cellular biology, and popular in high-throughput screening and drug discovery. However, FLuc assays have been plagued with positive and negative artefacts due to stabilisation and inhibition by small molecules from a range of chemical classes. Here we disclose Phase II clinical compound SMT C1100 for the treatment of Duchenne muscular dystrophy as an FLuc inhibitor (KD of 0.40 ±â€¯0.15 µM). Enzyme kinetic studies using SMT C1100 and other non-competitive inhibitors including resveratrol and NFκBAI4 identified previously undescribed modes of inhibition with respect to FLuc's luciferyl adenylate intermediate. Employing a photoaffinity strategy to identify SMT C1100's binding site, a photolabelled SMT C1100 probe instead underwent FLuc-dependent photooxidation. Our findings support novel binding sites on FLuc for non-competitive inhibitors.

Isolation, Structural Identification, Synthesis, and Pharmacological Profiling of 1,2-trans-Dihydro-1,2-diol Metabolites of the Utrophin Modulator Ezutromid.

5-(Ethylsulfonyl)-2-(naphthalen-2-yl)benzo[d]oxazole (ezutromid, 1) is a first-in-class utrophin modulator that has been evaluated in a phase 2 clinical study for the treatment of Duchenne muscular dystrophy (DMD). Ezutromid was found to undergo hepatic oxidation of its 2-naphthyl substituent to produce two regioisomeric 1,2-dihydronaphthalene-1,2-diols, DHD1 and DHD3, as the major metabolites after oral administration in humans and rodents. In many patients, plasma levels of the DHD metabolites were found to exceed those of ezutromid. Herein, we describe the structural elucidation of the main metabolites of ezutromid, the regio- and relative stereochemical assignments of DHD1 and DHD3, their de novo chemical synthesis, and their production in systems in vitro. We further elucidate the likely metabolic pathway and CYP isoforms responsible for DHD1 and DHD3 production and characterize their physicochemical, ADME, and pharmacological properties and their preliminary toxicological profiles.

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.

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. Here, we show that, in addition to its unclear mode of action, SJ-172550 is unstable in aqueous buffers, giving rise to side products of unknown biological activity. Using an SJ-172550-derived affinity probe, we observed promiscuous binding to cellular proteins whereas cellular thermal shift assays did not reveal a stabilizing effect on MDMX. Overall, our results raise further questions about the interpretation of data using SJ-172550 and related compounds to investigate cellular phenotypes.