Correction: Modified minimal-size fragments of heparan sulfate as inhibitors of endosulfatase-2 (Sulf-2).

Correction for 'Modified minimal-size fragments of heparan sulfate as inhibitors of endosulfatase-2 (Sulf-2)' by Alice Kennett et al., Chem. Commun., 2024, 60, 436-439, https://doi.org/10.1039/D3CC02565A.

Modified minimal-size fragments of heparan sulfate as inhibitors of endosulfatase-2 (Sulf-2).

Sulf-2 has been identified as a putative target for anticancer therapies. Here we report the design and synthesis of sulfated disaccharide inhibitors based on IdoA(2S)-GlcNS(6S). Trisulfated disaccharide inhibitor IdoA(2S)-GlcNS(6Sulfamate) demonstrated potent Sulf-2 inhibition. The IC50 value was determined to be 39.8 µM ± 18.3, which is comparable to a tetrasaccharide inhibitor of HSulf-1 reported in the literature. We propose that the disaccharide IdoA(2S)-GlcNS(6S) is the shortest fragment size required for effective inhibition of the Sulfs.

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.

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.

Biased agonists of GPR84 and insights into biological control.

GPR84 was first identified as an open reading frame encoding an orphan Class A G protein coupled receptor in 2001. Gpr84 mRNA is expressed in a limited number of cell types with the highest levels of expression being in innate immune cells, M1 polarised macrophages and neutrophils. The first reported ligands for this receptor were medium chain fatty acids with chain lengths between 9 and 12 carbons. Subsequently, a series of synthetic agonists that signal via the GPR84 receptor were identified. Radioligand binding assays and molecular modelling with site-directed mutagenesis suggest the presence of three ligand binding sites on the receptor, but the physiological agonist(s) of the receptor remain unidentified. Here, we review the effects of GPR84 agonists on innate immune cells following a series of chemical discoveries since 2001. The development of highly biased agonists has helped to probe receptor function in vitro, and the remaining challenge is to follow the effects of biased signalling to the physiological functions of innate immune cell types. LINKED ARTICLES: This article is part of a themed issue GPR84 Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.10/issuetoc.

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.

Developmental Expression of the Cell Cycle Regulator p16INK4a in Retinal Glial Cells: A Novel Marker for Immature Ocular Astrocytes?

Retinal astrocytes are vital for neuronal homeostasis in the retina. Together with Müller glia, they provide retinal cells with neurotrophic factors, antioxidative support, and defense mechanisms such as the formation of the blood-retinal barrier. Substantial heterogeneity of astrocyte morphology and function represents a challenge for identification of distinct subtypes which may be potential targets for therapeutic purposes. Hence, identification of novel markers of astrocyte subpopulations is highly relevant to better understand the molecular mechanisms involved in retinal development, homeostasis, and pathology. In this study, we observed that the cell cycle regulator, p16INK4a, is expressed in immature astrocytes in the mouse retina. Immunohistochemical analysis showed p16INK4a expression in the optic nerve of wild-type mice from 3 days to 3 months of age and in the nerve fiber layer of the adult mouse retina. Colocalization of p16INK4a expression and glial fibrillary acidic protein (immature/mature astrocyte marker) tends to decrease with age. However, colocalization of p16INK4a expression and vimentin (immature astrocyte marker) remains high in the optic nerve from the early postnatal period to adulthood. The observations from this study provide a valuable tool for further investigations of ocular astrocytes in the developing retina as well as in degenerative retinopathies.

A pharmacophore-based approach to demonstrating the scope of alcohol dehydrogenases.

Barriers to the ready adoption of biocatalysis into asymmetric synthesis for early stage medicinal chemistry are addressed, using ketone reduction by alcohol dehydrogenase as a model reaction. An efficient substrate screening approach is used to show the wide substrate scope of commercial alcohol dehydrogenase enzymes, with a high tolerance to chemical groups employed in drug discovery (heterocycle, trifluoromethyl and nitrile/nitro groups) observed. We use our screening data to build a preliminary predictive pharmacophore-based screening tool using Forge software, with a precision of 0.67/1, demonstrating the potential for developing substrate screening tools for commercially available enzymes without publicly available structures. We hope that this work will facilitate a culture shift towards adopting biocatalysis alongside traditional chemical catalytic methods in early stage drug discovery.

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.

Evaluating the efficacy and safety of a novel prophylactic nasal spray in the prevention of SARS-CoV-2 infection: A multi-centre, double blind, placebo-controlled, randomised trial.

Background The COVID-19 pandemic continues to devastate communities all over the world. The aim of this study was to evaluate the efficacy and safety of the test agent as a prophylaxis against SARS-CoV-2 infection in a population of high-risk healthcare workers. Methods The study was a multi-centre, prospective, double blind, randomized, placebo-controlled trial. Key eligibility criteria included absence of significant co-morbidity and no previous SARS-CoV-2 infection or vaccination. Participants were randomised to either the active agent nasal spray or placebo using computer generated random number tables. The nasal spray was administered 3 times daily over a 45 day course. The primary end point was the percentage of subjects who tested positive for IgGS (anti-spike, immunoglobulin G specific to the spike protein of SARS-CoV-2) at day 45. Results Between 16th April 2021 and 26th July 2021, 556 participants were analysed for the primary endpoint (275 Test; 281 Placebo). The test agent significantly reduced SARS-CoV-2 infection compared to placebo [36 cases (13.1%) Vs 97 cases (34.5%); OR 0.29 (95% CI; 0.18-0.45), p < 0.0001]. Fewer clinical symptoms were also seen in the test group [57 cases (17.6%) vs 112 cases (34.6%); OR 0.40, (95% CI; 0.27-0.59), p < 0.0001]. No harmful effects were associated with taking the test agent. Conclusion The test agent significantly reduced SARS-CoV-2 infection in healthcare workers, with 62% fewer infections when compared to placebo. It was found to be safe and well tolerated and offers a novel treatment option for prophylaxis against SARS-CoV-2 infection.

Statin activation of skeletal ryanodine receptors (RyR1) is a class effect but separable from HMG-CoA reductase inhibition.

BACKGROUND AND PURPOSE: Statins, inhibitors of HMG-CoA reductase, are mainstay treatment for hypercholesterolaemia. However, muscle pain and weakness prevent many patients from benefiting from their cardioprotective effects. We previously demonstrated that simvastatin activates skeletal ryanodine receptors (RyR1), an effect that could be important in initiating myopathy. Using a range of structurally diverse statin analogues, we examined structural features associated with RyR1 activation, aiming to identify statins lacking this property. EXPERIMENTAL APPROACH: Compounds were screened for RyR1 activity utilising [3 H]ryanodine binding. Mechanistic insight into RyR1 activity was studied by incorporating RyR1 channels from sheep, mouse or rabbit skeletal muscle into bilayers. KEY RESULTS: All UK-prescribed statins activated RyR1 at nanomolar concentrations. Cerivastatin, withdrawn from the market due to life-threatening muscle-related side effects, was more effective than currently-prescribed statins and possessed the unique ability to open RyR1 channels independently of cytosolic Ca2+ . We synthesised the one essential structural moiety that all statins must possess for HMG-CoA reductase inhibition, the R-3,5-dihydroxypentanoic acid unit, and it did not activate RyR1. We also identified five analogues retaining potent HMG-CoA reductase inhibition that inhibited RyR1 and four that lacked the ability to modulate RyR1. CONCLUSION AND IMPLICATIONS: That cerivastatin activates RyR1 most strongly supports the hypothesis that RyR1 activation is implicated in statin-induced myopathy. Demonstrating that statin regulation of RyR1 and HMG-CoA reductase are separable effects will allow the role of RyR1 in statin-induced myopathy to be further elucidated by the tool compounds we have identified, allowing development of effective cardioprotective statins with improved patient tolerance.

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.

Recent advances in small molecule stimulation of regeneration and repair.

Therapeutic approaches to stimulate regeneration and repair have the potential to transform healthcare and improve outcomes for patients suffering from numerous chronic degenerative diseases. To date most approaches have involved the transplantation of therapeutic cells, and while there have been a small number of clinical approvals, major hurdles exist to the routine adoption of such therapies. In recent years humans and other mammals have been shown to possess a regenerative capacity across multiple tissues and organs, and an innate regenerative and repair response has been shown to be activated in these organs in response to injury. These realisations have inspired a transformative approach in regenerative medicine: the development of new agents to directly target these innate regeneration and repair pathways. In this article we will review the current state of the art in the discovery of small molecule modulators of regeneration and their translation towards therapeutic agents, focussing specifically on the areas of neuroregeneration and cardiac regeneration.

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.

An outer-pore gate modulates the pharmacology of the TMEM16A channel.

TMEM16A Ca2+-activated chloride channels are involved in multiple cellular functions and are proposed targets for diseases such as hypertension, stroke, and cystic fibrosis. This therapeutic endeavor, however, suffers from paucity of selective and potent modulators. Here, exploiting a synthetic small molecule with a biphasic effect on the TMEM16A channel, anthracene-9-carboxylic acid (A9C), we shed light on sites of the channel amenable for pharmacological intervention. Mutant channels with the intracellular gate constitutively open were generated. These channels were entirely insensitive to extracellular A9C when intracellular Ca2+ was omitted. However, when physiological Ca2+ levels were reestablished, the mutants regained sensitivity to A9C. Thus, intracellular Ca2+ is mandatory for the channel response to an extracellular modulator. The underlying mechanism is a conformational change in the outer pore that enables A9C to enter the pore to reach its binding site. The explanation of this structural rearrangement highlights a critical site for pharmacological intervention and reveals an aspect of Ca2+ gating in the TMEM16A channel.

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.