Abstract Duchenne muscular dystrophy (DMD) presents a critical therapeutic gap in adolescent patients, where extensive fibro-fatty muscle replacement and depletion of the regenerative niche render existing interventions insufficient. Prostaglandin E2 signaling through the EP4 receptor stimulates bone and muscle regeneration and repair, but systemic off-target effects have limited the clinical translation of EP4 agonism in diseases such as DMD. We therefore evaluated irodanoprost (IROD), a bone-targeted prodrug of an EP4-selective agonist, in a DMD rat model, comparing early- and late-intervention cohorts. In adolescent rats, 8 weeks of treatment reduced body weight deficit by 41.4% and restored hindlimb muscle mass and maximum tetanic force to wild-type levels. IROD dose-dependently inhibited fibro-adipogenic progenitor differentiation into α-SMA⁺ myofibroblasts, facilitating active resolution of established fibrosis below pre-treatment baseline. This was accompanied by re-activation of a synchronized regenerative program marked by clustered eMHC⁺ fibers, restoring the total myofiber pool to wild-type levels. A strong linear correlation between intramuscular fat reduction and fibrosis resolution suggests that MRI-based fat imaging may serve as a non-invasive surrogate for monitoring anti-fibrotic efficacy. The efficacy of IROD is likely aided by the fact that while it selectively distributes to bone in healthy animals, we observed markedly enhanced accumulation of the drug in dystrophic muscle. These findings establish IROD as a pathology-targeted approach capable of resolving the fibro-fatty niche and restoring regenerative capacity in advanced DMD. Summary Pathology-targeted EP4 agonism resolves established fibrosis and restores myofiber regeneration in a rat model of adolescent Duchenne dystrophy. Graphic abstract

Inflammatory bowel diseases present with elevated levels of intestinal epithelial cell (IEC) death, which compromises the gut barrier, activating immune cells and triggering more IEC death. The endogenous signals that prevent IEC death and break this vicious cycle, allowing resolution of intestinal inflammation, remain largely unknown. Here we show that prostaglandin E2 signalling via the E-type prostanoid receptor 4 (EP4) on IECs represses epithelial necroptosis and induces resolution of colitis. We found that EP4 expression correlates with an improved IBD outcome and that EP4 activation induces a transcriptional signature consistent with resolution of intestinal inflammation. We further show that dysregulated necroptosis prevents resolution, and EP4 agonism suppresses necroptosis in human and mouse IECs. Mechanistically, EP4 signalling on IECs converges on receptor-interacting protein kinase 1 to suppress tumour necrosis factor-induced activation and membrane translocation of the necroptosis effector mixed-lineage kinase domain-like pseudokinase. In summary, our study indicates that EP4 promotes the resolution of colitis by suppressing IEC necroptosis.

The Hit to Lead (H2L) process is an integral part of contemporary drug discovery, encompassing the optimisation of validated Hit structures into Lead molecules. High quality leads build confidence, through activity and property profiles as well as preliminary biological data, which might include validating pharmacologic hypotheses along the way, indicating that further investment in the structure(s) and target would be worthwhile. Leads have line of sight to a development candidate and bring an understanding of what priorities Lead Optimisation should address. In this set of best practices, we detail the essential criteria that characterise a good lead, which include establishing SAR from analogues and assessing preliminary DMPK indicators, selectivity and early safety parameters. We highlight the importance of identifying liabilities of the lead series and demonstrating that each can be individually modulated whilst maintaining on target potency. We make the case for having physicochemical properties as critical optimisation parameters and how ligand efficiency metrics can enable this. Then we go over general tactics that can be used to convert hits into a lead series. These include essential steps that, when performed early, increase the chance of success such as deconstructive SAR, pharmacophore and bioactive conformation determination and scaffold optimisation. Finally, we suggest decision-making criteria to substantiate confidence in further investment or, as importantly, making a recommendation to cease further work on a series.

BACKGROUND:The main protease (Mpro) is one of the most attractive targets for antiviral drug discovery against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Mutations in Mpro have been linked to resistance against nirmatrelvir-ritonavir (NIR-RIT), an important therapy for SARS-CoV-2 infection. This study aimed to identify low-frequency antiviral resistance mutations in Mpro from NIR-RIT-treated patients and to analyze the enzymatic properties, inhibitor susceptibility, and structural features of new Mpro clinical variants. METHODS:We screened 1528 SARS-CoV-2-positive patients from 2 hospitals and identified 17 who remained positive after treatment. Whole-genome sequencing of nasopharyngeal specimens was conducted to identify Mpro clinical variants. The impact of these mutations on Mpro activity and inhibitor susceptibility was investigated using a fluorescent enzymatic biosensor in human cells, along with in vitro thermal stability and structure-based analyses of the Mpro mutants and Mpro-NIR complexes. RESULTS:The analysis identified 2 novel Mpro clinical variants: D48D/L58F/P132H (variant 1) and D48D/L67V/K90R/P132H (variant 2). Our data show that the selected clinical mutations are localized in the Mpro N-terminal domain, are far from the catalytic site, and strongly impact NIR resistance without affecting Mpro activity. Structural analysis and thermal denaturation analyses revealed that these mutations may disrupt the substrate binding site's structure and dynamics, reducing protein stability and potentially impacting substrate binding or dimerization without compromising catalytic activity. CONCLUSIONS:Our new Mpro clinical mutations that confer complete resistance to NIR were not identified during previous cell-culture-based studies. More research is needed to explore resistance mechanisms, providing insights into strategies that mitigate resistance and protect therapeutic efficacy.

Prostaglandin E2 receptor type 4 (EP4) agonists have been shown to be effective in treating experimental ulcerative colitis (UC) in animals and in human clinical trials, but their development has been impeded by unacceptable systemic side effects. In this study, a series of methylene phosphate prodrugs of a highly potent and selective prostaglandin EP4 receptor agonist were designed to target and remain localized in the gastrointestinal (GI) tract after either oral or rectal instillation. The prodrugs were designed to be converted to liberate active EP4 agonist by intestinal alkaline phosphate (IAP), a ubiquitous enzyme found at the luminal of the intestinal wall thus exposing the colon epithelial barrier while reducing systemic exposure to the active agonist. The prodrugs were shown to hydrolyze in plasma and after contact with GI tissue slices from ileum and colon. When optimized prodrugs were dosed orally, systemic peak exposure to the active agonist was not reduced, presumably due to IAP activity in the duodenum and small intestine. However, when dosed rectally, the prodrugs gave much reduced levels of EP4 agonist in the blood. An optimized prodrug was shown to be retained in the colon, when compared with free agonist after rectal administration in healthy mice and to be efficacious in a model of UC (the DSS mouse model). Plasma exposure to the active agonist was also much reduced in the mouse model of UC after 4 days of rectal dosing but after 7 days, one DSS mouse showed elevated systemic levels of the free agonist in the blood. The concept of efficacy and intestinal retention of an EP4 agonist-methylene phosphate prodrug was proven for rectal instillation but in DSS treated mice, severe disease appears to compromise the epithelia barrier sufficiently to allow some absorption of the prodrug to occur. Thus, further optimization of these prodrugs is required before a candidate can be selected for development for treating severe ulcerative colitis.

AbstractUp to 40% of patients with estrogen receptor (ER)-positive breast cancer will develop resistance against the majority of current ER-directed therapies. Resistance can arise through various mechanisms such as increased expression levels of coregulators, and key mutations acquired in the receptor’s ligand binding domain rendering it constitutively active. To overcome these resistance mechanisms, we explored targeting the ER Activation Function 2 (AF2) site, which is essential for coactivator binding and activation. Using artificial intelligence and the deep docking methodology, we virtually screened > 1 billion small molecules and identified 290 potential AF2 binders that were then characterized and validated through an iterative screening pipeline of cell-based and cell-free assays. We ranked the compounds based on their ability to reduce the transcriptional activity of the estrogen receptor and the viability of ER-positive breast cancer cells. We identified a lead compound, VPC-260724, which inhibits ER activity at low micromolar range. We confirmed its direct binding to the ER-AF2 site through a PGC1α peptide displacement experiment. Using proximity ligation assays, we showed that VPC-260724 disrupts the interaction between ER-AF2 and the coactivator SRC-3 and reduces the expression of ER target genes in various breast cancer models including the tamoxifen resistant cell line TamR3. In conclusion, we developed a novel ER-AF2 binder, VPC-260724, which shows antiproliferative activity in ER-positive breast cancer models. The use of an ER-AF2 inhibitor in combination with current treatments may provide a novel complementary therapeutic approach to target treatment resistance in ER-positive breast cancer.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) cleaves the N-terminal amino acids of peptides, which can then bind onto major histocompatibility class I (MHC-I) molecules for presentation onto the cell surface, driving the activation of adaptive immune responses. In cancer, overtrimming of mature antigenic peptides can reduce cytotoxic T-cell responses, and ERAP1 can generate self-antigenic peptides which contribute to autoimmune cellular responses. Therefore, modulation of ERAP1 activity has potential therapeutic indications for cancer immunotherapy and in autoimmune disease. Herein we describe the hit-to-lead optimization of a series of cyclohexyl acid ERAP1 inhibitors, found by X-ray crystallography to bind at an allosteric regulatory site. Structure-based drug design enabled a >1,000-fold increase in ERAP1 enzymatic and cellular activity, resulting in potent and selective tool molecules. For lead compound 7, rat pharmacokinetic properties showed moderate unbound clearance and oral bioavailability, thus highlighting the promise of the series for further optimization.

A highly curated dataset of published compounds from ChEMBL version 32 is used to seek evidence for the occurrence of ‘natural selection’ in drug discovery. Three complementary measures of natural product (NP) character were applied, to compare time- and target-matched compounds reaching the clinic (Clinical compounds in phase 1-4 development) with background compounds (Reference compounds). Pseudo-natural products (PNPs), containing NP fragments combined in ways inaccessible by Nature, are rapidly increasing over time, reaching 67% of Clinical compounds first disclosed since 2010. PNPs are 54% more likely to be found in post-2008 Clinical versus Reference compounds. The majority of target classes show increased Clinical compound NP character versus their Reference compounds. Only 176 NP fragments appear in >1000 Clinical compounds published since 2008, yet these make up on average 63% of the Clinical compound’s core scaffolds. There is huge untapped potential awaiting exploitation, by applying Nature’s building blocks - ‘Natural Intelligence’ - to drug design.

Antivirals with broad coronavirus activity are important for treating high-risk individuals exposed to the constantly evolving SARS-CoV-2 variants of concern (VOCs) as well as emerging drug-resistant variants. We developed and characterized a novel class of active-site-directed 3-chymotrypsin-like protease (3CLpro) inhibitors (C2-C5a). Our lead direct-acting antiviral (DAA), C5a, is a non-covalent, non-peptide with a dissociation constant of 170 nM against recombinant SARS-CoV-2 3CLpro. The compounds C2-C5a exhibit broad-spectrum activity against Omicron subvariants (BA.5, BQ.1.1, and XBB.1.5) and seasonal human coronavirus-229E infection in human cells. Notably, C5a has median effective concentrations of 30-50 nM against BQ.1.1 and XBB.1.5 in two different human cell lines. X-ray crystallography has confirmed the unique binding modes of C2-C5a to the 3CLpro, which can limit virus cross-resistance to emerging Paxlovid-resistant variants. We tested the effect of C5a with two of our newly discovered host-directed antivirals (HDAs): N-0385, a TMPRSS2 inhibitor, and bafilomycin D (BafD), a human vacuolar H+-ATPase [V-ATPase] inhibitor. We demonstrated a synergistic action of C5a in combination with N-0385 and BafD against Omicron BA.5 infection in human Calu-3 lung cells. Our findings underscore that a SARS-CoV-2 multi-targeted treatment for circulating Omicron subvariants based on DAAs (C5a) and HDAs (N-0385 or BafD) can lead to therapeutic benefits by enhancing treatment efficacy. Furthermore, the high-resolution structures of SARS-CoV-2 3CLpro in complex with C2-C5a will facilitate future rational optimization of our novel broad-spectrum active-site-directed 3C-like protease inhibitors.

The rapid global spread of the SARS-CoV-2 virus facilitated the development of novel direct-acting antiviral agents (DAAs). The papain-like protease (PLpro) has been proposed as one of the major SARS-CoV-2 targets for DAAs due to its dual role in processing viral proteins and facilitating the host's immune suppression. This dual role makes identifying small molecules that can effectively neutralize SARS-CoV-2 PLpro activity a high-priority task. However, PLpro drug discovery faces a significant challenge due to the high mobility and induced-fit effects in the protease's active site. Herein, we virtually screened the ZINC20 database with Deep Docking (DD) to identify prospective noncovalent PLpro binders and combined ultra-large consensus docking with two pharmacophore (ph4)-filtering strategies. The analysis of active compounds revealed their somewhat-limited diversity, likely attributed to the induced-fit nature of PLpro's active site in the crystal structures, and therefore, the use of rigid docking protocols poses inherited limitations. The top hits were assessed against recombinant viral proteins and live viruses, demonstrating desirable inhibitory activities. The best compound VPC-300195 (IC50: 15 μM) ranks among the top noncovalent PLpro inhibitors discovered through in silico methodologies. In the search for novel SARS-CoV-2 PLpro-specific chemotypes, the identified inhibitors could serve as diverse templates for the development of effective noncovalent PLpro inhibitors.

There is an urgent need for new treatments for Chagas disease, a parasitic infection which mostly impacts South and Central America. We previously reported on the discovery of GSK3494245/DDD01305143, a preclinical candidate for visceral leishmaniasis which acted through inhibition of the Leishmania proteasome. A related analogue, active against Trypanosoma cruzi, showed suboptimal efficacy in an animal model of Chagas disease, so alternative proteasome inhibitors were investigated. Screening a library of phenotypically active analogues against the T. cruzi proteasome identified an active, selective pyridazinone, the development of which is described herein. We obtained a cryo-EM co-structure of proteasome and a key inhibitor and used this to drive optimization of the compounds. Alongside this, optimization of the absorption, distribution, metabolism, and excretion (ADME) properties afforded a suitable compound for mouse efficacy studies. The outcome of these studies is discussed, alongside future plans to further understand the series and its potential to deliver a new treatment for Chagas disease.

The case for a renewed focus on Nature in drug discovery is reviewed; not in terms of natural product screening, but how and why biomimetic molecules, especially those produced by natural processes, should deliver in the age of artificial intelligence and screening of vast collections both in vitro and in silico. The declining natural product-likeness of licensed drugs and the consequent physicochemical implications of this trend in the context of current practices are noted. To arrest these trends, the logic of seeking new bioactive agents with enhanced natural mimicry is considered; notably that molecules constructed by proteins (enzymes) are more likely to interact with other proteins (e.g., targets and transporters), a notion validated by natural products. Nature's finite number of building blocks and their interactions necessarily reduce potential numbers of structures, yet these enable expansion of chemical space with their inherent diversity of physical characteristics, pertinent to property-based design. The feasible variations on natural motifs are considered and expanded to encompass pseudo-natural products, leading to the further logical step of harnessing bioprocessing routes to access them. Together, these offer opportunities for enhancing natural mimicry, thereby bringing innovation to drug synthesis exploiting the characteristics of natural recognition processes. The potential for computational guidance to help identifying binding commonalities in the route map is a logical opportunity to enable the design of tailored molecules, with a focus on "organic/biological" rather than purely "synthetic" structures. The design and synthesis of prototype structures should pay dividends in the disposition and efficacy of the molecules, while inherently enabling greener and more sustainable manufacturing techniques.

Approved drugs for the treatment of osteoporosis can prevent further bone loss but do not stimulate bone formation. Approaches that improve bone density in metabolic diseases are needed. Therapies that take advantage of the ability of mesenchymal stem cells (MSCs) to differentiate into various osteogenic lineages to treat bone disorders are of particular interest. Here we examine the ability of small interfering RNA (siRNA) to enhance osteoblast differentiation and bone formation by silencing the negative suppressor gene GNAS in bone MSCs. Using clinically validated lipid nanoparticle (LNP) siRNA delivery systems, we show that silencing the suppressor gene GNAS in vitro in MSCs leads to molecular and phenotypic changes similar to those seen in osteoblasts. Further, we demonstrate that these LNP-siRNAs can transfect a large proportion of mice MSCs in the compact bone following intravenous injection. Transfection of MSCs in various animal models led to silencing of GNAS and enhanced differentiation of MSCs into osteoblasts. These data demonstrate the potential for LNP delivery of siRNA to enhance the differentiation of MSCs into osteoblasts, and suggests that they are a promising approach for the treatment of osteoporosis and other bone diseases.

We describe a protocol to identify the binding site(s) for a drug called ivacaftor that potentiates the CFTR chloride channel. We use photoaffinity probes—based on the structure of ivacaftor—to covalently modify the CFTR protein at the region that constitutes the drug binding site(s). We define the methods for photo-labeling CFTR, its membrane extraction, and enzymatic digestion using trypsin. We then describe the experimental methods to identify the modified peptides by using mass spectrometry. For complete details on the use and execution of this protocol, please refer to Laselva et al. (2021).

Prostate cancer (PCa) patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer (CRPC). Targeting the androgen receptor (AR) Binding Function-3 (BF3) site offers a promising option to treat CRPC. However, BF3 inhibitors have been limited by poor potency or inadequate metabolic stability. Through extensive medicinal chemistry, molecular modeling, and biochemistry, we identified 2-(5,6,7-trifluoro-1H-Indol-3-yl)-quinoline-5-carboxamide (VPC-13789), a potent AR BF3 antagonist with markedly improved pharmacokinetic properties. We demonstrate that VPC-13789 suppresses AR-mediated transcription, chromatin binding, and recruitment of coregulatory proteins. This novel AR antagonist selectively reduces the growth of both androgen-dependent and enzalutamide-resistant PCa cell lines. Having demonstrated in vitro efficacy, we developed an orally bioavailable prodrug that reduced PSA production and tumor volume in animal models of CRPC with no observed toxicity. VPC-13789 is a potent, selective, and orally bioavailable antiandrogen with a distinct mode of action that has a potential as novel CRPC therapeutics.

Securing novel, safe, and effective medicines to treat Mycobacterium tuberculosis remains an elusive goal, particularly influenced by the largely impervious Mtb envelope that limits exposure and thus efficacy of inhibitors at their cellular and periplasmic targets. The impact of physicochemical properties on pharmacokinetic parameters that govern oral absorption and exposure at sites of infection is considered alongside how these properties influence penetration of the Mtb envelope, with the likely influence of transporter proteins. The findings are discussed to benchmark current drugs and the emerging pipeline, whilst considering tactics for future rational and targeted design strategies, based around emerging data on Mtb transporters and their structures and functions.

Ivacaftor (VX-770) was the first cystic fibrosis transmembrane conductance regulator (CFTR) modulatory drug approved for the treatment of patients with cystic fibrosis. Electron cryomicroscopy (cryo-EM) studies of detergent-solubilized CFTR indicated that VX-770 bound to a site at the interface between solvent and a hinge region in the CFTR protein conferred by transmembrane (tm) helices: tm4, tm5, and tm8. We re-evaluated VX-770 binding to CFTR in biological membranes using photoactivatable VX-770 probes. One such probe covalently labeled CFTR at two sites as determined following trypsin digestion and analysis by tandem-mass spectrometry. One labeled peptide resides in the cytosolic loop 4 of CFTR and the other is located in tm8, proximal to the site identified by cryo-EM. Complementary data from functional and molecular dynamic simulation studies support a model, where VX-770 mediates potentiation via multiple sites in the CFTR protein.

Objective: Osteogenesis imperfecta (OI) is a heritable bone fragility disorder that is usually caused by mutations affecting collagen type I synthesis in osteoblasts. Bisphosphonates are widely used to decrease fracture rate but are only partially effective. Bone anabolic compounds, such as prostaglandin E2 receptor 4 (EP4) agonists may be an alternative treatment approach. Here we assessed the effect of Mes-1007, a novel bone-targeted EP4 agonist in Jrt mice, a model of severe OI. Study design: Experimental study. Results: Male 8-week old wild type (WT) and OI mice were randomly assigned to 4 weeks of three intraperitoneal injections per week with Mes-1007 (25 mg per kg body mass), phosphate-buffered saline, zoledronate (5 mu g per kg), or a combination treatment of zoledronate and Mes-1007. Treatment with Mes-1007 alone did not lead to higher trabecular bone volume per tissue volume (BV/TV) in the distal femur or lumbar vertebra 4 in either WT or OI mice. Treatment with zoledronate alone was associated with a significant increase in distal femur and vertebra BV/TV in both genotypes. In zoledronate-treated WT and OI mice, Mes-1007 increased bone formation rate in vertebral trabecular bone and had an additive effect on BV/TV. Vertebral BV/TV in OI mice that received zoledronate or Mes-1007/zoledronate combination treatment was similar to untreated WT mice (p = 0.25). At the femoral midshaft, Mes-1007/zoledronate combination treatment increased cortical thickness in both genotypes and led to higher periosteal diameter in OI mice. Three-point bending tests of femurs showed that Mes1007/zoledronate combination treatment increased the stiffness, load at yield and maximal load in WT but not in OI mice. Conclusion: Dosing Mes-1007 in combination with zoledronate improved the bone properties in a manner that is consistent with a mechanism of action of EP4 agonists on bone and additive to effects of anti-resorptives typified by zoledronate.

A series of bone-targeting EP4 receptor agonist conjugate prodrugs were prepared wherein a potent EP4 receptor agonist was bound to a biologically inactive, bisphosphonate-based bone-targeting moiety. Singly and doubly radiolabeled conjugates were synthesized and were shown to be stable in blood, to be rapidly eliminated from the bloodstream, and to be effectively taken up into bone in vivo after intravenous dosing. From these preliminary studies a preferred conjugate 4 (also known as C3 and Mes-1007) was selected for follow up biodistribution and elimination studies. Doubly radiolabeled conjugate 4 was found to partition largely to the liver and bones, and both labels were eliminated from liver at the same rate indicating the conjugate was eliminated intact. Quantification of the labels in bones indicated that free EP4 agonist (EP4a)(2a) was released from bone-bound 4 with a half-time of about 7 days. When dosed orally, radiolabeled 4 was not absorbed and passed through the gastrointestinal tract essentially unchanged, and only traces of radiolabeled 4 were found in the liver, blood, or bones. 4 was found to bind rapidly and completely to powdered bone mineral or to various forms of calcium phosphate, forming a stable matrix suitable for implant and that could made into powders or solid forms and be sterilized without decomposition or release of 4. Basic hydrolysis released free EP4 agonist 2a quantitatively from the material.

The identification of a novel series of DprE1 inhibitors based on a 2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)-N-phenylpropanamide scaffold is described herein. SAR exploration around the HTS hit 1 led to the identification of multiple analogues with potent DprE1 inhibition and good whole-cell antimycobacterial activity.