An experimentally validated approach to automated biological evidence generation in drug discovery using knowledge graphs.

Explaining predictions for drug repositioning with biological knowledge graphs is a challenging problem. Graph completion methods using symbolic reasoning predict drug treatments and associated rules to generate evidence representing the therapeutic basis of the drug. Yet the vast amounts of generated paths that are biologically irrelevant or not mechanistically meaningful within the context of disease biology can limit utility. We use a reinforcement learning based knowledge graph completion model combined with an automatic filtering approach that produces the most relevant rules and biological paths explaining the predicted drug's therapeutic connection to the disease. In this work we validate the approach against preclinical experimental data for Fragile X syndrome demonstrating strong correlation between automatically extracted paths and experimentally derived transcriptional changes of selected genes and pathways of drug predictions Sulindac and Ibudilast. Additionally, we show it reduces the number of generated paths in two case studies, 85% for Cystic fibrosis and 95% for Parkinson's disease.

Computational drug discovery approaches identify mebendazole as a candidate treatment for autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a rare genetic disorder characterised by numerous renal cysts, the progressive expansion of which can impact kidney function and lead eventually to renal failure. Tolvaptan is the only disease-modifying drug approved for the treatment of ADPKD, however its poor side effect and safety profile necessitates the need for the development of new therapeutics in this area. Using a combination of transcriptomic and machine learning computational drug discovery tools, we predicted that a number of existing drugs could have utility in the treatment of ADPKD, and subsequently validated several of these drug predictions in established models of disease. We determined that the anthelmintic mebendazole was a potent anti-cystic agent in human cellular and in vivo models of ADPKD, and is likely acting through the inhibition of microtubule polymerisation and protein kinase activity. These findings demonstrate the utility of combining computational approaches to identify and understand potential new treatments for traditionally underserved rare diseases.

Identification of Novel Potent NSD2-PWWP1 Ligands Using Structure-Based Design and Computational Approaches.

Dysregulation of histone methyl transferase nuclear receptor-binding SET domain 2 (NSD2) has been implicated in several hematological and solid malignancies. NSD2 is a large multidomain protein that carries histone writing and histone reading functions. To date, identifying inhibitors of the enzymatic activity of NSD2 has proven challenging in terms of potency and SET domain selectivity. Inhibition of the NSD2-PWWP1 domain using small molecules has been considered as an alternative approach to reduce NSD2-unregulated activity. In this article, we present novel computational chemistry approaches, encompassing free energy perturbation coupled to machine learning (FEP/ML) models as well as virtual screening (VS) activities, to identify high-affinity NSD2 PWWP1 binders. Through these activities, we have identified the most potent NSD2-PWWP1 binder reported so far in the literature: compound 34 (pIC50 = 8.2). The compounds identified herein represent useful tools for studying the role of PWWP1 domains for inhibition of human NSD2.

A novel combination treatment for fragile X syndrome predicted using computational methods.

Fragile X syndrome is a neurodevelopmental disorder caused by silencing of the fragile X messenger ribonucleotide gene. Patients display a wide spectrum of symptoms ranging from intellectual and learning disabilities to behavioural challenges including autism spectrum disorder. In addition to this, patients also display a diversity of symptoms due to mosaicism. These factors make fragile X syndrome a difficult syndrome to manage and suggest that a single targeted therapeutic approach cannot address all the symptoms. To this end, we utilized Healx's data-driven drug discovery platform to identify a treatment strategy to address the wide range of diverse symptoms among patients. Computational methods identified the combination of ibudilast and gaboxadol as a treatment for several pathophysiological targets that could potentially reverse multiple symptoms associated with fragile X syndrome. Ibudilast is an approved broad-spectrum phosphodiesterase inhibitor, selective against both phosphodiesterase 4 and phosphodiesterase 10, and has demonstrated to have several beneficial effects in the brain. Gaboxadol is a GABAA receptor agonist, selective against the delta subunit, which has previously displayed encouraging results in a fragile X syndrome clinical trial. Alterations in GABA and cyclic adenosine monophosphate metabolism have long since been associated with the pathophysiology of fragile X syndrome; however, targeting both pathways simultaneously has never been investigated. Both drugs have a good safety and tolerability profile in the clinic making them attractive candidates for repurposing. We set out to explore whether the combination of ibudilast and gaboxadol could demonstrate therapeutic efficacy in a fragile X syndrome mouse model. We found that daily treatment with ibudilast significantly enhanced the ability of fragile X syndrome mice to perform a number of different cognitive assays while gaboxadol treatment improved behaviours such as hyperactivity, aggression, stereotypy and anxiety. Importantly, when ibudilast and gaboxadol were co-administered, the cognitive deficits as well as the aforementioned behaviours were rescued. Moreover, this combination treatment showed no evidence of tolerance, and no adverse effects were reported following chronic dosing. This work demonstrates for the first time that by targeting multiple pathways, with a combination treatment, we were able to rescue more phenotypes in a fragile X syndrome mouse model than either ibudilast or gaboxadol could achieve as monotherapies. This combination treatment approach holds promise for addressing the wide spectrum of diverse symptoms in this heterogeneous patient population and may have therapeutic potential for idiopathic autism.

Discovery and characterisation of quinazolines and 8-Azaquinazolines as NLRP3 agonists with oral bioavailability in mice.

The NLRP3 inflammasome is a multiprotein complex that plays a critical role in activating the immune system in response to danger signals. Small molecule agonists of NLRP3 may offer clinical benefits in cancer immunology either as a monotherapy or in combination with checkpoint blockade, where it is hypothesised that their application can help to initiate an antitumor immune response. In this study, we report the discovery of quinazolines and 8-azaquinazolines as NLRP3 agonists and their chemical optimization to afford compounds with oral bioavailability in mice. We confirm that these compounds engage the NLRP3 inflammasome by verifying their dependence upon lipopolysaccharide (LPS) priming for cytokine release and the activation of Caspase-1. We further demonstrate pathway engagement through loss of activity in an NLRP3-knockout THP1 cell line. Based on their pharmacokinetic profile and biological activity, these compounds represent valuable tools to evaluate the therapeutic potential of NLRP3 activation in a pre-clinical setting.

Discovery of a Potent and Orally Bioavailable Zwitterionic Series of Selective Estrogen Receptor Degrader-Antagonists.

Herein, we report the optimization of a meta-substituted series of selective estrogen receptor degrader (SERD) antagonists for the treatment of ER+ breast cancer. Structure-based design together with the use of modeling and NMR to favor the bioactive conformation led to a highly potent series of basic SERDs with promising physicochemical properties. Issues with hERG activity resulted in a strategy of zwitterion formation and ultimately in the identification of 38. This compound was shown to be a highly potent SERD capable of effectively degrading ERα in both MCF-7 and CAMA-1 cell lines. The low lipophilicity and zwitterionic nature led to a SERD with a clean secondary pharmacology profile and no hERG activity. Favorable physicochemical properties resulted in good oral bioavailability in preclinical species and potent in vivo activity in a mouse xenograft model.

Trends in Molecular Properties, Bioavailability, and Permeability across the Bayer Compound Collection.

For oral drugs, medicinal chemists aim to design compounds with high oral bioavailability, of which permeability is a key determinant. Taking advantage of >2000 compounds tested in rat bioavailability studies and >20,000 compounds tested in Caco2 assays at Bayer, we have examined the molecular properties governing bioavailability and permeability. In addition to classical parameters such as logD and molecular weight, we also investigated the relationship between calculated pKa and permeability. We find that neutral compounds retain permeability up to a molecular weight limit of 700, while stronger acids and bases are restricted to weights of 400-500. We also investigate trends for common properties such as hydrogen bond donors and acceptors, polar surface area, aromatic ring count, and rotatable bonds, including compounds which exceed Lipinski's rule of five (Ro5). These property-structure relationships are combined to provide design guidelines for bioavailable drugs in both traditional and "beyond rule of 5" (bRo5) chemical space.

Addressing the Reciprocal Crosstalk between the AR and the PI3K/AKT/mTOR Signaling Pathways for Prostate Cancer Treatment.

The reduction in androgen synthesis and the blockade of the androgen receptor (AR) function by chemical castration and AR signaling inhibitors represent the main treatment lines for the initial stages of prostate cancer. Unfortunately, resistance mechanisms ultimately develop due to alterations in the AR pathway, such as gene amplification or mutations, and also the emergence of alternative pathways that render the tumor less or, more rarely, completely independent of androgen activation. An essential oncogenic axis activated in prostate cancer is the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, as evidenced by the frequent alterations of the negative regulator phosphatase and tensin homolog (PTEN) and by the activating mutations in PI3K subunits. Additionally, crosstalk and reciprocal feedback loops between androgen signaling and the PI3K/AKT/mTOR signaling cascade that activate pro-survival signals and play an essential role in disease recurrence and progression have been evidenced. Inhibitors addressing different players of the PI3K/AKT/mTOR pathway have been evaluated in the clinic. Only a limited benefit has been reported in prostate cancer up to now due to the associated side effects, so novel combination approaches and biomarkers predictive of patient response are urgently needed. Here, we reviewed recent data on the crosstalk between AR signaling and the PI3K/AKT/mTOR pathway, the selective inhibitors identified, and the most advanced clinical studies, with a focus on combination treatments. A deeper understanding of the complex molecular mechanisms involved in disease progression and treatment resistance is essential to further guide therapeutic approaches with improved outcomes.

Retroperitoneal Compared to Transperitoneal Approach for Open Abdominal Aortic Aneurysm Repair Is Associated with Reduced Systemic Inflammation and Postoperative Morbidity.

BACKGROUND: In the United Kingdom, the most common surgical approach for repair of open abdominal aortic aneurysms (AAAs) is transperitoneal (TP). However, retroperitoneal (RP) approach is favored in those with more complex vascular anatomy often requiring a cross-clamp on the aorta superior to the renal arteries. This study compared these approaches in patients matched on all major demographic, comorbid, anatomic, and physiological variables. METHODS: Fifty-seven patients (TP: n = 24; RP: n = 33) unsuitable for endovascular aneurysm repair underwent preoperative cardiopulmonary exercise testing prior to open AAA repair. The surgical approach undertaken was dictated by individual surgeon preference. Postoperative mortality, complications, and length of hospital stay (LoS) were recorded. Patients were further stratified according to infrarenal (IR) or suprarenal/supraceliac (SR/SC) surgical clamping. Systemic inflammation (C-reactive protein) and renal function (serum creatinine and estimated glomerular filtration rate) were recorded. RESULTS: Twenty-three (96%) of TP patients only required an IR clamp compared with 12 (36%) in the RP group. Postoperative systemic inflammation was lower in RP patients (p = 0.002 vs. TP) and fewer reported pulmonary/gastrointestinal complications whereas renal impairment was more marked in those receiving SR/SC clamps (p < 0.001 vs. IR clamp). RP patients were defined by lower LoS (p = 0.001), while mid-/long-term mortality was low/comparable with TP, resulting in considerable cost savings. CONCLUSION: Despite the demands of more complicated vascular anatomy, the clinical and economic benefits highlighted by these findings justify the more routine adoption of the RP approach for complex AAA repair.

Attenuation of Human Growth Hormone-Induced Rash With Graded Dose Challenge.

Adult growth hormone (GH) deficiency is rare and requires replacement with extrinsic/synthetic injection. GH hypersensitivity has been reported; specifically, atopic patients may develop rashes from somatotropin therapy. Allergic and non-allergic skin reactions to recombinant human GH are uncommon and infrequently reported. We describe a graded-dose challenge with intravenous Norditropin® in a 65-year-old atopic adult woman who developed a severe whole-body rash with Norditropin FlexPro® administration on several occasions but was negative on skin-prick testing to Norditropin® percutaneously and intradermally, but the patch testing was positive for gold and nickel. The patient was registered as a direct admission to the emergency room at a university hospital for a rapid antigen coronavirus disease 2019 (COVID-19) testing after having received two COVID-19 vaccinations and re-testing four months after vaccination. She was then directly admitted to a non-COVID-19 intensive care unit with direct bedside supervision by a registered nurse and a physician board certified in internal medicine, allergy/immunology, and pulmonary diseases. The patient brought a Norditropin® pen which our pharmacy team attached to a compatible syringe for dilutions. A graded dose challenge at a final dosage of 0.1 mL was performed and the patient was monitored for allergic and other adverse drug reactions, which did not occur. At the time of writing this case report, the patient has been maintained on Norditropin FlexPro® 0.1 mL and has not experienced any adverse reactions, including recurrent skin eruptions. The case presented is the first to describe a patient who successfully tolerated a graded dose challenge of an adult patient to GH replacement therapy (as Norditropin®) under supervision in an intensive care unit, whereas prior to reporting of this case, a graded dose challenge to GH replacement therapy had only been successfully performed in a child using another formulation of somatotropin (Humatrope®). Hence, this case lends support that graded dose challenge with somatotropin analogs may be considered for patients with isolated GH deficiency such as in the case presented here.

A Simplified Modification of the Henderson Extensor Carpi Ulnaris Opposition Transfer.

PURPOSE: There are several potential donor muscle-tendon units for a thumb opposition transfer. The extensor carpi ulnaris (ECU) is useful when the more usual donor units are not available. The technique and results of a simplified ECU opposition transfer elongated with a free tendon graft are described. METHODS: Ten ECU opposition transfers were performed using this modification of Henderson technique in 5 adults after complex trauma, 3 adults with median and ulnar nerve palsies, and 2 children with congenital hand differences. RESULTS: Seven patients achieved a Kapandji opposition score of 6 to the distal phalanx of the small finger, and 3 patients achieved a Kapandji score of 5 to the distal phalanx of the ring finger. None of the patients required a secondary tenolysis or developed a radial deviation imbalance of their wrist. CONCLUSIONS: This modification of the Henderson technique using ECU elongated with a free tendon graft and inserted directly and only into the abductor pollicis brevis tendon is an effective method of restoring opposition to the thumb, especially when other conventional donor muscle-tendon units are not available. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic V.

Drug discovery for epigenetics targets.

Dysregulation of the epigenome is associated with the onset and progression of several diseases, including cancer, autoimmune, cardiovascular, and neurological disorders. Members from the three families of epigenetic proteins (readers, writers, and erasers) have been shown to be druggable using small-molecule inhibitors. Increasing knowledge of the role of epigenetics in disease and the reversibility of these modifications explain why pharmacological intervention is an attractive strategy for tackling epigenetic-based disease. In this review, we provide an overview of epigenetics drug targets, focus on approaches used for initial hit identification, and describe the subsequent role of structure-guided chemistry optimisation of initial hits to clinical candidates. We also highlight current challenges and future potential for epigenetics-based therapies.

Diverse, Potent, and Efficacious Inhibitors That Target the EED Subunit of the Polycomb Repressive Complex 2 Methyltransferase.

Aberrant activity of the histone methyltransferase polycomb repressive complex 2 (PRC2) has been linked to several cancers, with small-molecule inhibitors of the catalytic subunit of the PRC2 enhancer of zeste homologue 2 (EZH2) being recently approved for the treatment of epithelioid sarcoma (ES) and follicular lymphoma (FL). Compounds binding to the EED subunit of PRC2 have recently emerged as allosteric inhibitors of PRC2 methyltransferase activity. In contrast to orthosteric inhibitors that target EZH2, small molecules that bind to EED retain their efficacy in EZH2 inhibitor-resistant cell lines. In this paper we disclose the discovery of potent and orally bioavailable EED ligands with good solubilities. The solubility of the EED ligands was optimized through a variety of design tactics, with the resulting compounds exhibiting in vivo efficacy in EZH2-driven tumors.

Free energy perturbation in the design of EED ligands as inhibitors of polycomb repressive complex 2 (PRC2) methyltransferase.

Free Energy Perturbation (FEP) calculations can provide high-confidence predictions of the interaction strength between a ligand and its protein target. We sought to explore a series of triazolopyrimidines which bind to the EED subunit of the PRC2 complex as potential anticancer therapeutics, using FEP calculations to inform compound design. Combining FEP predictions with a late-stage functionalisation (LSF) inspired synthetic approach allowed us to rapidly evaluate structural modifications in a previously unexplored region of the EED binding site. This approach generated a series of novel triazolopyrimidine EED ligands with improved physicochemical properties and which inhibit PRC2 methyltransferase activity in a cancer-relevant G401 cell line.

Peptides as a platform for targeted therapeutics for cancer: peptide-drug conjugates (PDCs).

Peptides can offer the versatility needed for a successful oncology drug discovery approach. Peptide-drug conjugates (PDCs) are an emerging targeted therapeutic that present increased tumour penetration and selectivity. Despite these advantages, there are still limitations for the use of peptides as therapeutics exemplified through their slow progression to get into the clinic and limited oral bioavailability. New approaches to address these problems have been studied and successfully implemented to enhance the stability of peptides and their constructs. There is great promise for the future of PDCs with two molecules already on the market and many variations currently undergoing clinical trials, such as bicycle-toxin conjugates and peptide-dendrimer conjugates. This review summarises the entire process needed for the design and successful development of an oncology PDC including chemical and nanomaterial strategies to enhance peptide stability within circulation, the function of each component of a PDC construct, and current examples in clinical trials.

Targeting PRC2 for the treatment of cancer: an updated patent review (2016 - 2020).

INTRODUCTION: PRC2 is a histone methyltransferase complex associated with several cancer types. Tazemetostat was recently approved as the first inhibitor targeting the catalytic subunit EZH2 and several other EZH2 inhibitors are now under clinical evaluation. Beyond EZH2, researchers have also explored other approaches including PRC2 activators, dual agents inhibiting both EZH1 and EZH2, allosteric inhibitors binding to EED, and compounds which induce the degradation of PRC2 constituent proteins. AREAS COVERED: This review provides an overview of anticancer therapies targeting PRC2 during the period 2016-2020 including clinical trials, patents and the scientific literature. EXPERT OPINION: The approval of tazemetostat marks the clinical validation of EZH2 for the treatment of cancer. Despite this success many questions remain; for instance, tazemetostat was briefly placed on clinical hold for safety concerns, while another EZH2 inhibitor (GSK126) demonstrated insufficient efficacy during a Phase I/II trial. It is important to understand these risks as PRC2 therapies progress through clinic evaluation. Alternative approaches to target PRC2 may offer distinct advantages over the inhibition of EZH2, including the potential to overcome EZH2 resistance mutations. However, these emerging modalities may also incur new challenges as they progress toward the clinic. Nonetheless, the diversity of agents under development represents a wealth of therapeutic options for future patients.

Discovery of AZD9833, a Potent and Orally Bioavailable Selective Estrogen Receptor Degrader and Antagonist.

Herein we report the optimization of a series of tricyclic indazoles as selective estrogen receptor degraders (SERD) and antagonists for the treatment of ER+ breast cancer. Structure based design together with systematic investigation of each region of the molecular architecture led to the identification of N-[1-(3-fluoropropyl)azetidin-3-yl]-6-[(6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl]pyridin-3-amine (28). This compound was demonstrated to be a highly potent SERD that showed a pharmacological profile comparable to fulvestrant in its ability to degrade ERα in both MCF-7 and CAMA-1 cell lines. A stringent control of lipophilicity ensured that 28 had favorable physicochemical and preclinical pharmacokinetic properties for oral administration. This, combined with demonstration of potent in vivo activity in mouse xenograft models, resulted in progression of this compound, also known as AZD9833, into clinical trials.

The kinetics of inorganic phosphate excretion in the acidotic rabbit during intravenous phosphate loading: a pseudo-ruminant model.

The rabbit is a much-used experimental animal in renal tubule physiology studies. Although a monogastric mammal, the rabbit is a known hindgut fermenter. That ruminant species excrete inorganic phosphate (Pi) mainly through the digestive system while non-ruminants eliminate surplus phosphate primarily through the renal system are acknowledged facts. To understand phosphate homeostasis in the acidotic rabbit, anaesthetized animals were infused with hydrochloric acid, after which they underwent intravenous phosphate loading. Biofluids were collected during the infusion process for analysis. Plasma Pi increased (7.9 ± 1.7 mmoles.Litre-1 (N = 5) vs 2.2 ± 0.4 mmoles.Litre-1 (N = 10) pre-infusion, (p < 0.001)), while urinary phosphate excretion was also enhanced (74.4 ± 15.3 from a control value of 4.7 ± 3 µmol.min-1 (N = 9), pre-infusion, p < 0.001)) over an 82.5 minute Pi loading period. However, the fractional excretion of Pi (FePi) only increased from 14.2 ± 5.4% to a maximum of 61.7 ± 19% (N = 5) over the infusion period. Furthermore, the renal tubular maximum reabsorption rate of phosphate to glomerular filtration rate (TmPi/GFR) computed to 3.5 mmol.L-1, while a reading of 23.2 µmol.min-1.Kg.0.75 was obtained for the transport maximum for Pi (TmPi). The high reabsorptivity of the rabbit nephrons coupled with possibly a high secretory capacity of the salivary glands for Pi, may constitute a unique physiological mechanism that ensures the rabbit hindgut receives adequate phosphate to regulate caecal pH in favour of the resident metabolically - active microbiota. The handling of Pi by the rabbit is in keeping with the description of this animal as a monogastric, pseudo-ruminant herbivore.

Demonstration of the utility of DOS-derived fragment libraries for rapid hit derivatisation in a multidirectional fashion.

Organic synthesis underpins the evolution of weak fragment hits into potent lead compounds. Deficiencies within current screening collections often result in the requirement of significant synthetic investment to enable multidirectional fragment growth, limiting the efficiency of the hit evolution process. Diversity-oriented synthesis (DOS)-derived fragment libraries are constructed in an efficient and modular fashion and thus are well-suited to address this challenge. To demonstrate the effective nature of such libraries within fragment-based drug discovery, we herein describe the screening of a 40-member DOS library against three functionally distinct biological targets using X-Ray crystallography. Firstly, we demonstrate the importance for diversity in aiding hit identification with four fragment binders resulting from these efforts. Moreover, we also exemplify the ability to readily access a library of analogues from cheap commercially available materials, which ultimately enabled the exploration of a minimum of four synthetic vectors from each molecule. In total, 10-14 analogues of each hit were rapidly accessed in three to six synthetic steps. Thus, we showcase how DOS-derived fragment libraries enable efficient hit derivatisation and can be utilised to remove the synthetic limitations encountered in early stage fragment-based drug discovery.

The Next Generation of Pattern Recognition Receptor Agonists: Improving Response Rates in Cancer Immunotherapy.

The recent success of checkpoint blocking antibodies has sparked a revolution in cancer immunotherapy. Checkpoint inhibition activates the adaptive immune system leading to durable responses across a range of tumor types, although this response is limited to patient populations with pre-existing tumor-infiltrating T cells. Strategies to stimulate the immune system to prime an antitumor response are of intense interest and several groups are now working to develop agents to activate the Pattern Recognition Receptors (PRRs), proteins which detect pathogenic and damageassociated molecules and respond by activating the innate immune response. Although early efforts focused on the Toll-like Receptor (TLR) family of membrane-bound PRRs, TLR activation has been associated with both pro- and antitumor effects. Nonetheless, TLR agonists have been deployed as potential anticancer agents in a range of clinical trials. More recently, the cytosolic PRR Stimulator of IFN Genes (STING) has attracted attention as another promising target for anticancer drug development, with early clinical data beginning to emerge. Besides STING, several other cytosolic PRR targets have likewise captured the interest of the drug discovery community, including the RIG-Ilike Receptors (RLRs) and NOD-like Receptors (NLRs). In this review, we describe the outlook for activators of PRRs as anticancer therapeutic agents and contrast the earlier generation of TLR agonists with the emerging focus on cytosolic PRR activators, both as single agents and in combination with other cancer immunotherapies.