Surveying GPCR solubilisation conditions using surface plasmon resonance.

Biophysical screening techniques, such as surface plasmon resonance, enable detailed kinetic analysis of ligands binding to solubilised G-protein coupled receptors. The activity of a receptor solubilised out of the membrane is crucially dependent on the environment in which it is suspended. Finding the right conditions is challenging due to the number of variables to investigate in order to determine the optimum solubilisation buffer for any given receptor. In this study we used surface plasmon resonance technology to screen a variety of solubilisation conditions including buffers and detergents for two model receptors: CXCR4 and CCR5. We tested 950 different combinations of solubilisation conditions for both receptors. The activity of both receptors was monitored by using conformation dependent monoclonal antibodies and the binding of small molecule ligands. Despite both receptors belonging to the chemokine receptor family they show some differences in their preference for solubilisation conditions that provide the highest level of binding for both the conformation dependent antibodies and small molecules. The study described here is focused not only on finding the best solubilisation conditions for each receptor, but also on factors that determine the sensitivity of the assay for each receptor. We also suggest how these data about different buffers and detergents can be used as a guide for selecting solubilisation conditions for other membrane proteins.

Automated design of ligands to polypharmacological profiles.

The clinical efficacy and safety of a drug is determined by its activity profile across many proteins in the proteome. However, designing drugs with a specific multi-target profile is both complex and difficult. Therefore methods to design drugs rationally a priori against profiles of several proteins would have immense value in drug discovery. Here we describe a new approach for the automated design of ligands against profiles of multiple drug targets. The method is demonstrated by the evolution of an approved acetylcholinesterase inhibitor drug into brain-penetrable ligands with either specific polypharmacology or exquisite selectivity profiles for G-protein-coupled receptors. Overall, 800 ligand-target predictions of prospectively designed ligands were tested experimentally, of which 75% were confirmed to be correct. We also demonstrate target engagement in vivo. The approach can be a useful source of drug leads when multi-target profiles are required to achieve either selectivity over other drug targets or a desired polypharmacology.

Multi-Center Retrospective Evaluation of Screw and Polymethylmethacrylate Constructs for Atlantoaxial Fixation in Dogs.

OBJECTIVE: To evaluate outcome and adverse events following ventral stabilization of the atlantoaxial (AA) joint in dogs with clinical AA subluxation using screw/polymethymethacrylate (PMMA) constructs in a retrospective, multi-center cohort study. STUDY DESIGN: Historical cohort study. ANIMALS: 35 client-owned dogs. METHODS: Medical records from 3 institutions were reviewed to identify dogs with AA subluxation treated with ventral screw and PMMA constructs. Data on signalment, pre- and postoperative neurologic status, imaging performed, and adverse events were retrieved. Neurologic examination data were abstracted to generate a modified Frankel score at admission, discharge, and re-examination. Telephone interview of owners >180 days postoperative was conducted. RESULTS: Thirty-five dogs with AA subluxation treated with ventral screw/PMMA constructs were included. Most dogs were young (median age 1 year), small breed dogs with acute onset of neurologic signs (median duration 22.5 hours). Most dogs were non-ambulatory at the time of admission (median modified Frankel score 3). Adverse events were identified in 15/35 dogs including 9 dogs with major adverse events. Four dogs required a second surgery due to vertebral canal violation (n = 2) or implant failure (n = 2). Re-examination at 4-6 weeks postoperative reported 15/28 dogs with improved neurologic status and 19/28 dogs were ambulatory. Telephone follow-up was available for 23/35 dogs with 23/23 reported as ambulatory (median follow-up 390 days). CONCLUSIONS: Ventral application of screw and PMMA constructs for AA subluxation, as described here, is associated with clinical improvement in the majority of dog. Major adverse events are infrequent and the technique is considered relatively safe.

A crowdsourcing evaluation of the NIH chemical probes.

Between 2004 and 2008, the US National Institutes of Health Molecular Libraries and Imaging initiative pilot phase funded 10 high-throughput screening centers, resulting in the deposition of 691 assays into PubChem and the nomination of 64 chemical probes. We crowdsourced the Molecular Libraries and Imaging initiative output to 11 experts, who expressed medium or high levels of confidence in 48 of these 64 probes.

Bispecific repurposed medicines targeting the viral and immunological arms of COVID-19.

Effective agents to treat coronavirus infection are urgently required, not only to treat COVID-19, but to prepare for future outbreaks. Repurposed anti-virals such as remdesivir and human anti-inflammatories such as barcitinib have received emergency approval but their overall benefits remain unclear. Vaccines are the most promising prospect for COVID-19, but will need to be redeveloped for any future coronavirus outbreak. Protecting against future outbreaks requires the identification of targets that are conserved between coronavirus strains and amenable to drug discovery. Two such targets are the main protease (Mpro) and the papain-like protease (PLpro) which are essential for the coronavirus replication cycle. We describe the discovery of two non-antiviral therapeutic agents, the caspase-1 inhibitor SDZ 224015 and Tarloxotinib that target Mpro and PLpro, respectively. These were identified through extensive experimental screens of the drug repurposing ReFRAME library of 12,000 therapeutic agents. The caspase-1 inhibitor SDZ 224015, was found to be a potent irreversible inhibitor of Mpro (IC50 30 nM) while Tarloxotinib, a clinical stage epidermal growth factor receptor inhibitor, is a sub micromolar inhibitor of PLpro (IC50 300 nM, Ki 200 nM) and is the first reported PLpro inhibitor with drug-like properties. SDZ 224015 and Tarloxotinib have both undergone safety evaluation in humans and hence are candidates for COVID-19 clinical evaluation.

Network pharmacology: the next paradigm in drug discovery.

The dominant paradigm in drug discovery is the concept of designing maximally selective ligands to act on individual drug targets. However, many effective drugs act via modulation of multiple proteins rather than single targets. Advances in systems biology are revealing a phenotypic robustness and a network structure that strongly suggests that exquisitely selective compounds, compared with multitarget drugs, may exhibit lower than desired clinical efficacy. This new appreciation of the role of polypharmacology has significant implications for tackling the two major sources of attrition in drug development--efficacy and toxicity. Integrating network biology and polypharmacology holds the promise of expanding the current opportunity space for druggable targets. However, the rational design of polypharmacology faces considerable challenges in the need for new methods to validate target combinations and optimize multiple structure-activity relationships while maintaining drug-like properties. Advances in these areas are creating the foundation of the next paradigm in drug discovery: network pharmacology.

Evaluation of treatment with a combination of azathioprine and prednisone in dogs with meningoencephalomyelitis of undetermined etiology: 40 cases (2000-2007).

OBJECTIVE: To evaluate outcome of treatment with a combination of azathioprine and prednisone in dogs with meningoencephalomyelitis of undetermined etiology (MUE). DESIGN: Retrospective case series. ANIMALS: 40 dogs. PROCEDURES: Medical records of dogs with MUE treated with prednisone and azathioprine were evaluated with regard to response, survival, and adverse effects. RESULTS: All dogs improved during treatment. Twenty-four (60%) dogs had a complete response (resolution of clinical signs), and the other 16 (40%) dogs had a partial response (improvement but not resolution of signs). Most dogs that achieved a complete response remained neurologically normal. Six dogs remained stable after a partial response. Eleven dogs had a relapse of clinical signs. Twenty dogs died during the study period, 18 survived, and 2 were lost to follow-up monitoring. Median survival time was 1,834 days (range, 50 to 2,469 days). Survival time was significantly longer for dogs that had a complete response than for those that did not. Survival time was significantly shorter for dogs that relapsed than for those that did not. The most common adverse effects included weight gain, thinning of the hair, and elevated activities of liver enzymes, all of which may have been attributed to concurrent corticosteroid administration. Less common adverse effects included diabetes mellitus, keratoconjunctivitis sicca, mammary gland adenoma, lymphoma, and hepatic masses. CONCLUSIONS AND CLINICAL RELEVANCE: Azathioprine appeared to be a safe and potentially effective adjunct to prednisone for treatment of dogs with MUE. Prospective, double-blinded, controlled studies with histologic confirmation are warranted to substantiate these findings.

Can we rationally design promiscuous drugs?

Structure-based drug design is now used widely in modern medicinal chemistry. The application of structural biology to medicinal chemistry has heralded the "rational drug design" vision of discovering exquisitely selective ligands. However, recent advances in post-genomic biology are indicating that polypharmacology may be a necessary trait for the efficacy of many drugs, therefore questioning the "one drug, one target" assumption of current rational drug design. By combining advances in chemoinformatics and structural biology, it might be possible to rationally design the next generation of promiscuous drugs with polypharmacology.

Global mapping of pharmacological space.

We present the global mapping of pharmacological space by the integration of several vast sources of medicinal chemistry structure-activity relationships (SAR) data. Our comprehensive mapping of pharmacological space enables us to identify confidently the human targets for which chemical tools and drugs have been discovered to date. The integration of SAR data from diverse sources by unique canonical chemical structure, protein sequence and disease indication enables the construction of a ligand-target matrix to explore the global relationships between chemical structure and biological targets. Using the data matrix, we are able to catalog the links between proteins in chemical space as a polypharmacology interaction network. We demonstrate that probabilistic models can be used to predict pharmacology from a large knowledge base. The relationships between proteins, chemical structures and drug-like properties provide a framework for developing a probabilistic approach to drug discovery that can be exploited to increase research productivity.

How many drug targets are there?

For the past decade, the number of molecular targets for approved drugs has been debated. Here, we reconcile apparently contradictory previous reports into a comprehensive survey, and propose a consensus number of current drug targets for all classes of approved therapeutic drugs. One striking feature is the relatively constant historical rate of target innovation (the rate at which drugs against new targets are launched); however, the rate of developing drugs against new families is significantly lower. The recent approval of drugs that target protein kinases highlights two additional trends: an emerging realization of the importance of polypharmacology, and also the power of a gene-family-led approach in generating novel and important therapies.

The druggable genome.

An assessment of the number of molecular targets that represent an opportunity for therapeutic intervention is crucial to the development of post-genomic research strategies within the pharmaceutical industry. Now that we know the size of the human genome, it is interesting to consider just how many molecular targets this opportunity represents. We start from the position that we understand the properties that are required for a good drug, and therefore must be able to understand what makes a good drug target.

Discovery of New Bromodomain Scaffolds by Biosensor Fragment Screening.

The discovery of novel bromodomain inhibitors by fragment screening is complicated by the presence of dimethyl sulfoxide (DMSO), an acetyl-lysine mimetic, that can compromise the detection of low affinity fragments. We demonstrate surface plasmon resonance as a primary fragment screening approach for the discovery of novel bromodomain scaffolds, by describing a protocol to overcome the DMSO interference issue. We describe the discovery of several novel small molecules scaffolds that inhibit the bromodomains PCAF, BRD4, and CREBBP, representing canonical members of three out of the seven subfamilies of bromodomains. High-resolution crystal structures of the complexes of key fragments binding to BRD4(1), CREBBP, and PCAF were determined to provide binding mode data to aid the development of potent and selective inhibitors of PCAF, CREBBP, and BRD4.

Surface plasmon resonance analysis of seven-transmembrane receptors.

G-protein coupled receptors (GPCRs) are the primary target class of currently marketed drugs, accounting for around a third of all drug targets of approved medicines. However, almost all the screening efforts for novel ligand discovery rely exclusively on cellular systems overexpressing the receptors. Current receptor assay systems are based on measurement of either ligand displacement or downstream functional responses, rather than direct observation of ligand binding. Issues of allosteric modulation, probe dependence, and functional selectivity create challenges in selecting suitable assay formats. Therefore, a method that directly measures GPCR-ligand interactions, independent of binding site, probe, and signaling pathway would be a useful primary and orthogonal screening method. An alternative ligand discovery strategy would be the direct measurement of GPCR-ligand interactions by label-free technologies, such as surface plasmon resonance (SPR). In this chapter, we summarize overview of the SPR technology and development of applications for detection of ligand binding to GPCRs using wild-type and thermostabilized receptors. We discuss the utilization of SPR as a biophysical screening method for fragment-based drug discovery for GPCRs. In particular, we show how SPR screening can detect both orthosteric and allosteric ligands with the appropriate experimental design.

The Joint European Compound Library: boosting precompetitive research.

The Joint European Compound Library (JECL) is a new high-throughput screening collection aimed at driving precompetitive drug discovery and target validation. The JECL has been established with a core of over 321,000 compounds from the proprietary collections of seven pharmaceutical companies and will expand to around 500,000 compounds. Here, we analyse the physicochemical profile and chemical diversity of the core collection, showing that the collection is diverse and has a broad spectrum of predicted biological activity. We also describe a model for sharing compound information from multiple proprietary collections, enabling diversity and quality analysis without disclosing structures. The JECL is available for screening at no cost to European academic laboratories and SMEs through the IMI European Lead Factory (http://www.europeanleadfactory.eu/).

Application of RNAi to Genomic Drug Target Validation in Schistosomes.

Concerns over the possibility of resistance developing to praziquantel (PZQ), has stimulated efforts to develop new drugs for schistosomiasis. In addition to the development of improved whole organism screens, the success of RNA interference (RNAi) in schistosomes offers great promise for the identification of potential drug targets to initiate drug discovery. In this study we set out to contribute to RNAi based validation of putative drug targets. Initially a list of 24 target candidates was compiled based on the identification of putative essential genes in schistosomes orthologous of C. elegans essential genes. Knockdown of Calmodulin (Smp_026560.2) (Sm-Calm), that topped this list, produced a phenotype characterised by waves of contraction in adult worms but no phenotype in schistosomula. Knockdown of the atypical Protein Kinase C (Smp_096310) (Sm-aPKC) resulted in loss of viability in both schistosomula and adults and led us to focus our attention on other kinase genes that were identified in the above list and through whole organism screening of known kinase inhibitor sets followed by chemogenomic evaluation. RNAi knockdown of these kinase genes failed to affect adult worm viability but, like Sm-aPKC, knockdown of Polo-like kinase 1, Sm-PLK1 (Smp_009600) and p38-MAPK, Sm-MAPK p38 (Smp_133020) resulted in an increased mortality of schistosomula after 2-3 weeks, an effect more marked in the presence of human red blood cells (hRBC). For Sm-PLK-1 the same effects were seen with the specific inhibitor, BI2536, which also affected viable egg production in adult worms. For Sm-PLK-1 and Sm-aPKC the in vitro effects were reflected in lower recoveries in vivo. We conclude that the use of RNAi combined with culture with hRBC is a reliable method for evaluating genes important for larval development. However, in view of the slow manifestation of the effects of Sm-aPKC knockdown in adults and the lack of effects of Sm-PLK-1 and Sm-MAPK p38 on adult viability, these kinases may not represent suitable drug targets.