Application of an NMR/Crystallography Fragment Screening Platform for the Assessment and Rapid Discovery of New HIV-CA Binding Fragments.

Identification and assessment of novel targets is essential to combat drug resistance in the treatment of HIV/AIDS. HIV Capsid (HIV-CA), the protein playing a major role in both the early and late stages of the viral life cycle, has emerged as an important target. We have applied an NMR fragment screening platform and identified molecules that bind to the N-terminal domain (NTD) of HIV-CA at a site close to the interface with the C-terminal domain (CTD). Using X-ray crystallography, we have been able to obtain crystal structures to identify the binding mode of these compounds. This allowed for rapid progression of the initial, weak binding, fragment starting points to compounds 37 and 38, which have 19F-pKi values of 5.3 and 5.4 respectively.

Correction: Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies.

[This corrects the article DOI: 10.1371/journal.pone.0253364.].

Encouraging Student Attendance and Engagement in Lectures & Workshops in the Pre- and Post-Covid World.

In the post-Covid era, second year chemistry lectures are fully flipped with all content being online. All the live lecture sessions are used for group work and are fully interactive. Students have agency in the lectures by directing what is taught in these student-led sessions. Students find the sessions very engaging and respond positively. In particular they value the agency they are given. In a second study that took place pre-Covid, workshops are changed from 1-hour to 2-hour sessions but with half the number and a much simplified timetable for students. Group work and peer-assessment with marking criteria help make the sessions engaging for students and more useful. The increased level of attendance from less than 20% to more than 70% (in the best case) is evidence of increased value to the students and success of the new format.

Design of the Global Health chemical diversity library v2 for screening against infectious diseases.

There is a need for novel chemical matter for phenotypic and target-based screens to find starting points for drug discovery programmes in neglected infectious diseases and non-hormonal contraceptives that disproportionately affect Low- and Middle-Income Countries (LMICs). In some disease areas multiple screens of corporate and other libraries have been carried out, giving rise to some valuable starting points and leading to preclinical candidates. Whilst in other disease areas, little screening has been carried out. Much screening against pathogens has been conducted phenotypically as there are few robustly validated protein targets. However, many of the active compound series identified share the same molecular targets. To address the need for new chemical material, in this article we describe the design of a new library, designed for screening in drug discovery programmes for neglected infectious diseases. The compounds have been selected from the Enamine REAL (REadily AccessibLe) library, a virtual library which contains approximately 4.5 billion molecules. The molecules theoretically can be synthesized quickly using commercially available intermediates and building blocks. The vast majority of these have not been prepared before, so this is a source of novel compounds. In this paper we describe the design of a diverse library of 30,000 compounds from this collection (graphical abstract). The new library will be made available to laboratories working in neglected infectious diseases, subject to a review process. The project has been supported by the Bill & Melinda Gates Foundation and the Wellcome Trust (Wellcome).

Correction to "Bespoke Drug Discovery Training for Low-Middle Income Countries".

[This corrects the article DOI: 10.1021/acsmedchemlett.3c00215.].

Identification and Optimization of Novel Inhibitors of the Polyketide Synthase 13 Thioesterase Domain with Antitubercular Activity.

There is an urgent need for new tuberculosis (TB) treatments, with novel modes of action, to reduce the incidence/mortality of TB and to combat resistance to current treatments. Through both chemical and genetic methodologies, polyketide synthase 13 (Pks13) has been validated as essential for mycobacterial survival and as an attractive target for Mycobacterium tuberculosis growth inhibitors. A benzofuran series of inhibitors that targeted the Pks13 thioesterase domain, failed to progress to preclinical development due to concerns over cardiotoxicity. Herein, we report the identification of a novel oxadiazole series of Pks13 inhibitors, derived from a high-throughput screening hit and structure-guided optimization. This new series binds in the Pks13 thioesterase domain, with a distinct binding mode compared to the benzofuran series. Through iterative rounds of design, assisted by structural information, lead compounds were identified with improved antitubercular potencies (MIC < 1 µM) and in vitro ADMET profiles.

Development of a 2,4-Diaminothiazole Series for the Treatment of Human African Trypanosomiasis Highlights the Importance of Static-Cidal Screening of Analogues.

While treatment options for human African trypanosomiasis (HAT) have improved significantly, there is still a need for new drugs with eradication now a realistic possibility. Here, we report the development of 2,4-diaminothiazoles that demonstrate significant potency against Trypanosoma brucei, the causative agent of HAT. Using phenotypic screening to guide structure-activity relationships, potent drug-like inhibitors were developed. Proof of concept was established in an animal model of the hemolymphatic stage of HAT. To treat the meningoencephalitic stage of infection, compounds were optimized for pharmacokinetic properties, including blood-brain barrier penetration. However, in vivo efficacy was not achieved, in part due to compounds evolving from a cytocidal to a cytostatic mechanism of action. Subsequent studies identified a nonessential kinase involved in the inositol biosynthesis pathway as the molecular target of these cytostatic compounds. These studies highlight the need for cytocidal drugs for the treatment of HAT and the importance of static-cidal screening of analogues.

Lysyl-tRNA synthetase, a target for urgently needed M. tuberculosis drugs.

Tuberculosis is a major global cause of both mortality and financial burden mainly in low and middle-income countries. Given the significant and ongoing rise of drug-resistant strains of Mycobacterium tuberculosis within the clinical setting, there is an urgent need for the development of new, safe and effective treatments. Here the development of a drug-like series based on a fused dihydropyrrolidino-pyrimidine scaffold is described. The series has been developed against M. tuberculosis lysyl-tRNA synthetase (LysRS) and cellular studies support this mechanism of action. DDD02049209, the lead compound, is efficacious in mouse models of acute and chronic tuberculosis and has suitable physicochemical, pharmacokinetic properties and an in vitro safety profile that supports further development. Importantly, preliminary analysis using clinical resistant strains shows no pre-existing clinical resistance towards this scaffold.

Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity.

During RNA replication, coronaviruses require proofreading to maintain the integrity of their large genomes. Nsp14 associates with viral polymerase complex to excise the mismatched nucleotides. Aside from the exonuclease activity, nsp14 methyltransferase domain mediates cap methylation, facilitating translation initiation and protecting viral RNA from recognition by the innate immune sensors. The nsp14 exonuclease activity is modulated by a protein co-factor nsp10. While the nsp10/nsp14 complex structure is available, the mechanistic basis for nsp10-mediated modulation remains unclear in the absence of the nsp14 structure. Here, we provide a crystal structure of nsp14 in an apo-form. Comparative analysis of the apo- and nsp10-bound structures explain the modulatory role of the co-factor protein and reveal the allosteric nsp14 control mechanism essential for drug discovery. Further, the flexibility of the N-terminal lid of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp14 structure presented in this study rationalizes the recently proposed idea of nsp14/nsp10/nsp16 ternary complex.

Identification and development of a series of disubstituted piperazines for the treatment of Chagas disease.

Approximately 6-7 million people around the world are estimated to be infected with Trypanosoma cruzi, the causative agent of Chagas disease. The current treatments are inadequate and therefore new medical interventions are urgently needed. In this paper we describe the identification of a series of disubstituted piperazines which shows good potency against the target parasite but is hampered by poor metabolic stability. We outline the strategies used to mitigate this issue such as lowering logD, bioisosteric replacements of the metabolically labile piperazine ring and use of plate-based arrays for quick diversity scoping. We discuss the success of these strategies within the context of this series and highlight the challenges faced in phenotypic programs when attempting to improve the pharmacokinetic profile of compounds whilst maintaining potency against the desired target.

Optimization of TAM16, a Benzofuran That Inhibits the Thioesterase Activity of Pks13; Evaluation toward a Preclinical Candidate for a Novel Antituberculosis Clinical Target.

With increasing drug resistance in tuberculosis (TB) patient populations, there is an urgent need for new drugs. Ideally, new agents should work through novel targets so that they are unencumbered by preexisting clinical resistance to current treatments. Benzofuran 1 was identified as a potential lead for TB inhibiting a novel target, the thioesterase domain of Pks13. Although, having promising activity against Mycobacterium tuberculosis, its main liability was inhibition of the hERG cardiac ion channel. This article describes the optimization of the series toward a preclinical candidate. Despite improvements in the hERG liability in vitro, when new compounds were assessed in ex vivo cardiotoxicity models, they still induced cardiac irregularities. Further series development was stopped because of concerns around an insufficient safety window. However, the demonstration of in vivo activity for multiple series members further validates Pks13 as an attractive novel target for antitubercular drugs and supports development of alternative chemotypes.

Ligand binding: evaluating the contribution of the water molecules network using the Fragment Molecular Orbital method.

Water molecules play a crucial role in protein-ligand binding, and many tools exist that aim to predict the position and relative energies of these important, but challenging participants of biomolecular recognition. The available tools are, in general, capable of predicting the location of water molecules. However, predicting the effects of their displacement is still very challenging. In this work, a linear-scaling quantum mechanics-based approach was used to assess water network energetics and the changes in network stability upon ligand structural modifications. This approach offers a valuable way to improve understanding of SAR data and help guide compound design.

Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies.

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

Targeting Mycobacterium tuberculosis CoaBC through Chemical Inhibition of 4'-Phosphopantothenoyl-l-cysteine Synthetase (CoaB) Activity.

Coenzyme A (CoA) is a ubiquitous cofactor present in all living cells and estimated to be required for up to 9% of intracellular enzymatic reactions. Mycobacterium tuberculosis (Mtb) relies on its own ability to biosynthesize CoA to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the pathway to CoA biosynthesis is recognized as a potential source of novel tuberculosis drug targets. In prior work, we genetically validated CoaBC as a bactericidal drug target in Mtb in vitro and in vivo. Here, we describe the identification of compound 1f, a small molecule inhibitor of the 4'-phosphopantothenoyl-l-cysteine synthetase (PPCS; CoaB) domain of the bifunctional Mtb CoaBC, and show that this compound displays on-target activity in Mtb. Compound 1f was found to inhibit CoaBC uncompetitively with respect to 4'-phosphopantothenate, the substrate for the CoaB-catalyzed reaction. Furthermore, metabolomic profiling of wild-type Mtb H37Rv following exposure to compound 1f produced a signature consistent with perturbations in pantothenate and CoA biosynthesis. As the first report of a direct small molecule inhibitor of Mtb CoaBC displaying target-selective whole-cell activity, this study confirms the druggability of CoaBC and chemically validates this target.

Scaffold-Hopping Strategy on a Series of Proteasome Inhibitors Led to a Preclinical Candidate for the Treatment of Visceral Leishmaniasis.

There is an urgent need for new treatments for visceral leishmaniasis (VL), a parasitic infection which impacts heavily large areas of East Africa, Asia, and South America. We previously reported on the discovery of GSK3494245/DDD01305143 (1) as a preclinical candidate for VL and, herein, we report on the medicinal chemistry program that led to its identification. A hit from a phenotypic screen was optimized to give a compound with in vivo efficacy, which was hampered by poor solubility and genotoxicity. The work on the original scaffold failed to lead to developable compounds, so an extensive scaffold-hopping exercise involving medicinal chemistry design, in silico profiling, and subsequent synthesis was utilized, leading to the preclinical candidate. The compound was shown to act via proteasome inhibition, and we report on the modeling of different scaffolds into a cryo-EM structure and the impact this has on our understanding of the series' structure-activity relationships.

Resistance of Mycobacterium tuberculosis to indole 4-carboxamides occurs through alterations in drug metabolism and tryptophan biosynthesis.

Tryptophan biosynthesis represents an important potential drug target for new anti-TB drugs. We identified a series of indole-4-carboxamides with potent antitubercular activity. In vitro, Mycobacterium tuberculosis (Mtb) acquired resistance to these compounds through three discrete mechanisms: (1) a decrease in drug metabolism via loss-of-function mutations in the amidase that hydrolyses these carboxamides, (2) an increased biosynthetic rate of tryptophan precursors via loss of allosteric feedback inhibition of anthranilate synthase (TrpE), and (3) mutation of tryptophan synthase (TrpAB) that decreased incorporation of 4-aminoindole into 4-aminotryptophan. Thus, these indole-4-carboxamides act as prodrugs of a tryptophan antimetabolite, 4-aminoindole.

Active Learning: From Flipped Lectures to the Covid-19 Era.

The use of flipped lectures and Active Learning has been explored in first year lectures for several years. Learning Catalytics software has been used to facilitate Peer-2-Peer learning in the Active Learning sessions. Several lessons were learned during these sessions which were important as we moved into delivering online material much more comprehensively. Bringing interaction to the lectures that were delivered live (synchronous sessions) was important to staff. Several technical solutions for producing online videos of high quality were brought into broader use. New teaching formats including a 'radio show' have been introduced to ensure that rich interaction with the students is possible with large groups.

Spirocycle MmpL3 Inhibitors with Improved hERG and Cytotoxicity Profiles as Inhibitors of Mycobacterium tuberculosis Growth.

With the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, there is a pressing need for new oral drugs with novel mechanisms of action. A number of scaffolds with potent anti-tubercular in vitro activity have been identified from phenotypic screening that appear to target MmpL3. However, the scaffolds are typically lipophilic, which facilitates partitioning into hydrophobic membranes, and several contain basic amine groups. Highly lipophilic basic amines are typically cytotoxic against mammalian cell lines and have associated off-target risks, such as inhibition of human ether-à-go-go related gene (hERG) and IKr potassium current modulation. The spirocycle compound 3 was reported to target MmpL3 and displayed promising efficacy in a murine model of acute tuberculosis (TB) infection. However, this highly lipophilic monobasic amine was cytotoxic and inhibited the hERG ion channel. Herein, the related spirocycles (1-2) are described, which were identified following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis. The novel N-alkylated pyrazole portion offered improved physicochemical properties, and optimization led to identification of a zwitterion series, exemplified by lead 29, with decreased HepG2 cytotoxicity as well as limited hERG ion channel inhibition. Strains with mutations in MmpL3 were resistant to 29, and under replicating conditions, 29 demonstrated bactericidal activity against M. tuberculosis. Unfortunately, compound 29 had no efficacy in an acute model of TB infection; this was most likely due to the in vivo exposure remaining above the minimal inhibitory concentration for only a limited time.