Convergence of immune escape strategies highlights plasticity of SARS-CoV-2 spike.

The global spread of the SARS-CoV-2 virus has resulted in emergence of lineages which impact the effectiveness of immunotherapies and vaccines that are based on the early Wuhan isolate. All currently approved vaccines employ the spike protein S, as it is the target for neutralizing antibodies. Here we describe two SARS-CoV-2 isolates with unusually large deletions in the N-terminal domain (NTD) of the spike. Cryo-EM structural analysis shows that the deletions result in complete reshaping of the NTD supersite, an antigenically important region of the NTD. For both spike variants the remodeling of the NTD negatively affects binding of all tested NTD-specific antibodies in and outside of the NTD supersite. For one of the variants, we observed a P9L mediated shift of the signal peptide cleavage site resulting in the loss of a disulfide-bridge; a unique escape mechanism with high antigenic impact. Although the observed deletions and disulfide mutations are rare, similar modifications have become independently established in several other lineages, indicating a possibility to become more dominant in the future. The observed plasticity of the NTD foreshadows its broad potential for immune escape with the continued spread of SARS-CoV-2.

From Oxetane to Thietane: Extending the Antiviral Spectrum of 2'-Spirocyclic Uridines by Substituting Oxygen with Sulfur.

In continuation of our efforts of finding novel nucleoside inhibitors for the treatment of viral diseases, we initiated a discovery research program aimed at identifying novel nucleos(t)ide inhibitors for emerging diseases like Dengue and Chikungunya. Based on the previously reported 2'-spiro-oxetane uridine derivatives active against Hepatitis C Virus (HCV), we envisaged its sulfur analogue as an interesting congener both from a synthetic as well as biological point of view. Surprisingly, we found the 2'-spirothietane uridine derivatives not only to be active against HCV and Dengue virus (DENV), viruses belonging to the flavivirus family, but also to demonstrate activity against alphaviruses like Chikungunya virus (CHIKV) and Sindbis virus (SINV).

A Therapeutic Hepatitis B Virus DNA Vaccine Induces Specific Immune Responses in Mice and Non-Human Primates.

Despite the availability of an effective prophylactic vaccine for more than 30 years, nearly 300 million people worldwide are chronically infected with the hepatitis B virus (HBV), leading to 1 death every 30 s mainly from viral hepatitis-related cirrhosis and liver cancer. Chronic HBV patients exhibit weak, transient, or dysfunctional CD8+ T-cell responses to HBV, which contrasts with high CD8+ T-cell responses seen for resolvers of acute HBV infection. Therefore, a therapeutic DNA vaccine was designed, expressing both HBV core and polymerase proteins, and was sequence optimized to ensure high protein expression and secretion. Although the vaccine, administered intramuscularly via electroporation, had no effect on plasma viral parameters in a mouse model of persistent HBV infection, it did induce robust HBV-specific immune responses in healthy and adeno-associated hepatitis B virus (AAV-HBV) infected mice as well as in healthy non-human primates.

Tales of 1,008 small molecules: phenomic profiling through live-cell imaging in a panel of reporter cell lines.

Phenomic profiles are high-dimensional sets of readouts that can comprehensively capture the biological impact of chemical and genetic perturbations in cellular assay systems. Phenomic profiling of compound libraries can be used for compound target identification or mechanism of action (MoA) prediction and other applications in drug discovery. To devise an economical set of phenomic profiling assays, we assembled a library of 1,008 approved drugs and well-characterized tool compounds manually annotated to 218 unique MoAs, and we profiled each compound at four concentrations in live-cell, high-content imaging screens against a panel of 15 reporter cell lines, which expressed a diverse set of fluorescent organelle and pathway markers in three distinct cell lineages. For 41 of 83 testable MoAs, phenomic profiles accurately ranked the reference compounds (AUC-ROC ≥ 0.9). MoAs could be better resolved by screening compounds at multiple concentrations than by including replicates at a single concentration. Screening additional cell lineages and fluorescent markers increased the number of distinguishable MoAs but this effect quickly plateaued. There remains a substantial number of MoAs that were hard to distinguish from others under the current study's conditions. We discuss ways to close this gap, which will inform the design of future phenomic profiling efforts.

Anti-HBV activity of the HBV capsid assembly modulator JNJ-56136379 across full-length genotype A-H clinical isolates and core site-directed mutants in vitro.

OBJECTIVES: To characterize antiviral activity of the capsid assembly modulator (CAM-N) JNJ-56136379 against HBV genotypes and variants carrying amino acid substitutions in the core protein. METHODS: Anti-HBV activity of JNJ-56136379 was investigated against a diverse panel of 53 HBV clinical isolates (genotypes A-H). The impact of core amino acid substitutions using site-directed mutants (SDMs) was assessed in a transient replication assay. RESULTS: JNJ-56136379 median 50% effective concentration (EC50) values across all genotypes were 10-33 nM versus 17 nM (genotype D reference). JNJ-56136379 remained active against isolates carrying nucleos(t)ide analogue resistance mutations (median EC50 2-25 nM) or basal core promoter (BCP) ± precore (PC) mutations (median EC50 13-20 nM) or PC mutations (median EC50 11 nM), representing activity against isolates from HBeAg-positive and -negative hepatitis B patients. Core amino acid substitutions in the CAM-binding pocket, when tested as SDMs at positions 23, 25, 30, 33, 37, 106, 110, 118, 124, 127 and 128, reduced JNJ-56136379 anti-HBV activity; EC50 fold increases ranged from 3.0 (S106T) to 85 (T33N). All substitutions were rare in a public database of >7600 HBV core sequences (frequencies 0.01%-0.3%). Nucleos(t)ide analogues retained full activity against these core SDMs. CONCLUSIONS: JNJ-56136379, a potent HBV CAM-N, currently in Phase 2 clinical development, was generally fully active against an extensive panel of genotype A-H clinical isolates, regardless of the presence of nucleos(t)ide analogue resistance or BCP/PC mutations. JNJ-56136379 activity was reduced by some core amino acid substitutions in the CAM-binding pocket.

Protocols for the Design of Kinase-focused Compound Libraries.

Protocols for the design of kinase-focused compound libraries are presented. Kinase-focused compound libraries can be differentiated based on the design goal. Depending on whether the library should be a discovery library specific for one particular kinase, a general discovery library for multiple distinct kinase projects, or even phenotypic screening, there exists today a variety of in silico methods to design candidate compound libraries. We address the following scenarios: 1) Datamining of SAR databases and kinase focused vendor catalogues; 2) Predictions and virtual screening; 3) Structure-based design of combinatorial kinase inhibitors; 4) Design of covalent kinase inhibitors; 5) Design of macrocyclic kinase inhibitors; and 6) Design of allosteric kinase inhibitors and activators.

Extending kinome coverage by analysis of kinase inhibitor broad profiling data.

The explored kinome was extended with broad profiling using the DiscoveRx and Millipore assay panels. The analysis of the profiling of 3368 selected inhibitors on 456 kinases in the DiscoveRx format delivered several insights. First, the coverage depended on the threshold of the selectivity parameter. Second, the relation between hit confirmation rates and inhibitor selectivity showed unexpectedly that higher selectivity can increase the likelihood of false positives. Third, comparing the coverage of a focused to that of a random library showed that the design based on a maximum number of scaffolds was superior to a limited number of scaffolds. Therefore, selective compounds can be used in target validation, enable the jumpstarting of new kinase drug discovery projects, and chart new biological space via phenotypic screening.

The application of the open pharmacological concepts triple store (open PHACTS) to support drug discovery research.

Integration of open access, curated, high-quality information from multiple disciplines in the Life and Biomedical Sciences provides a holistic understanding of the domain. Additionally, the effective linking of diverse data sources can unearth hidden relationships and guide potential research strategies. However, given the lack of consistency between descriptors and identifiers used in different resources and the absence of a simple mechanism to link them, gathering and combining relevant, comprehensive information from diverse databases remains a challenge. The Open Pharmacological Concepts Triple Store (Open PHACTS) is an Innovative Medicines Initiative project that uses semantic web technology approaches to enable scientists to easily access and process data from multiple sources to solve real-world drug discovery problems. The project draws together sources of publicly-available pharmacological, physicochemical and biomolecular data, represents it in a stable infrastructure and provides well-defined information exploration and retrieval methods. Here, we highlight the utility of this platform in conjunction with workflow tools to solve pharmacological research questions that require interoperability between target, compound, and pathway data. Use cases presented herein cover 1) the comprehensive identification of chemical matter for a dopamine receptor drug discovery program 2) the identification of compounds active against all targets in the Epidermal growth factor receptor (ErbB) signaling pathway that have a relevance to disease and 3) the evaluation of established targets in the Vitamin D metabolism pathway to aid novel Vitamin D analogue design. The example workflows presented illustrate how the Open PHACTS Discovery Platform can be used to exploit existing knowledge and generate new hypotheses in the process of drug discovery.

Delayed bactericidal response of Mycobacterium tuberculosis to bedaquiline involves remodelling of bacterial metabolism.

Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multidrug-resistant tuberculosis in decades. Though BDQ has shown excellent efficacy in clinical trials, its early bactericidal activity during the first week of chemotherapy is minimal. Here, using microfluidic devices and time-lapse microscopy of Mycobacterium tuberculosis, we confirm the absence of significant bacteriolytic activity during the first 3-4 days of exposure to BDQ. BDQ-induced inhibition of ATP synthesis leads to bacteriostasis within hours after drug addition. Transcriptional and proteomic analyses reveal that M. tuberculosis responds to BDQ by induction of the dormancy regulon and activation of ATP-generating pathways, thereby maintaining bacterial viability during initial drug exposure. BDQ-induced bacterial killing is significantly enhanced when the mycobacteria are grown on non-fermentable energy sources such as lipids (impeding ATP synthesis via glycolysis). Our results show that BDQ exposure triggers a metabolic remodelling in mycobacteria, thereby enabling transient bacterial survival.

Nociceptive and behavioural sensitisation by protein kinase Cepsilon signalling in the CNS.

Despite the apparent homology in the protein kinase C (PKC) family, it has become clear that slight structural differences are sufficient to have unique signalling properties for each individual isoform. For PKCepsilon in depth investigation of these aspects revealed unique actions in the CNS and lead to development of specific modulators with clinical perspective. In this review, we describe to which extent PKCepsilon is distinct from other isoforms on the level of tissue expression and protein structure. As this kinase is highly expressed in the brain, we outline three main aspects of PKCepsilon signalling in the CNS. First, its ability to alter the permeability of N-type Ca2+ channels in dorsal root ganglia has been shown to enhance nociception. Secondly, PKCepsilon increases anxiety by diminishing GABA(A)R-induced inhibitory post-synaptic currents in the prefrontal cortex. Another important aspect of the latter inhibition is the reduced sensitivity of GABA(A) receptors to ethanol, a mechanism potentially contributing to abuse. A third signalling cascade improves cognitive functions by facilitating cholinergic signalling in the hippocampus. Collectively, these findings point to a physical and behavioural sensitising role for this kinase.

Downregulation of two novel genes in Sl/Sld and W(LacZ)/Wv mouse jejunum.

Interstitial cells of Cajal (ICC) are the so-called pacemaker cells of the gut. W(LacZ)/Wv and Sl/Sld mice lack ICC surrounding the myenteric plexus (MP) in the jejunum. We compared the gene expression profile of wild type (WT) and W(LacZ)/Wv and Sl/Sld mice using suppression subtractive hybridization (SSH), generating a cDNA library of 1303 clones from which 48 unique sequences were differentially expressed with Southern blot. Among them, we identified heme oxygenase2, TROY, and phospholamban in ICC using immunohistochemistry. Using RT-qPCR, c-Kit and two new transcripts Dithp and prenylcysteine oxidase1 were significantly lower expressed in Sl/Sld and W(LacZ)/Wv versus WT. Prenylcysteine oxidase1 appeared cytotoxic for COS-7 cells and was highly expressed in liver while Dithp was mainly expressed in small intestine. The combination of SSH, Southern blot, RT-qPCR, and immunohistochemistry turned out to be a useful approach to identify rarely expressed genes and genes with small differences in expression.

A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis.

The incidence of tuberculosis has been increasing substantially on a worldwide basis over the past decade, but no tuberculosis-specific drugs have been discovered in 40 years. We identified a diarylquinoline, R207910, that potently inhibits both drug-sensitive and drug-resistant Mycobacterium tuberculosis in vitro (minimum inhibitory concentration 0.06 mug/ml). In mice, R207910 exceeded the bactericidal activities of isoniazid and rifampin by at least 1 log unit. Substitution of drugs included in the World Health Organization's first-line tuberculosis treatment regimen (rifampin, isoniazid, and pyrazinamide) with R207910 accelerated bactericidal activity, leading to complete culture conversion after 2 months of treatment in some combinations. A single dose of R207910 inhibited mycobacterial growth for 1 week. Plasma levels associated with efficacy in mice were well tolerated in healthy human volunteers. Mutants selected in vitro suggest that the drug targets the proton pump of adenosine triphosphate (ATP) synthase.