Deletion of endocannabinoid synthesizing enzyme DAGLα from cerebellar Purkinje cells decreases social preference and elevates anxiety.

The endocannabinoid (eCB) signaling system is robustly expressed in the cerebellum starting from the embryonic developmental stages to adulthood. There it plays a key role in regulating cerebellar synaptic plasticity and excitability, suggesting that impaired eCB signaling will lead to deficits in cerebellar adjustments of ongoing behaviors and cerebellar learning. Indeed, human mutations in DAGLα are associated with neurodevelopmental disorders. In this study, we show that selective deletion of the eCB synthesizing enzyme diacylglycerol lipase alpha (Daglα) from mouse cerebellar Purkinje cells (PCs) alters motor and social behaviors, disrupts short-term synaptic plasticity in both excitatory and inhibitory synapses, and reduces Purkinje cell activity during social exploration. Our results provide the first evidence for cerebellar-specific eCB regulation of social behaviors and implicate eCB regulation of synaptic plasticity and PC activity as the neural substrates contributing to these deficits.

Mapping protein binding sites by photoreactive fragment pharmacophores.

Fragment screening is a popular strategy of generating viable chemical starting points especially for challenging targets. Although fragments provide a better coverage of chemical space and they have typically higher chance of binding, their weak affinity necessitates highly sensitive biophysical assays. Here, we introduce a screening concept that combines evolutionary optimized fragment pharmacophores with the use of a photoaffinity handle that enables high hit rates by LC-MS-based detection. The sensitivity of our screening protocol was further improved by a target-conjugated photocatalyst. We have designed, synthesized, and screened 100 diazirine-tagged fragments against three benchmark and three therapeutically relevant protein targets of different tractability. Our therapeutic targets included a conventional enzyme, the first bromodomain of BRD4, a protein-protein interaction represented by the oncogenic KRasG12D protein, and the yet unliganded N-terminal domain of the STAT5B transcription factor. We have discovered several fragment hits against all three targets and identified their binding sites via enzymatic digestion, structural studies and modeling. Our results revealed that this protocol outperforms screening traditional fully functionalized and photoaffinity fragments in better exploration of the available binding sites and higher hit rates observed for even difficult targets.

The impact of SARS-CoV-2 spike mutation on peptide presentation is HLA allomorph-specific.

CD8+ T cells are crucial for viral elimination and recovery from viral infection. Nonetheless, the current understanding of the T cell response to SARS-CoV-2 at the antigen level remains limited. The Spike protein is an external structural protein that is prone to mutations, threatening the efficacy of current vaccines. Therefore, we have characterised the immune response towards the immunogenic Spike-derived peptide (S976-984, VLNDILSRL), restricted to the HLA-A*02:01 molecule, which is mutated in both Alpha (S982A) and Omicron BA.1 (L981F) variants of concern. We determined that the mutation in the Alpha variant (S982A) impacted both the stability and conformation of the peptide, bound to HLA-A*02:01, in comparison to the original S976-984. We identified a longer and overlapping immunogenic peptide (S975-984, SVLNDILSRL) that could be presented by HLA-A*02:01, HLA-A*11:01 and HLA-B*13:01 allomorphs. We showed that S975-specific CD8+ T cells were weakly cross-reactive to the mutant peptides despite their similar conformations when presented by HLA-A*11:01. Altogether, our results show that the impact of SARS-CoV-2 mutations on peptide presentation is HLA allomorph-specific, and that post vaccination there are T cells able to react and cross-react towards the variant of concern peptides.

Characterisation of novel influenza-derived HLA-B*18:01-restricted epitopes.

Objectives: Seasonal influenza viruses cause roughly 650 000 deaths annually despite available vaccines. CD8+ T cells typically recognise influenza-derived peptides from internal structural and non-structural influenza proteins and are an attractive avenue for future vaccine design as they could reduce the severity of disease following infection with diverse influenza strains. CD8+ T cells recognise peptides presented by the highly polymorphic Human Leukocyte Antigens class I molecules (HLA-I). Each HLA-I variant has distinct peptide binding preferences, representing a significant obstacle for designing vaccines that elicit CD8+ T cell responses across broad populations. Consequently, the rational design of a CD8+ T cell-mediated vaccine would require the identification of highly immunogenic peptides restricted to a range of different HLA molecules. Methods: Here, we assessed the immunogenicity of six recently published novel influenza-derived peptides identified by mass-spectrometry and predicted to bind to the prevalent HLA-B*18:01 molecule. Results: Using CD8+ T cell activation assays and protein biochemistry, we showed that 3/6 of the novel peptides were immunogenic in several HLA-B*18:01+ individuals and confirmed their HLA-B*18:01 restriction. We subsequently compared CD8+ T cell responses towards the previously identified highly immunogenic HLA-B*18:01-restricted NP219 peptide. Using X-ray crystallography, we solved the first crystal structures of HLA-B*18:01 presenting immunogenic influenza-derived peptides. Finally, we dissected the first TCR repertoires specific for HLA-B*18:01 restricted pathogen-derived peptides, identifying private and restricted repertoires against each of the four peptides. Conclusion: Overall the characterisation of these novel immunogenic peptides provides additional HLA-B*18:01-restricted vaccine targets derived from the Matrix protein 1 and potentially the non-structural protein and the RNA polymerase catalytic subunit of influenza viruses.

Novel WRN Helicase Inhibitors Selectively Target Microsatellite-Unstable Cancer Cells.

Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumors, and WRN inhibitors are in development. In this study, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth in vitro and in vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic lethal targeting of WRN in MSI cancer and tools to dissect WRN biology. Significance: We report the discovery and characterization of potent, selective WRN helicase inhibitors for MSI cancer treatment, with biomarker analysis and evaluation of efficacy in vivo and in immunotherapy-refractory preclinical models. These findings pave the way to translate WRN inhibition into MSI cancer therapies and provide tools to investigate WRN biology. See related commentary by Wainberg, p. 1369.

Fighting flu: novel CD8+ T-cell targets are required for future influenza vaccines.

Seasonal influenza viruses continue to cause severe medical and financial complications annually. Although there are many licenced influenza vaccines, there are billions of cases of influenza infection every year, resulting in the death of over half a million individuals. Furthermore, these figures can rise in the event of a pandemic, as seen throughout history, like the 1918 Spanish influenza pandemic (50 million deaths) and the 1968 Hong Kong influenza pandemic (~4 million deaths). In this review, we have summarised many of the currently licenced influenza vaccines available across the world and current vaccines in clinical trials. We then briefly discuss the important role of CD8+ T cells during influenza infection and why future influenza vaccines should consider targeting CD8+ T cells. Finally, we assess the current landscape of known immunogenic CD8+ T-cell epitopes and highlight the knowledge gaps required to be filled for the design of rational future influenza vaccines that incorporate CD8+ T cells.

Increasing HbA1c is associated with reduced CD8+ T cell functionality in response to influenza virus in a TCR-dependent manner in individuals with diabetes mellitus.

Diabetes mellitus is on the rise globally and is a known susceptibility factor for severe influenza virus infections. However, the mechanisms by which diabetes increases the severity of an influenza virus infection are yet to be fully defined. Diabetes mellitus is hallmarked by high glucose concentrations in the blood. We hypothesized that these high glucose concentrations affect the functionality of CD8+ T cells, which play a key role eliminating virus-infected cells and have been shown to decrease influenza disease severity. To study the effect of hyperglycemia on CD8+ T cell function, we stimulated peripheral blood mononuclear cells (PBMCs) from donors with and without diabetes with influenza A virus, anti-CD3/anti-CD28-coated beads, PMA and ionomycin (PMA/I), or an influenza viral peptide pool. After stimulation, cells were assessed for functionality [as defined by expression of IFN-γ, TNF-α, macrophage inflammatory protein (MIP)-1ß, and lysosomal-associated membrane protein-1 (CD107a)] using flow cytometry. Our results showed that increasing HbA1c correlated with a reduction in TNF-α production by CD8+ T cells in response to influenza stimulation in a TCR-specific manner. This was not associated with any changes to CD8+ T cell subsets. We conclude that hyperglycemia impairs CD8+ T cell function to influenza virus infection, which may be linked with the increased risk of severe influenza in patients with diabetes.

Covalent targeting of non-cysteine residues in PI4KIIIß.

The synthesis and characterisation of fluorosulfate covalent inhibitors of the lipid kinase PI4KIIIß is described. The conserved lysine residue located within the ATP binding site was targeted, and optimised compounds based upon reversible inhibitors with good activity and physicochemical profile showed strong reversible interactions and slow onset times for the covalent inhibition, resulting in an excellent selectivity profile for the lipid kinase target. X-Ray crystallography demonstrated a distal tyrosine residue could also be targeted using a fluorosulfate strategy. Combination of this knowledge showed that a dual covalent inhibitor could be developed which reveals potential in addressing the challenges associated with drug resistant mutations.

Mutate and Conjugate: A Method to Enable Rapid In-Cell Target Validation.

Target validation remains a challenge in drug discovery, which leads to a high attrition rate in the drug discovery process, particularly in Phase II clinical trials. Consequently, new approaches to enhance target validation are valuable tools to improve the drug discovery process. Here, we report the combination of site-directed mutagenesis and electrophilic fragments to enable the rapid identification of small molecules that selectively inhibit the mutant protein. Using the bromodomain-containing protein BRD4 as an example, we employed a structure-based approach to identify the L94C mutation in the first bromodomain of BRD4 [BRD4(1)] as having a minimal effect on BRD4(1) function. We then screened a focused, KAc mimic-containing fragment set and a diverse fragment library against the mutant and wild-type proteins and identified a series of fragments that showed high selectivity for the mutant protein. These compounds were elaborated to include an alkyne click tag to enable the attachment of a fluorescent dye. These clickable compounds were then assessed in HEK293T cells, transiently expressing BRD4(1)WT or BRD4(1)L94C, to determine their selectivity for BRD4(1)L94C over other possible cellular targets. One compound was identified that shows very high selectivity for BRD4(1)L94C over all other proteins. This work provides a proof-of-concept that the combination of site-directed mutagenesis and electrophilic fragments, in a mutate and conjugate approach, can enable rapid identification of small molecule inhibitors for an appropriately mutated protein of interest. This technology can be used to assess the cellular phenotype of inhibiting the protein of interest, and the electrophilic ligand provides a starting point for noncovalent ligand development.

Reactive fragments targeting carboxylate residues employing direct to biology, high-throughput chemistry.

The screening of covalent or 'reactive' fragment libraries against proteins is becoming an integral approach in hit identification, enabling the development of targeted covalent inhibitors and tools. To date, reactive fragment screening has been limited to targeting cysteine residues, thus restricting applicability across the proteome. Carboxylate residues present a unique opportunity to expand the accessible residues due to high proteome occurrence (∼12%). Herein, we present the development of a carboxylate-targeting reactive fragment screening platform utilising 2-aryl-5-carboxytetrazole (ACT) as the photoreactive functionality. The utility of ACT photoreactive fragments (ACT-PhABits) was evaluated by screening a 546-membered library with a small panel of purified proteins. Hits identified for BCL6 and KRASG12D were characterised by LC-MS/MS studies, revealing the selectivity of the ACT group. Finally, a photosensitised approach to ACT activation was developed, obviating the need for high energy UV-B light.

Determination of Intracellular Esterase Activity Using Ratiometric Raman Sensing and Spectral Phasor Analysis.

Carboxylesterases (CEs) are a class of enzymes that catalyze the hydrolysis of esters in a variety of endogenous and exogenous molecules. CEs play an important role in drug metabolism, in the onset and progression of disease, and can be harnessed for prodrug activation strategies. As such, the regulation of CEs is an important clinical and pharmaceutical consideration. Here, we report the first ratiometric sensor for CE activity using Raman spectroscopy based on a bisarylbutadiyne scaffold. The sensor was shown to be highly sensitive and specific for CE detection and had low cellular cytotoxicity. In hepatocyte cells, the ratiometric detection of esterase activity was possible, and the result was validated by multimodal imaging with standard viability stains used for fluorescence microscopy within the same cell population. In addition, we show that the detection of localized ultraviolet damage in a mixed cell population was possible using stimulated Raman scattering microscopy coupled with spectral phasor analysis. This sensor demonstrates the practical advantages of low molecular weight sensors that are detected using ratiometric Raman imaging and will have applications in drug discovery and biomedical research.

Profiling Sulfur(VI) Fluorides as Reactive Functionalities for Chemical Biology Tools and Expansion of the Ligandable Proteome.

Here, we report a comprehensive profiling of sulfur(VI) fluorides (SVI-Fs) as reactive groups for chemical biology applications. SVI-Fs are reactive functionalities that modify lysine, tyrosine, histidine, and serine sidechains. A panel of SVI-Fs were studied with respect to hydrolytic stability and reactivity with nucleophilic amino acid sidechains. The use of SVI-Fs to covalently modify carbonic anhydrase II (CAII) and a range of kinases was then investigated. Finally, the SVI-F panel was used in live cell chemoproteomic workflows, identifying novel protein targets based on the type of SVI-F used. This work highlights how SVI-F reactivity can be used as a tool to expand the liganded proteome.

Homologous peptides derived from influenza A, B and C viruses induce variable CD8+ T cell responses with cross-reactive potential.

Objective: Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally, infecting humans and causing widespread morbidity and mortality. Here, we investigate the T cell response towards an immunodominant IAV epitope, NP265-273, and its IBV and ICV homologues, presented by HLA-A*03:01 molecule expressed in ~ 4% of the global population (~ 300 million people). Methods: We assessed the magnitude (tetramer staining) and quality of the CD8+ T cell response (intracellular cytokine staining) towards NP265-IAV and described the T cell receptor (TCR) repertoire used to recognise this immunodominant epitope. We next assessed the immunogenicity of NP265-IAV homologue peptides from IBV and ICV and the ability of CD8+ T cells to cross-react towards these homologous peptides. Furthermore, we determined the structures of NP265-IAV and NP323-IBV peptides in complex with HLA-A*03:01 by X-ray crystallography. Results: Our study provides a detailed characterisation of the CD8+ T cell response towards NP265-IAV and its IBV and ICV homologues. The data revealed a diverse repertoire for NP265-IAV that is associated with superior anti-viral protection. Evidence of cross-reactivity between the three different influenza virus strain-derived epitopes was observed, indicating the discovery of a potential vaccination target that is broad enough to cover all three influenza strains. Conclusion: We show that while there is a potential to cross-protect against distinct influenza virus lineages, the T cell response was stronger against the IAV peptide than IBV or ICV, which is an important consideration when choosing targets for future vaccine design.

Protocol for generation of human peptide-specific primary CD8+ T cell lines.

Directly ex vivo, peptide-specific CD8+ T cells are present at relatively low frequency and are typically in a resting state. This protocol details the expansion of memory peptide-specific CD8+ T cells by in vitro stimulation, which can be subsequently characterized using a range of assays including tetramer staining and intracellular cytokine staining. For complete details on the use and execution of this protocol, please refer to Lineburg et al. (2021).

Progesterone receptor expression contributes to gemcitabine resistance at higher ECM stiffness in breast cancer cell lines.

Chemoresistance poses a great barrier to breast cancer treatment and is thought to correlate with increased matrix stiffness. We developed two-dimensional (2D) polyacrylamide (PAA) and three-dimensional (3D) alginate in vitro models of tissue stiffness that mimic the stiffness of normal breast and breast cancer. We then used these to compare cell viability in response to chemotherapeutic treatment. In both 2D and 3D we observed that breast cancer cell growth and size was increased at a higher stiffness corresponding to tumours compared to normal tissue. When chemotherapeutic response was measured, a specific differential response in cell viability was observed for gemcitabine in 2 of the 7 breast cancer cell lines investigated. MCF7 and T-47D cell lines showed gemcitabine resistance at 4 kPa compared to 500 Pa. These cell lines share a common phenotype of progesterone receptor (PGR) expression and, indeed, pre-treatment with the selective progesterone receptor modulator (SPRM) mifepristone abolished resistance to gemcitabine at high stiffness. Our data reveals that combined treatment with SPRMs may therefore help in reducing resistance to gemcitabine in stiffer breast tumours which are PGR positive.

Limited Recognition of Highly Conserved Regions of SARS-CoV-2.

Understanding the immune response to severe acute respiratory syndrome coronavirus (SARS-CoV-2) is critical to overcome the current coronavirus disease (COVID-19) pandemic. Efforts are being made to understand the potential cross-protective immunity of memory T cells, induced by prior encounters with seasonal coronaviruses, in providing protection against severe COVID-19. In this study we assessed T-cell responses directed against highly conserved regions of SARS-CoV-2. Epitope mapping revealed 16 CD8+ T-cell epitopes across the nucleocapsid (N), spike (S), and open reading frame (ORF)3a proteins of SARS-CoV-2 and five CD8+ T-cell epitopes encoded within the highly conserved regions of the ORF1ab polyprotein of SARS-CoV-2. Comparative sequence analysis showed high conservation of SARS-CoV-2 ORF1ab T-cell epitopes in seasonal coronaviruses. Paradoxically, the immune responses directed against the conserved ORF1ab epitopes were infrequent and subdominant in both convalescent and unexposed participants. This subdominant immune response was consistent with a low abundance of ORF1ab encoded proteins in SARS-CoV-2 infected cells. Overall, these observations suggest that while cross-reactive CD8+ T cells likely exist in unexposed individuals, they are not common and therefore are unlikely to play a significant role in providing broad preexisting immunity in the community. IMPORTANCE T cells play a critical role in protection against SARS-CoV-2. Despite being highly topical, the protective role of preexisting memory CD8+ T cells, induced by prior exposure to circulating common coronavirus strains, remains less clear. In this study, we established a robust approach to specifically assess T cell responses to highly conserved regions within SARS-CoV-2. Consistent with recent observations we demonstrate that recognition of these highly conserved regions is associated with an increased likelihood of milder disease. However, extending these observations we observed that recognition of these conserved regions is rare in both exposed and unexposed volunteers, which we believe is associated with the low abundance of these proteins in SARS-CoV-2 infected cells. These observations have important implications for the likely role preexisting immunity plays in controlling severe disease, further emphasizing the importance of vaccination to generate the immunodominant T cells required for immune protection.

A direct-to-biology high-throughput chemistry approach to reactive fragment screening.

Methods for rapid identification of chemical tools are essential for the validation of emerging targets and to provide medicinal chemistry starting points for the development of new medicines. Here, we report a screening platform that combines 'direct-to-biology' high-throughput chemistry (D2B-HTC) with photoreactive fragments. The platform enabled the rapid synthesis of >1000 PhotoAffinity Bits (HTC-PhABits) in 384-well plates in 24 h and their subsequent screening as crude reaction products with a protein target without purification. Screening the HTC-PhABit library with carbonic anhydrase I (CAI) afforded 7 hits (0.7% hit rate), which were found to covalently crosslink in the Zn2+ binding pocket. A powerful advantage of the D2B-HTC screening platform is the ability to rapidly perform iterative design-make-test cycles, accelerating the development and optimisation of chemical tools and medicinal chemistry starting points with little investment of resource.

Molecular Basis of a Dominant SARS-CoV-2 Spike-Derived Epitope Presented by HLA-A*02:01 Recognised by a Public TCR.

The data currently available on how the immune system recognises the SARS-CoV-2 virus is growing rapidly. While there are structures of some SARS-CoV-2 proteins in complex with antibodies, which helps us understand how the immune system is able to recognise this new virus; however, we lack data on how T cells are able to recognise this virus. T cells, especially the cytotoxic CD8+ T cells, are critical for viral recognition and clearance. Here we report the X-ray crystallography structure of a T cell receptor, shared among unrelated individuals (public TCR) in complex with a dominant spike-derived CD8+ T cell epitope (YLQ peptide). We show that YLQ activates a polyfunctional CD8+ T cell response in COVID-19 recovered patients. We detail the molecular basis for the shared TCR gene usage observed in HLA-A*02:01+ individuals, providing an understanding of TCR recognition towards a SARS-CoV-2 epitope. Interestingly, the YLQ peptide conformation did not change upon TCR binding, facilitating the high-affinity interaction observed.

The role of T-cell immunity in COVID-19 severity amongst people living with type II diabetes.

The COVID-19 pandemic has highlighted the vulnerability of people with diabetes mellitus (DM) to respiratory viral infections. Despite the short history of COVID-19, various studies have shown that patients with DM are more likely to have increased hospitalisation and mortality rates as compared to patients without. At present, the mechanisms underlying this susceptibility are unclear. However, prior studies show that the course of COVID-19 disease is linked to the efficacy of the host's T-cell responses. Healthy individuals who can elicit a robust T-cell response are more likely to limit the severity of COVID-19. Here, we investigate the hypothesis that an impaired T-cell response in patients with type 2 diabetes mellitus (T2DM) drives the severity of COVID-19 in this patient population. While there is currently a limited amount of information that specifically addresses T-cell responses in COVID-19 patients with T2DM, there is a wealth of evidence from other infectious diseases that T-cell immunity is impaired in patients with T2DM. The reasons for this are likely multifactorial, including the presence of hyperglycaemia, glycaemic variability and metformin use. This review emphasises the need for further research into T-cell responses of COVID-19 patients with T2DM in order to better inform our response to COVID-19 and future disease outbreaks.

Diet and Exercise for FRAILty (DEFRAIL): protocol for a study to examine the effect of a novel community-based group exercise and nutritional intervention, designed to reverse frailty in older adults.

INTRODUCTION: Frailty refers to a multifaceted age-related loss of physiological reserve. Aside from the immediate challenges it presents, it is also associated with various adverse health outcomes. Given our ageing population, the healthcare and societal costs resulting from frailty present a significant and growing public health challenge. Rapidly accumulating evidence suggests that resistance exercise combined with protein supplementation can reverse frailty in older adults. However, translation of these findings into practice has proven difficult, due to either a lack of clarity regarding the interventions used or the use of interventions not suitable for widespread implementation. There remains an absence of evidence-based programmes suitable for delivery to frail older adults in the community. METHODS AND ANALYSIS: This paper outlines the protocol for a study to examine the effect of a novel programme of exercise and protein supplementation. This intervention has been developed by an expert consensus group, specifically for delivery to frail older adults in a group setting in the community. The study will take the form of a within-subjects non-randomised trial. Participants will be assessed at baseline, then following an 8-week period of regular activity, then following the 8-week intervention. Frailty (according to the Fried Frailty criteria) will be the primary outcome measure, along with a range of secondary outcome measures (including physical performance measures, body mass composition, psychosocial assessments and frailty-related biomarkers). If shown to be feasible to implement and effective at reversing frailty, the Diet and Exercise for FRAILty (DEFRAIL) intervention may facilitate more widespread participation in resistance exercise for frail older adults. ETHICS AND DISSEMINATION: This study received ethical approval from the Research Ethics committees of both the Health Service Executive South-Eastern Area and Waterford Institute of Technology. Its findings will be disseminated through journal publications, conference presentations and other forms of public engagement. TRIAL REGISTRATION NUMBER: ISRCTN46458028; Pre-results.