Covalent fragment libraries in drug discovery-Design, synthesis, and screening methods.

As the development of drugs with a covalent mode of action is becoming increasingly popular, well-validated covalent fragment-based drug discovery (FBDD) methods have been comparatively slow to keep up with the demand. In this chapter the principles of covalent fragment reactivity, library design, synthesis, and screening methods are explored in depth, focussing on literature examples with direct applications to practical covalent fragment library design and screening. Further, questions about the future of the field are explored and potential useful advances are proposed.

Implementation of 'One Health' approach in Kerala state, India - A systematic review.

With humans, animals and the environment being as interconnected as they are, the science describing their interactions needs to cut across disciplinary boundaries. Systems research at the interface between the three goes by several names, such as 'Eco-Health' and 'Planetary Health', each with a varied focus, but the concept of 'One Health' (OH) has stood out as the most popular one. COVID-19 has reiterated the importance of OH in response to health challenges. This review aimed to assess the OH approach integration and implementation level in Kerala state, India, in the context of emerging zoonotic diseases. A systematic literature review was conducted by searching for relevant articles with specific keywords across six electronic databases. This involved screening the initial hits for titles and abstracts, then systematic sorting to identify the ones that met the criteria, followed by more thorough scrutiny to finally shortlist the six studies to be included in the review. We found that OH in Kerala has made good progress, as evident from a few recent examples, but has a long way to go with significant challenges. In line with the study's aim, identifying and analysing what is already done, what is missing and what needs to be done can have wider implications for future OH implementation. Relevant threats and opportunities were identified, with lessons for Kerala and India and broader applications.

Impact of an educational intervention for advanced cancer patients referred for early phase clinical trials.

A 2017 service evaluation identified a lack of information and knowledge among patients who were referred on to early phase oncology clinical trials (Hood, 2020). An educational booklet was developed to improve patients' knowledge and experience. To build upon this work, a patient co-designed website was developed. This study examined the impact, if any, of a patient co-designed educational intervention within the clinical pathway for patients who are referred for an early phase oncology clinical trial at an experimental cancer medicine centre (ECMC). AIMS: 1. To understand the experiences of patients who have been referred to an ECMC for an early phase clinical trial pre- and post-intervention. 2. To investigate if the intervention reduced anxiety levels in newly referred patients. METHOD: A convergent mixed-methods design was used in this study, to collect quantitative and qualitative data in parallel. OUTCOMES: This study examined the experiences of advanced cancer patients who attended their initial research outpatient appointment to discuss the possibility of taking part in an early phase clinical trial and the impact of an educational resource.

Infection prevention and control measures for preterm infants discharged into the community: a scoping review protocol.

BACKGROUND: Infection prevention and control (IPC) is an evidence-based and practical approach to prevention of harm by infection (Infection prevention and control https://www.who.int/health-topics/infection-prevention-and-control#tab=tab_1 ). IPC recommendations targeted at community-acquired infection aim to prevent illness and subsequent hospital readmission. Cohesive guidance for parents of preterm infants has not been clearly established. The review objectives are to identify and map the global characteristics of IPC measures/recommendations for parents of preterm infants discharged home to the community. METHODS: The scoping review will be conducted using the JBI methodological approach for scoping reviews and reported following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Scoping Review extension (PRISMA ScR) and the PRISMA extension for reporting literature searches in systematic reviews. Electronic databases will be searched and limited by publication year (2013-present day). Grey literature, reference lists and expert-provided sources will be searched against predetermined criteria. A minimum of two authors will independently screen evidence sources and chart evidence on a predetermined charting form. Sources including IPC measures, or recommendations for parents of preterm infants during discharge planning or in the community/home, will be permitted within inclusion criteria. Limits include human studies only and evidence from 2013-present day. Recommendations aimed at professional implementation will be excluded. A descriptive summary of findings will be presented, with diagrammatic and tabular representation. DISCUSSION: Collated evidence will guide future research which will subsequently aim to develop policy and enhance clinical approaches. SYSTEMATIC REVIEW REGISTRATION: This review has been registered on the Open Science Framework (OSF) 4th May 2021, available at https://osf.io/9yhzk .

Application of occupational justice concepts to children who are born preterm or admitted to neonatal intensive care and their parents: a scoping review protocol.

OBJECTIVE: This review aims to identify and map the usage, application, and context of occupational justice concepts and related terms by occupational therapists and occupational scientists in relation to parents and children when children are born preterm or admitted to a neonatal intensive care unit. INTRODUCTION: Occupational justice concepts and related terms can inform occupational therapy practice at the individual level or as a wider social approach. However, the extent to which these concepts have been applied to parents and children, when children are born preterm or admitted to neonatal intensive care, is unknown. INCLUSION CRITERIA: Studies must include 1 or more occupational justice concepts or associated terms in relation to the named population groups. Sources must be related to occupational therapy or occupational science. METHODS: The review will follow the JBI methodology for scoping reviews and will be reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) and the PRISMA-S extension for reporting literature searches in systematic reviews. Several electronic databases and sources of gray literature will be searched, limited by publication year (2000 till the present day). The review will only include human studies and studies with a title or abstract in English. Book chapters will be excluded. Reference lists of included studies will be searched against pre-determined criteria. Evidence sources will be independently screened by a minimum of 2 authors, and evidence will be mapped on a pre-determined template. DETAILS OF THE REVIEW AVAILABLE AT: Open Science Framework https://osf.io/fgd7n.

Uncovering the Most Kinetically Influential Reaction Pathway Driving the Generation of HCN from Oxyma/DIC Adduct: A Theoretical Study.

The combination of ethyl (hydroxyimino)cyanoacetate (Oxyma) and diisopropylcarbodiimide (DIC) has demonstrated superior performance in amino acid activation for peptide synthesis. However, it was recently reported that Oxyma and DIC could react to generate undesired hydrogen cyanide (HCN) at 20 °C, raising safety concerns for the practical use of this activation strategy. To help minimize the risks, there is a need for a comprehensive investigation of the mechanism and kinetics of the generation of HCN. Here we show the results of the first systematic computational study of the underpinning mechanism, including comparisons with experimental data. Two pathways for the decomposition of the Oxyma/DIC adduct are derived to account for the generation of HCN and its accompanying cyclic product. These two mechanisms differ in the electrophilic carbon atom attacked by the nucleophilic sp2-nitrogen in the cyclization step and in the cyclic product generated. On the basis of computed "observed" activation energies, ΔG obs ⧧, the mechanism that proceeds via the attack of the sp2-nitrogen at the oxime carbon is identified as the most kinetically favorable one, a conclusion that is supported by closer agreement between predicted and experimental 13C NMR data. These results can provide a theoretical basis to develop a design strategy for suppressing HCN generation when using Oxyma/DIC for amino acid activation.

Effectiveness of a neonatal COVID-19 response project: A mixed-methods evaluation using the Donabedian model.

Objective: This article outlines notable findings of a service evaluation of a COVID-19 response project, the Nurture Project (July 2020-March 2021). Method: The Donabedian structure-process-outcome model was used. Mixed-methods online surveys and organisational data were analysed using reflexive thematic analysis and statistical analysis methods. Results: Most staff and service users were satisfied with the project, reporting positive benefits to mental health, child development, and wellbeing. However, project outcome measures (Generalised Anxiety Disorder Scale GAD-7 and the Patient Health Questionnaire PHQ-9) were statistically non-significant. Conclusion: Although the project was considered successful, recommendations for future service evaluation methods, outcome measurement, and future research are provided.

Synthesis of Sulfoximine Propargyl Carbamates under Improved Conditions for Rhodium Catalyzed Carbamate Transfer to Sulfoxides.

Sulfoximines provide aza-analogues of sulfones, with potentially improved properties for medicinal chemistry. The sulfoximine nitrogen also provides an additional vector for the inclusion of other functionality. Here, we report improved conditions for rhodium catalyzed synthesis of sulfoximine (and sulfilimine) carbamates, especially for previously low-yielding carbamates containing π-functionality. Notably we report the preparation of propargyl sulfoximine carbamates to provide an alkyne as a potential click handle. Using Rh2(esp)2 as catalyst and a DOE optimization approach provided considerably increased yields.

Acrylamide fragment inhibitors that induce unprecedented conformational distortions in enterovirus 71 3C and SARS-CoV-2 main protease.

RNA viruses are critically dependent upon virally encoded proteases to cleave the viral polyproteins into functional proteins. Many of these proteases exhibit a similar fold and contain an essential catalytic cysteine, offering the opportunity to inhibit these enzymes with electrophilic small molecules. Here we describe the successful application of quantitative irreversible tethering (qIT) to identify acrylamide fragments that target the active site cysteine of the 3C protease (3Cpro) of Enterovirus 71, the causative agent of hand, foot and mouth disease in humans, altering the substrate binding region. Further, we re-purpose these hits towards the main protease (Mpro) of SARS-CoV-2 which shares the 3C-like fold and a similar active site. The hit fragments covalently link to the catalytic cysteine of Mpro to inhibit its activity. We demonstrate that targeting the active site cysteine of Mpro can have profound allosteric effects, distorting secondary structures to disrupt the active dimeric unit.

Identification of the first structurally validated covalent ligands of the small GTPase RAB27A.

Rab27A is a small GTPase, which mediates transport and docking of secretory vesicles at the plasma membrane via protein-protein interactions (PPIs) with effector proteins. Rab27A promotes the growth and invasion of multiple cancer types such as breast, lung and pancreatic, by enhancing secretion of chemokines, metalloproteases and exosomes. The significant role of Rab27A in multiple cancer types and the minor role in adults suggest that Rab27A may be a suitable target to disrupt cancer metastasis. Similar to many GTPases, the flat topology of the Rab27A-effector PPI interface and the high affinity for GTP make it a challenging target for inhibition by small molecules. Reported co-crystal structures show that several effectors of Rab27A interact with the Rab27A SF4 pocket ('WF-binding pocket') via a conserved tryptophan-phenylalanine (WF) dipeptide motif. To obtain structural insight into the ligandability of this pocket, a novel construct was designed fusing Rab27A to part of an effector protein (fRab27A), allowing crystallisation of Rab27A in high throughput. The paradigm of KRas covalent inhibitor development highlights the challenge presented by GTPase proteins as targets. However, taking advantage of two cysteine residues, C123 and C188, that flank the WF pocket and are unique to Rab27A and Rab27B among the >60 Rab family proteins, we used the quantitative Irreversible Tethering (qIT) assay to identify the first covalent ligands for native Rab27A. The binding modes of two hits were elucidated by co-crystallisation with fRab27A, exemplifying a platform for identifying suitable lead fragments for future development of competitive inhibitors of the Rab27A-effector interaction interface, corroborating the use of covalent libraries to tackle challenging targets.

A genetically-encoded crosslinker screen identifies SERBP1 as a PKCε substrate influencing translation and cell division.

The PKCε-regulated genome protective pathway provides transformed cells a failsafe to successfully complete mitosis. Despite the necessary role for Aurora B in this programme, it is unclear whether its requirement is sufficient or if other PKCε cell cycle targets are involved. To address this, we developed a trapping strategy using UV-photocrosslinkable amino acids encoded in the PKCε kinase domain. The validation of the mRNA binding protein SERBP1 as a PKCε substrate revealed a series of mitotic events controlled by the catalytic form of PKCε. PKCε represses protein translation, altering SERBP1 binding to the 40 S ribosomal subunit and promoting the assembly of ribonucleoprotein granules containing SERBP1, termed M-bodies. Independent of Aurora B, SERBP1 is shown to be necessary for chromosome segregation and successful cell division, correlating with M-body formation. This requirement for SERBP1 demonstrates that Aurora B acts in concert with translational regulation in the PKCε-controlled pathway exerting genome protection.

Co-ordinated control of the Aurora B abscission checkpoint by PKCε complex assembly, midbody recruitment and retention.

A requirement for PKCε in exiting from the Aurora B dependent abscission checkpoint is associated with events at the midbody, however, the recruitment, retention and action of PKCε in this compartment are poorly understood. Here, the prerequisite for 14-3-3 complex assembly in this pathway is directly linked to the phosphorylation of Aurora B S227 at the midbody. However, while essential for PKCε control of Aurora B, 14-3-3 association is shown to be unnecessary for the activity-dependent enrichment of PKCε at the midbody. This localisation is demonstrated to be an autonomous property of the inactive PKCε D532N mutant, consistent with activity-dependent dissociation. The C1A and C1B domains are necessary for this localisation, while the C2 domain and inter-C1 domain (IC1D) are necessary for retention at the midbody. Furthermore, it is shown that while the IC1D mutant retains 14-3-3 complex proficiency, it does not support Aurora B phosphorylation, nor rescues division failure observed with knockdown of endogenous PKCε. It is concluded that the concerted action of multiple independent events facilitates PKCε phosphorylation of Aurora B at the midbody to control exit from the abscission checkpoint.

Synthesis and Configurational Assignment of Vinyl Sulfoximines and Sulfonimidamides.

Vinyl sulfones and sulfonamides are valued for their use as electrophilic warheads in covalent protein inhibitors. Conversely, the S(VI) aza-isosteres thereof, vinyl sulfoximines and sulfonimidamides, are far less studied and have yet to be applied to the field of protein bioconjugation. Herein, we report a range of different synthetic methodologies for constructing vinyl sulfoximine and vinyl sulfonimidamide architectures that allows access to new areas of electrophilic chemical space. We demonstrate how late-stage functionalization can be applied to these motifs to incorporate alkyne tags, generating fully functionalized probes for future chemical biology applications. Finally, we establish a workflow for determining the absolute configuration of enantioenriched vinyl sulfoximines and sulfonimidamides by comparing experimentally and computationally determined electronic circular dichroism spectra, enabling access to configurationally assigned enantiomeric pairs by separation.

Multiparameter Kinetic Analysis for Covalent Fragment Optimization by Using Quantitative Irreversible Tethering (qIT).

Chemical probes that covalently modify cysteine residues in a protein-specific manner are valuable tools for biological investigations. Covalent fragments are increasingly implemented as probe starting points, but the complex relationship between fragment structure and binding kinetics makes covalent fragment optimization uniquely challenging. We describe a new technique in covalent probe discovery that enables data-driven optimization of covalent fragment potency and selectivity. This platform extends beyond the existing repertoire of methods for identifying covalent fragment hits by facilitating rapid multiparameter kinetic analysis of covalent structure-activity relationships through the simultaneous determination of Ki , kinact and intrinsic reactivity. By applying this approach to develop novel probes against electrophile-sensitive kinases, we showcase the utility of the platform in hit identification and highlight how multiparameter kinetic analysis enabled a successful fragment-merging strategy.

Quantitative Irreversible Tethering (qIT) for Target-directed Covalent Fragment Screening.

Small molecules that react to form covalent bonds with proteins are widely used as biological tools and therapeutic agents. Screening cysteine-reactive fragments against a protein target is an efficient way to identify chemical starting points for covalent probe development. Mass spectrometry is often used to identify the site and degree of covalent fragment binding. However, robust hit identification requires characterization of the kinetics of covalent binding that can be readily achieved using quantitative irreversible tethering. This screening platform uses a non-specific cysteine-reactive fluorogenic probe to monitor the rate of reaction between covalent fragments and cysteine containing biomolecules. Fragment libraries are simultaneously screened against the target protein and glutathione, which functions as a control, to identify hit fragments with kinetic selectivity for covalent modification of the target. Screening by quantitative irreversible tethering accounts for variations in the intrinsic reactivity of individual fragments enabling robust hit identification and ranking.

An augmented reality approach for ADL support in Alzheimer's disease: a crossover trial.

BACKGROUND: Dementia of the Alzheimer's type can impair the performance of activities of daily living and therefore severely impact independent living. Assistive technologies can support such patients when carrying out daily tasks. METHODS: In this crossover study, we used an augmented reality approach using a Microsoft HoloLens to support patients in a tea making task. During task execution, subjects received three-dimensional dynamic holograms of the sub-steps necessary to complete the task. Ten patients suffering from Alzheimer's disease were tested and post-hoc semi-structured interviews were conducted to assess usability. RESULTS: The patients committed errors when executing the task with and without holographic assistance. No differences in success rates or error frequencies were observed (psuccess = .250, perrors = .887). Patients revealed prolonged trial durations (Glass' Δ = 1.475) when wearing the augmented reality headset. A model of multiple linear regression (R2adjusted = .958) revealed an influence of the errors in the control condition and a moderation by the errors in the experimental condition. Patients with more severe problems in the natural performance of the task showed lower increases in trial durations when wearing the HoloLens. CONCLUSIONS: We assume that the application was a secondary task requesting its own resources and impairing performance on its own. The regression suggests however that the given assistance was compensating these additional costs in patients with stronger needs of support. Interview data on usability revealed an overall positive feedback towards the application although the hardware was considered uncomfortable and too large. We conclude that the approach proved feasible and the acceptability was overall high, although advances in hardware and the patient-interface are necessary to assist patients suffering from Alzheimer's disease in daily activities. TRIAL REGISTRATION: DRKS, DRKS00014870. Registered 11 June 2018 - Retrospectively registered, TrialID = DRKS00014870 .

Exploitation of Antibiotic Resistance as a Novel Drug Target: Development of a ß-Lactamase-Activated Antibacterial Prodrug.

Expression of ß-lactamase is the single most prevalent determinant of antibiotic resistance, rendering bacteria resistant to ß-lactam antibiotics. In this article, we describe the development of an antibiotic prodrug that combines ciprofloxacin with a ß-lactamase-cleavable motif. The prodrug is only bactericidal after activation by ß-lactamase. Bactericidal activity comparable to ciprofloxacin is demonstrated against clinically relevant E. coli isolates expressing diverse ß-lactamases; bactericidal activity was not observed in strains without ß-lactamase. These findings demonstrate that it is possible to exploit antibiotic resistance to selectively target ß-lactamase-producing bacteria using our prodrug approach, without adversely affecting bacteria that do not produce ß-lactamase. This paves the way for selective targeting of drug-resistant pathogens without disrupting or selecting for resistance within the microbiota, reducing the rate of secondary infections and subsequent antibiotic use.

Vinyl sulfonamide synthesis for irreversible tethering via a novel α-selenoether protection strategy.

Vinyl sulfonamides are valuable electrophiles for targeted protein modification and inhibition. We describe a novel approach to the synthesis of terminal vinyl sulfonamides which uses mild oxidative conditions to induce elimination of an α-selenoether masking group. The method complements traditional synthetic approaches and typically yields vinyl sulfonamides in high purity after aqueous work-up without requiring column chromatography of the final electrophilic product. The methodology is applied to the synthesis of covalent fragments for use in irreversible protein tethering and crucially enables the attachment of diverse fragments to the vinyl sulfonamide warhead via a chemical linker. Using thymidylate synthase as a model system, ethylene glycol is identified as an effective linker for irreversible protein tethering.

An Explorative Note on Apraxia Tests.

Apraxia is stated independent of primary motor disorders. However, patient groups suffering from stroke or dementia can reveal motor impairments. In this study we examined the dependence of apraxia tests of imitation and pantomime on a latent motor component using a principal component analysis. With samples sizes of 11 patients suffering from dementia of the Alzheimer's type and 15 healthy control subjects, clear limitations concerning the validity of the results are given. Nevertheless, we could observe strong dependence of the three apraxia tests, especially the imitation of finger and hand gestures, on a latent motor component in this preliminary examination. We suggest confirmation by larger samples sizes and to control for the basic motor capacity when testing for signs of apraxia in such patient samples.

Step by Step: Kinematics of the Reciprocal Trail Making Task Predict Slowness of Activities of Daily Living Performance in Alzheimer's Disease.

Dementia impairs the ability to perform everyday activities. Reduced motor capacity and executive functions as well as loss of memory function and forms of apraxia and action disorganization syndrome can be reasons for such impairments. In this study, an analysis of the hand trajectories during the sequential movements in an adapted version of the trail making task, the reciprocal trail making task (RTMT), was used to predict performance in activities of daily living (ADL) of patients suffering from mild cognitive impairment and dementia. 1 patient with dementia of the Alzheimer's type and 15 healthy, age-matched adults were tested in the standardized ADL of tea making and document filing. The characteristics of the kinematic performance in the RTMT were assessed, and models of multiple linear regression were computed to predict the durations of the ADL. Patients showed increased trial durations (TDs) in the ADL (Cohen's d: tea making 1.64, document filing 1.25). Parameters and explained variability differed across patients and control as well as between different activities. The models for the patient sample were stronger and particularly high for the document filing task for which kinematics explained 71% of the variance ([Formula: see text]: tea making 0.62, document filing 0.71; both tasks combined patients 0.55, controls 0.25). The most relevant factors for the models were the TD and a parameter characterizing movement fluency and variability ("movement harmonicity") in the RTMT. The models of multiple linear regression suggested that the patients' activity of daily living performance was limited by cognitive demands, namely, identifying the varying targets during sequencing and the healthy controls' performance by their motor capacity. Such models could be used to estimate the severity of ADL impairments in patients.