The state of the art in secondary pharmacology and its impact on the safety of new medicines.

Secondary pharmacology screening of investigational small-molecule drugs for potentially adverse off-target activities has become standard practice in pharmaceutical research and development, and regulatory agencies are increasingly requesting data on activity against targets with recognized adverse effect relationships. However, the screening strategies and target panels used by pharmaceutical companies may vary substantially. To help identify commonalities and differences, as well as to highlight opportunities for further optimization of secondary pharmacology assessment, we conducted a broad-ranging survey across 18 companies under the auspices of the DruSafe leadership group of the International Consortium for Innovation and Quality in Pharmaceutical Development. Based on our analysis of this survey and discussions and additional research within the group, we present here an overview of the current state of the art in secondary pharmacology screening. We discuss best practices, including additional safety-associated targets not covered by most current screening panels, and present approaches for interpreting and reporting off-target activities. We also provide an assessment of the safety impact of secondary pharmacology screening, and a perspective on opportunities and challenges in this rapidly developing field.

A single amino acid in the Salmonella effector SarA/SteE triggers supraphysiological activation of STAT3 for anti-inflammatory target gene expression.

Non-typhoidal Salmonella enterica cause an estimated 1 million cases of gastroenteritis annually in the United States. These serovars use secreted protein effectors to mimic and reprogram host cellular functions. We previously discovered that the secreted effector SarA (Salmonella anti-inflammatory response activator; also known as SteE) was required for increased intracellular replication of S. Typhimurium and production of the anti-inflammatory cytokine interleukin-10 (IL-10). SarA facilitates phosphorylation of STAT3 through a region of homology with the host cytokine receptor gp130. Here, we demonstrate that a single amino acid difference between SarA and gp130 is critical for the anti-inflammatory bias of SarA-STAT3 signaling. An isoleucine at the pY+1 position of the YxxQ motif in SarA (which binds the SH2 domain in STAT3) causes increased STAT3 phosphorylation and expression of anti-inflammatory target genes. This isoleucine, completely conserved in ~4000 Salmonella isolates, renders SarA a better substrate for tyrosine phosphorylation by GSK-3. GSK-3 is canonically a serine/threonine kinase that nonetheless undergoes tyrosine autophosphorylation at a motif that has an invariant isoleucine at the pY+1 position. Our results provide a molecular basis for how a Salmonella secreted effector achieves supraphysiological levels of STAT3 activation to control host genes during infection.

Hormonal steroids induce multidrug resistance and stress response genes in Neisseria gonorrhoeae by binding to MtrR.

Transcriptional regulator MtrR inhibits the expression of the multidrug efflux pump operon mtrCDE in the pathogenic bacterium Neisseria gonorrhoeae. Here, we show that MtrR binds the hormonal steroids progesterone, ß-estradiol, and testosterone, which are present at urogenital infection sites, as well as ethinyl estrogen, a component of some hormonal contraceptives. Steroid binding leads to the decreased affinity of MtrR for cognate DNA, increased mtrCDE expression, and enhanced antimicrobial resistance. Furthermore, we solve crystal structures of MtrR bound to each steroid, thus revealing their binding mechanisms and the conformational changes that induce MtrR.

A Predictive Model of Vaccine Reactogenicity Using Data from an In Vitro Human Innate Immunity Assay System.

A primary concern in vaccine development is safety, particularly avoiding an excessive immune reaction in an otherwise healthy individual. An accurate prediction of vaccine reactogenicity using in vitro assays and computational models would facilitate screening and prioritization of novel candidates early in the vaccine development process. Using the modular in vitro immune construct model of human innate immunity, PBMCs from 40 healthy donors were treated with 10 different vaccines of varying reactogenicity profiles and then cell culture supernatants were analyzed via flow cytometry and a multichemokine/cytokine assay. Differential response profiles of innate activity and cell viability were observed in the system. In parallel, an extensive adverse event (AE) dataset for the vaccines was assembled from clinical trial data. A novel reactogenicity scoring framework accounting for the frequency and severity of local and systemic AEs was applied to the clinical data, and a machine learning approach was employed to predict the incidence of clinical AEs from the in vitro assay data. Biomarker analysis suggested that the relative levels of IL-1B, IL-6, IL-10, and CCL4 have higher predictive importance for AE risk. Predictive models were developed for local reactogenicity, systemic reactogenicity, and specific individual AEs. A forward-validation study was performed with a vaccine not used in model development, Trumenba (meningococcal group B vaccine). The clinically observed Trumenba local and systemic reactogenicity fell on the 26th and 93rd percentiles of the ranges predicted by the respective models. Models predicting specific AEs were less accurate. Our study presents a useful framework for the further development of vaccine reactogenicity predictive models.

Medical Oxygen as a Life-Saving Medicine: A rapid review of the oxygen landscape and innovative efforts in the World Health Organization Eastern Mediterranean Region in Response to COVID-19 and Beyond.

Background: Medical oxygen is an essential treatment for life-threatening hypoxemic conditions and is commonly indicated for the clinical management of many leading causes of mortality. Many countries of the World Health Organization (WHO) Eastern Mediterranean Region (EMR) lacked robust medical oxygen systems prior to the COVID-19 (corona virus disease) pandemic and this situation was exacerbated by increased needs, particularly in remote and rural health facilities, resulting in many unfortunate deaths. The aim of this article is to describe the oxygen landscape in the region and the regional initiatives undertaken by countries and WHO. Methodology: We conducted a rapid review to synthesize the available literature on the needs and availability of oxygen and its related resources and the regional initiatives undertaken. We conducted search in PubMed, relevant WHO and World Bank websites, and in general using google to understand the health of conditions that could benefit from the availability of medical oxygen, oxygen related resources including health workforce available for support and usage of medical oxygen, and the initiatives by WHO, countries and partners to improve the situation. We used a snowballing technique and reviewed all available databases for reports, surveys, assessments, and studies related to medical oxygen, besides WHO internal records, assessments, and consultation reports. Results: The data on oxygen availability, supply demand gap, infrastructure facilities, and human resources were sparse. The regional initiatives have led to increase in resources, including human resources and oxygen production infrastructure. The Live Oxygen Platform (LOP), contributed to improved availability of quality data needed for supply demand assessments. Conclusion: A regional enterprise strategy to promote sustainable, decentralized, and contextualized production, supply, and monitoring of oxygen together with human resource support including training and placement by WHO, partners, and governments contributed to improved availability of oxygen in the region. Additionally, with the LOP, governments, WHO, and partners have access to better data availability for policy decision making and timely resource allocation.

Hormonal steroids bind the Neisseria gonorrhoeae multidrug resistance regulator, MtrR, to induce a multidrug binding efflux pump and stress-response sigma factor.

Overexpression of the multidrug efflux pump MtrCDE, a critical factor of multidrug-resistance in Neisseria gonorrhoeae , the causative agent of gonorrheae, is repressed by the transcriptional regulator, MtrR (multiple transferable resistance repressor). Here, we report the results from a series of in vitro experiments to identify innate, human inducers of MtrR and to understand the biochemical and structural mechanisms of the gene regulatory function of MtrR. Isothermal titration calorimetry experiments reveal that MtrR binds the hormonal steroids progesterone, ß-estradiol, and testosterone, all of which are present at significant concentrations at urogenital infection sites as well as ethinyl estrogen, a component of some birth control pills. Binding of these steroids results in decreased affinity of MtrR for cognate DNA, as demonstrated by fluorescence polarization-based assays. The crystal structures of MtrR bound to each steroid provided insight into the flexibility of the binding pocket, elucidated specific residue-ligand interactions, and revealed the conformational consequences of the induction mechanism of MtrR. Three residues, D171, W136 and R176 are key to the specific binding of these gonadal steroids. These studies provide a molecular understanding of the transcriptional regulation by MtrR that promotes N. gonorrhoeae survival in its human host.

Cholera prevention, control strategies, challenges and World Health Organization initiatives in the Eastern Mediterranean Region: A narrative review.

The resurgence of cholera is presenting unusual challenges in the Eastern Mediterranean Region (EMR), where it is considered endemic in nine-member states. The risk of a cholera outbreak spreading to non-endemic countries remains high. We discuss the regional trends of cholera, regional burden, and challenges with a focus on World Health Organization (WHO) initiatives in the region that could be useful in preventing and controlling the disease in similar contexts. Despite significant progress in the control of cholera worldwide, the disease continues to be a major public health problem across the region, where it constitutes both an emerging and re-emerging threat. Recurring cholera outbreaks are an indication of deprived water and sanitation conditions as well as weak health systems, contributing to the transmission and spread of the cholera infection. We note that despite the challenges in eliminating cholera in the region, effective implementation of the proposed WHO EMR Strategic framework, among other measures, could sustain the region's cholera prevention, preparedness, and response needs.

Strengthening hospital resilience in the Eastern Mediterranean Region.

"Adaptation is surviving but resilience is for thriving."In recent years, the multiple threats of COVID-19 and other disease outbreaks, intensified climate change and severe weather events, and increasing conflicts and humanitarian emergencies have highlighted the need to strengthen resilience in the different sectors, including social, economic, environment, and health. Resilience is the ability of a system, community or society exposed to hazards to resist, absorb, accommodate, adapt to, transform, and recover from the effects of a hazard in a timely and efficient manner, including through the preservation and restoration of its essential basic structures and functions through risk management.

Structures of trehalose-6-phosphate synthase, Tps1, from the fungal pathogen Cryptococcus neoformans: a target for novel antifungals.

Invasive fungal diseases are a major threat to human health, resulting in more than 1.5 million deaths worldwide each year. Yet the arsenal of antifungal therapeutics remains limited and is in dire need of novel drugs that target additional fungal-specific biosynthetic pathways. One such pathway involves the biosynthesis of trehalose. Trehalose is a non-reducing disaccharide composed of two molecules of glucose that is required for pathogenic fungi, including Candida albicans and Cryptococcus neoformans, to survive in their human hosts. Trehalose biosynthesis is a two-step process in fungal pathogens. Trehalose-6-phosphate synthase (Tps1) converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate (T6P). Subsequently, trehalose-6-phosphate phosphatase (Tps2) converts T6P to trehalose. The trehalose biosynthesis pathway has been identified as a top candidate for novel antifungal development based on quality, occurrence, specificity, and assay development. However, there are currently no known antifungal agents that target this pathway. As initial steps to develop Tps1 from Cryptococcus neoformans (CnTps1) as a drug target, we report the structures of full-length apo CnTps1 and CnTps1 in complex with uridine diphosphate (UDP) and glucose-6-phosphate (G6P). Both CnTps1 structures are tetramers and display D2 (222) molecular symmetry. Comparison of these two structures reveals significant movement towards the catalytic pocket by the N-terminus upon ligand binding and identifies key residues required for substrate-binding, which are conserved amongst other Tps1 enzymes, as well as residues that stabilize the tetramer. Intriguingly, an intrinsically disordered domain (IDD), encompassing residues M209 to I300, which is conserved amongst Cryptococcal species and closely related Basidiomycetes, extends from each subunit of the tetramer into the "solvent" but is not visible in the density maps. Although, activity assays revealed that the highly conserved IDD is not required for catalysis in vitro, we hypothesize that the IDD is required for C. neoformans Tps1-dependent thermotolerance and osmotic stress survival. Characterization of the substrate specificity of CnTps1 revealed that UDP-galactose, an epimer of UDP-glucose, is a very poor substrate and inhibitor of the enzyme and highlights the exquisite substrate specificity of Tps1. In toto, these studies expand our knowledge of trehalose biosynthesis in Cryptococcus and highlight the potential of developing antifungal therapeutics that disrupt the synthesis of this disaccharide or the formation of a functional tetramer and the use of cryo-EM in the structural characterization of CnTps1-ligand/drug complexes.

Mitigating the resurgence of cholera in the Eastern Mediterranean Region.

Countries in the WHO Eastern Mediterranean Region (EMR) are currently experiencing a resurgence of cholera. As of 31 December 2022, 8 of the 22 Member States in the region - Afghanistan, Islamic Republic of Iran, Iraq, Lebanon, Pakistan, Somalia, Syria, and Yemen - were grappling with outbreaks of cholera and acute watery diarrhoea (AWD). More than 1 000 000 suspected AWD/cholera cases, more than 7500 laboratory-confirmed cases, and 375 cholera-associated deaths were reported across the region in 2022.

Gaza disaster: we need a permanent ceasefire, now!

Another year ends with multiple humanitarian crises ongoing simultaneously across the globe. In the Eastern Mediterranean Region, 9 of the 22 Member States and territory are considered fragile or conflict-affected, with severe health consequences for the population.

Genomic sequencing for epidemic and pandemic preparedness and response: EMRO's vision and strategic interventions.

Whole-genome sequencing (WGS) is an approach for studying and analysing the entire genomic sequence of pathogens. It provides the most comprehensive characterization of an organism's genetic make-up. In January 2020, scientists used next-generation sequencing (NGS) as one of several sequencing technologies to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to reveal the first genetic makeup of this new virus only 11 days after the first cluster of cases due to the COVID-19 pandemic was reported. This crucial information was vital to the subsequent rapid development of test kits, vaccines and treatment regimens to respond to the pandemic. The sequencing information, later as the pandemic evolved, became essential for informing public health policies through monitoring and characterization of SARS-CoV-2 variants.

Single-cell genome-wide association reveals that a nonsynonymous variant in ERAP1 confers increased susceptibility to influenza virus.

During pandemics, individuals exhibit differences in risk and clinical outcomes. Here, we developed single-cell high-throughput human in vitro susceptibility testing (scHi-HOST), a method for rapidly identifying genetic variants that confer resistance and susceptibility. We applied this method to influenza A virus (IAV), the cause of four pandemics since the start of the 20th century. scHi-HOST leverages single-cell RNA sequencing (scRNA-seq) to simultaneously assign genetic identity to cells in mixed infections of cell lines of European, African, and Asian origin, reveal associated genetic variants for viral burden, and identify expression quantitative trait loci. Integration of scHi-HOST with human challenge and experimental validation demonstrated that a missense variant in endoplasmic reticulum aminopeptidase 1 (ERAP1; rs27895) increased IAV burden in cells and human volunteers. rs27895 exhibits population differentiation, likely contributing to greater permissivity of cells from African populations to IAV. scHi-HOST is a broadly applicable method and resource for decoding infectious-disease genetics.

Monkeypox outbreak and response efforts in the Eastern Mediterranean Region.

Monkeypox (MPX) is a viral zoonotic disease that is endemic in some countries of Central and Western Africa. Since 1 January 2022, cases of MPX have been reported to WHO by 74 Member States across all 6 WHO regions. As of 21 July 2022, a total of 15 328 laboratory confirmed cases and 72 probable cases, including 5 deaths, have been reported to WHO. Most (11 638/15 328, 76%) of the laboratory-confirmed cases were reported by countries of the WHO European Region, 22% (3316/15 328) by the Region of the Americas, 2% (301/15 328) by the African Region, less than 1% (53/15 328) by the Western Pacific Region, less than 1% (18/15 328) by the Eastern Mediterranean Region (EMR) and less than 1% (2/15 328) by the Region of South-East Asia. All five deaths were reported by the African Region.

Molecular dissection of the glutamine synthetase-GlnR nitrogen regulatory circuitry in Gram-positive bacteria.

How bacteria sense and respond to nitrogen levels are central questions in microbial physiology. In Gram-positive bacteria, nitrogen homeostasis is controlled by an operon encoding glutamine synthetase (GS), a dodecameric machine that assimilates ammonium into glutamine, and the GlnR repressor. GlnR detects nitrogen excess indirectly by binding glutamine-feedback-inhibited-GS (FBI-GS), which activates its transcription-repression function. The molecular mechanisms behind this regulatory circuitry, however, are unknown. Here we describe biochemical and structural analyses of GS and FBI-GS-GlnR complexes from pathogenic and non-pathogenic Gram-positive bacteria. The structures show FBI-GS binds the GlnR C-terminal domain within its active-site cavity, juxtaposing two GlnR monomers to form a DNA-binding-competent GlnR dimer. The FBI-GS-GlnR interaction stabilizes the inactive GS conformation. Strikingly, this interaction also favors a remarkable dodecamer to tetradecamer transition in some GS, breaking the paradigm that all bacterial GS are dodecamers. These data thus unveil unique structural mechanisms of transcription and enzymatic regulation.