PCSK9 inhibition attenuates alcohol-associated neuronal oxidative stress and cellular injury.

Alcohol Use Disorder (AUD) is a persistent condition linked to neuroinflammation, neuronal oxidative stress, and neurodegenerative processes. While the inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has demonstrated effectiveness in reducing liver inflammation associated with alcohol, its impact on the brain remains largely unexplored. This study aimed to assess the effects of alirocumab, a monoclonal antibody targeting PCSK9 to lower systemic low-density lipoprotein cholesterol (LDL-C), on central nervous system (CNS) pathology in a rat model of chronic alcohol exposure. Alirocumab (50 mg/kg) or vehicle was administered weekly for six weeks in 32 male rats subjected to a 35 % ethanol liquid diet or a control liquid diet (n = 8 per group). The study evaluated PCSK9 expression, LDL receptor (LDLR) expression, oxidative stress, and neuroinflammatory markers in brain tissues. Chronic ethanol exposure increased PCSK9 expression in the brain, while alirocumab treatment significantly upregulated neuronal LDLR and reduced oxidative stress in neurons and brain vasculature (3-NT, p22phox). Alirocumab also mitigated ethanol-induced microglia recruitment in the cortex and hippocampus (Iba1). Additionally, alirocumab decreased the expression of pro-inflammatory cytokines and chemokines (TNF, CCL2, CXCL3) in whole brain tissue and attenuated the upregulation of adhesion molecules in brain vasculature (ICAM1, VCAM1, eSelectin). This study presents novel evidence that alirocumab diminishes oxidative stress and modifies neuroimmune interactions in the brain elicited by chronic ethanol exposure. Further investigation is needed to elucidate the mechanisms by which PCSK9 signaling influences the brain in the context of chronic ethanol exposure.

Plate-based diversity subset screening generation 2: an improved paradigm for high-throughput screening of large compound files.

High-throughput screening (HTS) is an effective method for lead and probe discovery that is widely used in industry and academia to identify novel chemical matter and to initiate the drug discovery process. However, HTS can be time consuming and costly and the use of subsets as an efficient alternative to screening entire compound collections has been investigated. Subsets may be selected on the basis of chemical diversity, molecular properties, biological activity diversity or biological target focus. Previously, we described a novel form of subset screening: plate-based diversity subset (PBDS) screening, in which the screening subset is constructed by plate selection (rather than individual compound cherry-picking), using algorithms that select for compound quality and chemical diversity on a plate basis. In this paper, we describe a second-generation approach to the construction of an updated subset: PBDS2, using both plate and individual compound selection, that has an improved coverage of the chemical space of the screening file, whilst only selecting the same number of plates for screening. We describe the validation of PBDS2 and its successful use in hit and lead discovery. PBDS2 screening became the default mode of singleton (one compound per well) HTS for lead discovery in Pfizer.

Aggressive chemotherapy and the selection of drug resistant pathogens.

Drug resistant pathogens are one of the key public health challenges of the 21st century. There is a widespread belief that resistance is best managed by using drugs to rapidly eliminate target pathogens from patients so as to minimize the probability that pathogens acquire resistance de novo. Yet strong drug pressure imposes intense selection in favor of resistance through alleviation of competition with wild-type populations. Aggressive chemotherapy thus generates opposing evolutionary forces which together determine the rate of drug resistance emergence. Identifying treatment regimens which best retard resistance evolution while maximizing health gains and minimizing disease transmission requires empirical analysis of resistance evolution in vivo in conjunction with measures of clinical outcomes and infectiousness. Using rodent malaria in laboratory mice, we found that less aggressive chemotherapeutic regimens substantially reduced the probability of onward transmission of resistance (by >150-fold), without compromising health outcomes. Our experiments suggest that there may be cases where resistance evolution can be managed more effectively with treatment regimens other than those which reduce pathogen burdens as fast as possible.

The evolutionary consequences of blood-stage vaccination on the rodent malaria Plasmodium chabaudi.

Malaria vaccine developers are concerned that antigenic escape will erode vaccine efficacy. Evolutionary theorists have raised the possibility that some types of vaccine could also create conditions favoring the evolution of more virulent pathogens. Such evolution would put unvaccinated people at greater risk of severe disease. Here we test the impact of vaccination with a single highly purified antigen on the malaria parasite Plasmodium chabaudi evolving in laboratory mice. The antigen we used, AMA-1, is a component of several candidate malaria vaccines currently in various stages of trials in humans. We first found that a more virulent clone was less readily controlled by AMA-1-induced immunity than its less virulent progenitor. Replicated parasites were then serially passaged through control or AMA-1 vaccinated mice and evaluated after 10 and 21 rounds of selection. We found no evidence of evolution at the ama-1 locus. Instead, virulence evolved; AMA-1-selected parasites induced greater anemia in naïve mice than both control and ancestral parasites. Our data suggest that recombinant blood stage malaria vaccines can drive the evolution of more virulent malaria parasites.

N-Myristoyltransferase as a potential drug target in malaria and leishmaniasis.

Infections caused by protozoan parasites are among the most widespread and intractable transmissible diseases affecting the developing world, with malaria and leishmaniasis being the most costly in terms of morbidity and mortality. Although new drugs are urgently required against both diseases in the face of ever-rising resistance to frontline therapies, very few candidates passing through development pipelines possess a known and novel mode of action. Set in the context of drugs currently in use and under development, we present the evidence for N-myristoyltransferase (NMT), an enzyme that N-terminally lipidates a wide range of specific target proteins through post-translational modification, as a potential drug target in malaria and the leishmaniases. We discuss the limitations of current knowledge regarding the downstream targets of this enzyme in protozoa, and our recent progress towards potent cell-active NMT inhibitors against the most clinically-relevant species of parasite. Finally, we outline the next steps required in terms of both tools to understand N-myristoylation in protozoan parasites, and the generation of potential development candidates based on the output of our recently-reported high-throughput screens.

Assessing the Impact of PCSK9 and HMGCR Inhibition on Liver Function: Drug-Target Mendelian Randomization Analyses in Four Ancestries.

BACKGROUND & AIMS: Observational studies have linked lipid-lowering drug targets pro-protein convertase subtilisin/kexin 9 (PCSK9) and HMG-CoA reductase (HMGCR) with adverse liver outcomes; however, liver disease incidence varies across diverse populations, and the long-term hepatic impact of these lipid-lowering drugs among non-white Europeans remains largely unknown. METHODS: We use single nucleotide polymorphisms (SNPs) in PCSK9 and HMGCR loci from genome-wide association study data of low-density lipoprotein cholesterol in 4 populations (East Asian [EAS], South Asian [SAS], African [AFR], and European [EUR]) to perform drug-target Mendelian randomization investigating relationships between PCSK9 and HMGCR inhibition and alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), and bilirubin. RESULTS: Analyses of PCSK9 instruments, including functional variants R46L and E670G, failed to find evidence for relationships of low-density lipoprotein cholesterol lowering via PCSK9 variants and adverse effects on ALT, AST, GGT, or ALP among the cohorts. PCSK9 inhibition was associated with increased direct bilirubin levels in EUR (ß = 0.089; P value = 5.69 × 10-6) and, nominally, in AFR (ß = 0.181; P value = .044). HMGCR inhibition was associated with reduced AST in SAS (ß = -0.705; P value = .005) and, nominally, reduced AST in EAS (ß = -0.096; P value = .03), reduced ALP in EUR (ß = -2.078; P value = .014), and increased direct bilirubin in EUR (ß = 0.071; P value = .032). Sensitivity analyses using genetic instruments derived from circulating PCSK9 protein levels, tissue-specific PCSK9 expression, and HMGCR expression were in alignment, strengthening causal inference. CONCLUSIONS: We did not find ALT, AST, GGT, or ALP associated with genetically proxied PCSK9 and HMGCR inhibition across ancestries. We identified possible relationships in several ancestries between PCSK9 and increased direct and total bilirubin and between HMGCR and reduced AST. These findings support long-term safety profiles and low hepatotoxic risk of PCSK9 and HMGCR inhibition in diverse populations.

Major Psychiatric Disorders, Substance Use Behaviors, and Longevity.

Importance: Observational studies suggest that major psychiatric disorders and substance use behaviors reduce longevity, making it difficult to disentangle their relationships with aging-related outcomes. Objective: To evaluate the associations between the genetic liabilities for major psychiatric disorders, substance use behaviors (smoking and alcohol consumption), and longevity. Design, Settings, and Participants: This 2-sample mendelian randomization (MR) study assessed associations between psychiatric disorders, substance use behaviors, and longevity using single-variable and multivariable models. Multiomics analyses were performed elucidating transcriptomic underpinnings of the MR associations and identifying potential proteomic therapeutic targets. This study sourced summary-level genome-wide association study (GWAS) data, gene expression, and proteomic data from cohorts of European ancestry. Analyses were performed from May 2022 to November 2023. Exposures: Genetic susceptibility for major depression (n = 500 199), bipolar disorder (n = 413 466), schizophrenia (n = 127 906), problematic alcohol use (n = 435 563), weekly alcohol consumption (n = 666 978), and lifetime smoking index (n = 462 690). Main Outcomes and Measures: The main outcome encompassed aspects of health span, lifespan, and exceptional longevity. Additional outcomes were epigenetic age acceleration (EAA) clocks. Results: Findings from multivariable MR models simultaneously assessing psychiatric disorders and substance use behaviorsm suggest a negative association between smoking and longevity in cohorts of European ancestry (n = 709 709; 431 503 [60.8%] female; ß, -0.33; 95% CI, -0.38 to -0.28; P = 4.59 × 10-34) and with increased EAA (n = 34 449; 18 017 [52.3%] female; eg, PhenoAge: ß, 1.76; 95% CI, 0.72 to 2.79; P = 8.83 × 10-4). Transcriptomic imputation and colocalization identified 249 genes associated with smoking, including 36 novel genes not captured by the original smoking GWAS. Enriched pathways included chromatin remodeling and telomere assembly and maintenance. The transcriptome-wide signature of smoking was inversely associated with longevity, and estimates of individual smoking-associated genes, eg, XRCC3 and PRMT6, aligned with the smoking-longevity MR analyses, suggesting underlying transcriptomic mediators. Cis-instrument MR prioritized brain proteins associated with smoking behavior, including LY6H (ß, 0.02; 95% CI, 0.01 to 0.03; P = 2.37 × 10-6) and RIT2 (ß, 0.02; 95% CI, 0.01 to 0.03; P = 1.05 × 10-5), which had favorable adverse-effect profiles across 367 traits evaluated in phenome-wide MR. Conclusions: The findings suggest that the genetic liability of smoking, but not of psychiatric disorders, is associated with longevity. Transcriptomic associations offer insights into smoking-related pathways, and identified proteomic targets may inform therapeutic development for smoking cessation strategies.

Plate-based diversity subset screening: an efficient paradigm for high throughput screening of a large screening file.

The screening files of many large companies, including Pfizer, have grown considerably due to internal chemistry efforts, company mergers and acquisitions, external contracted synthesis, or compound purchase schemes. In order to screen the targets of interest in a cost-effective fashion, we devised an easy-to-assemble, plate-based diversity subset (PBDS) that represents almost the entire computed chemical space of the screening file whilst comprising only a fraction of the plates in the collection. In order to create this file, we developed new design principles for the quality assessment of screening plates: the Rule of 40 (Ro40) and a plate selection process that insured excellent coverage of both library chemistry and legacy chemistry space. This paper describes the rationale, design, construction, and performance of the PBDS, that has evolved into the standard paradigm for singleton (one compound per well) high-throughput screening in Pfizer since its introduction in 2006.

Influence of climate on malaria transmission depends on daily temperature variation.

Malaria transmission is strongly influenced by environmental temperature, but the biological drivers remain poorly quantified. Most studies analyzing malaria-temperature relations, including those investigating malaria risk and the possible impacts of climate change, are based solely on mean temperatures and extrapolate from functions determined under unrealistic laboratory conditions. Here, we present empirical evidence to show that, in addition to mean temperatures, daily fluctuations in temperature affect parasite infection, the rate of parasite development, and the essential elements of mosquito biology that combine to determine malaria transmission intensity. In general, we find that, compared with rates at equivalent constant mean temperatures, temperature fluctuation around low mean temperatures acts to speed up rate processes, whereas fluctuation around high mean temperatures acts to slow processes down. At the extremes (conditions representative of the fringes of malaria transmission, where range expansions or contractions will occur), fluctuation makes transmission possible at lower mean temperatures than currently predicted and can potentially block transmission at higher mean temperatures. If we are to optimize control efforts and develop appropriate adaptation or mitigation strategies for future climates, we need to incorporate into predictive models the effects of daily temperature variation and how that variation is altered by climate change.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) in the central nervous system.

The gene encoding proprotein convertase subtilisin/kexin type 9 (PCSK9) and its protein product have been widely studied for their role in cholesterol and lipid metabolism. PCSK9 increases the rate of metabolic degradation of low-density lipoprotein receptors, preventing the diffusion of low-density lipoprotein (LDL) from plasma into cells and contributes to high lipoprotein-bound cholesterol levels in the plasma. While most research has focused on the regulation and disease relevance of PCSK9 to the cardiovascular system and lipid metabolism, there is a growing body of evidence that PCSK9 plays a crucial role in pathogenic processes in other organ systems, including the central nervous system. PCSK9's impact on the brain is not yet fully understood, though several recent studies have sought to illuminate its impact on various neurodegenerative and psychiatric disorders, as well as its connection with ischemic stroke. Cerebral PCSK9 expression is low but is highly upregulated during disease states. Among others, PCSK9 is known to play a role in neurogenesis, neural cell differentiation, central LDL receptor metabolism, neural cell apoptosis, neuroinflammation, Alzheimer's Disease, Alcohol Use Disorder, and stroke. The PCSK9 gene contains several polymorphisms, including both gain-of-function and loss-of-function mutations which profoundly impact normal PCSK9 signaling and cholesterol metabolism. Gain-of-function mutations lead to persistent hypercholesterolemia and poor health outcomes, while loss-of-function mutations generally lead to hypocholesterolemia and may serve as a protective factor against diseases of the liver, cardiovascular system, and central nervous system. Recent genomic studies have sought to identify the end-organ effects of such mutations and continue to identify evidence of a much broader role for PCSK9 in extrahepatic organ systems. Despite this, there remain large gaps in our understanding of PCSK9, its regulation, and its effects on disease risk outside the liver. This review, which incorporates data from a wide range of scientific disciplines and experimental paradigms, is intended to describe PCSK9's role in the central nervous system as it relates to cerebral disease and neuropsychiatric disorders, and to examine the clinical potential of PCSK9 inhibitors and genetic variation in the PCSK9 gene on disease outcomes, including neurological and neuropsychiatric disease.

Multi-omic underpinnings of epigenetic aging and human longevity.

Biological aging is accompanied by increasing morbidity, mortality, and healthcare costs; however, its molecular mechanisms are poorly understood. Here, we use multi-omic methods to integrate genomic, transcriptomic, and metabolomic data and identify biological associations with four measures of epigenetic age acceleration and a human longevity phenotype comprising healthspan, lifespan, and exceptional longevity (multivariate longevity). Using transcriptomic imputation, fine-mapping, and conditional analysis, we identify 22 high confidence associations with epigenetic age acceleration and seven with multivariate longevity. FLOT1, KPNA4, and TMX2 are novel, high confidence genes associated with epigenetic age acceleration. In parallel, cis-instrument Mendelian randomization of the druggable genome associates TPMT and NHLRC1 with epigenetic aging, supporting transcriptomic imputation findings. Metabolomics Mendelian randomization identifies a negative effect of non-high-density lipoprotein cholesterol and associated lipoproteins on multivariate longevity, but not epigenetic age acceleration. Finally, cell-type enrichment analysis implicates immune cells and precursors in epigenetic age acceleration and, more modestly, multivariate longevity. Follow-up Mendelian randomization of immune cell traits suggests lymphocyte subpopulations and lymphocytic surface molecules affect multivariate longevity and epigenetic age acceleration. Our results highlight druggable targets and biological pathways involved in aging and facilitate multi-omic comparisons of epigenetic clocks and human longevity.

Multivariate genome-wide analysis of aging-related traits identifies novel loci and new drug targets for healthy aging.

The concept of aging is complex, including many related phenotypes such as healthspan, lifespan, extreme longevity, frailty and epigenetic aging, suggesting shared biological underpinnings; however, aging-related endpoints have been primarily assessed individually. Using data from these traits and multivariate genome-wide association study methods, we modeled their underlying genetic factor ('mvAge'). mvAge (effective n = ~1.9 million participants of European ancestry) identified 52 independent variants in 38 genomic loci. Twenty variants were novel (not reported in input genome-wide association studies). Transcriptomic imputation identified age-relevant genes, including VEGFA and PHB1. Drug-target Mendelian randomization with metformin target genes showed a beneficial impact on mvAge (P value = 8.41 × 10-5). Similarly, genetically proxied thiazolidinediones (P value = 3.50 × 10-10), proprotein convertase subtilisin/kexin 9 inhibition (P value = 1.62 × 10-6), angiopoietin-like protein 4, beta blockers and calcium channel blockers also had beneficial Mendelian randomization estimates. Extending the drug-target Mendelian randomization framework to 3,947 protein-coding genes prioritized 122 targets. Together, these findings will inform future studies aimed at improving healthy aging.

Additive Effects of Stress and Alcohol Exposure on Accelerated Epigenetic Aging in Alcohol Use Disorder.

BACKGROUND: Stress contributes to premature aging and susceptibility to alcohol use disorder (AUD), and AUD itself is a factor in premature aging; however, the interrelationships of stress, AUD, and premature aging are poorly understood. METHODS: We constructed a composite score of stress from 13 stress-related outcomes in a discovery cohort of 317 individuals with AUD and control subjects. We then developed a novel methylation score of stress (MS stress) as a proxy of composite score of stress comprising 211 CpGs selected using a penalized regression model. The effects of MS stress on health outcomes and epigenetic aging were assessed in a sample of 615 patients with AUD and control subjects using epigenetic clocks and DNA methylation-based telomere length. Statistical analysis with an additive model using MS stress and a MS for alcohol consumption (MS alcohol) was conducted. Results were replicated in 2 independent cohorts (Generation Scotland, N = 7028 and the Grady Trauma Project, N = 795). RESULTS: Composite score of stress and MS stress were strongly associated with heavy alcohol consumption, trauma experience, epigenetic age acceleration (EAA), and shortened DNA methylation-based telomere length in AUD. Together, MS stress and MS alcohol additively showed strong stepwise increases in EAA. Replication analyses showed robust association between MS stress and EAA in the Generation Scotland and Grady Trauma Project cohorts. CONCLUSIONS: A methylation-derived score tracking stress exposure is associated with various stress-related phenotypes and EAA. Stress and alcohol have additive effects on aging, offering new insights into the pathophysiology of premature aging in AUD and, potentially, other aspects of gene dysregulation in this disorder.

Virulence, drug sensitivity and transmission success in the rodent malaria, Plasmodium chabaudi.

Here, we test the hypothesis that virulent malaria parasites are less susceptible to drug treatment than less virulent parasites. If true, drug treatment might promote the evolution of more virulent parasites (defined here as those doing more harm to hosts). Drug-resistance mechanisms that protect parasites through interactions with drug molecules at the sub-cellular level are well known. However, parasite phenotypes associated with virulence might also help parasites survive in the presence of drugs. For example, rapidly replicating parasites might be better able to recover in the host if drug treatment fails to eliminate parasites. We quantified the effects of drug treatment on the in-host survival and between-host transmission of rodent malaria (Plasmodium chabaudi) parasites which differed in virulence and had never been previously exposed to drugs. In all our treatment regimens and in single- and mixed-genotype infections, virulent parasites were less sensitive to pyrimethamine and artemisinin, the two antimalarial drugs we tested. Virulent parasites also achieved disproportionately greater transmission when exposed to pyrimethamine. Overall, our data suggest that drug treatment can select for more virulent parasites. Drugs targeting transmission stages (such as artemisinin) may minimize the evolutionary advantage of virulence in drug-treated infections.

Complex effects of temperature on mosquito immune function.

Over the last 20 years, ecological immunology has provided much insight into how environmental factors shape host immunity and host-parasite interactions. Currently, the application of this thinking to the study of mosquito immunology has been limited. Mechanistic investigations are nearly always conducted under one set of conditions, yet vectors and parasites associate in a variable world. We highlight how environmental temperature shapes cellular and humoral immune responses (melanization, phagocytosis and transcription of immune genes) in the malaria vector, Anopheles stephensi. Nitric oxide synthase expression peaked at 30°C, cecropin expression showed no main effect of temperature and humoral melanization, and phagocytosis and defensin expression peaked around 18°C. Further, immune responses did not simply scale with temperature, but showed complex interactions between temperature, time and nature of immune challenge. Thus, immune patterns observed under one set of conditions provide little basis for predicting patterns under even marginally different conditions. These quantitative and qualitative effects of temperature have largely been overlooked in vector biology but have significant implications for extrapolating natural/transgenic resistance mechanisms from laboratory to field and for the efficacy of various vector control tools.

Shaping a screening file for maximal lead discovery efficiency and effectiveness: elimination of molecular redundancy.

High Throughput Screening (HTS) is a successful strategy for finding hits and leads that have the opportunity to be converted into drugs. In this paper we highlight novel computational methods used to select compounds to build a new screening file at Pfizer and the analytical methods we used to assess their quality. We also introduce the novel concept of molecular redundancy to help decide on the density of compounds required in any region of chemical space in order to be confident of running successful HTS campaigns.

PCSK9 Base Editing Therapeutics and Ischemic Stroke.

Individuals with genetic gain-of-function variation in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene are at an increased risk of cardiovascular disease, including ischemic stroke. While PCSK9 inhibitors (PCSK9i) are effective in reducing cardiovascular disease risk and ischemic stroke risk, novel genomic technologies including the use of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 complex-mediated delivery and adenine base editing (ABE) enable promising new therapeutic and preventative approaches. In this paper we discuss ongoing work into PCSK9 base editing and highlight future directions relevant to cardiovascular disease and ischemic stroke.

Mendelian Randomization Study of PCSK9 and HMG-CoA Reductase Inhibition and Cognitive Function.

BACKGROUND: Lipid-lowering therapy with statins and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition are effective strategies in reducing cardiovascular disease risk; however, concerns remain about potential long-term adverse neurocognitive effects. OBJECTIVES: This genetics-based study aimed to evaluate the relationships of long-term PCSK9 inhibition and statin use on neurocognitive outcomes. METHODS: We extracted single-nucleotide polymorphisms in 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) and PCSK9 from predominantly European ancestry-based genome-wide association studies summary-level statistics of low-density lipoprotein cholesterol and performed drug-target Mendelian randomization, proxying the potential neurocognitive impact of drug-based PCSK9 and HMGCR inhibition using a range of outcomes to capture the complex facets of cognition and dementia. RESULTS: Using data from a combined sample of ∼740,000 participants, we observed a neutral cognitive profile related to genetic PCSK9 inhibition, with no significant effects on cognitive performance, memory performance, or cortical surface area. Conversely, we observed several adverse associations for HMGCR inhibition with lowered cognitive performance (beta: -0.082; 95% CI: -0.16 to -0.0080; P = 0.03), reaction time (beta = 0.00064; 95% CI: 0.00030-0.00098; P = 0.0002), and cortical surface area (beta = -0.18; 95% CI: -0.35 to -0.014; P = 0.03). Neither PCSK9 nor HMGCR inhibition impacted biomarkers of Alzheimer's disease progression or Lewy body dementia risk. Consistency of findings across Mendelian randomization methods accommodating different assumptions about genetic pleiotropy strengthens causal inference. CONCLUSIONS: Using a wide range of cognitive function and dementia endpoints, we failed to find genetic evidence of an adverse PCSK9-related impact, suggesting a neutral cognitive profile. In contrast, we observed adverse neurocognitive effects related to HMGCR inhibition, which may well be outweighed by the cardiovascular benefits of statin use, but nonetheless may warrant pharmacovigilance.

Comparing the Relationships of Genetically Proxied PCSK9 Inhibition With Mood Disorders, Cognition, and Dementia Between Men and Women: A Drug-Target Mendelian Randomization Study.

Background PCSK9 (proprotein convertase subtilisin/kexin type 9) inhibitors are important therapeutic options for reducing cardiovascular disease risk; however, questions remain regarding potential differences in the neuropsychiatric impact of long-term PCSK9 inhibition between men and women. Methods and Results Using PCSK9 gene single-nucleotide polymorphisms from European ancestry-based genome-wide association studies of low-density lipoprotein cholesterol (N=1 320 016), circulating PCSK9 protein levels (N=10 186), tissue-specific PCSK9 gene expression, sex-specific genome-wide association studies of anxiety, depression, cognition, insomnia, and dementia (ranging from 54 321 to 194 174), we used drug-target inverse variance-weighted Mendelian randomization (MR) and complementary MR methods (MR Egger, weighted median, and weighted mode) to investigate potential neuropsychiatric consequences of genetically proxied PCSK9 inhibition in men and women. We failed to find evidence surpassing correction for multiple comparisons of relationships between genetically proxied PCSK9 inhibition and the risk for the 12 neuropsychiatric end points in either men or women. Drug-target analyses were generally well-powered to detect effect estimates at several hypothesized thresholds for both combined-sex and sex-specific end points, especially analyses using PCSK9 instruments derived from protein and expression quantitative trait loci. Further, MR estimates across complementary MR methods and additional models using genetic instruments derived from circulating PCSK9 protein levels and tissue-specific PCSK9 expression were in alignment, strengthening causal inference. Conclusions Genetically proxied PCSK9 inhibition showed a neutral neuropsychiatric side effect profile with no major sex-specific differences. Given statistical power considerations, replication with larger samples, as well as data from other ancestral populations, are necessary. These findings may have important clinical implications for lipid-lowering drug-prescribing practices and side effect monitoring of approved and future PCSK9 therapies.

Challenges of drug discovery in novel target space. The discovery and evaluation of PF-3893787: a novel histamine H4 receptor antagonist.

We describe the development of novel benzimidazoles as small molecule histamine H4 receptor (H4R) antagonists and their profiling in rat early toxicity studies. The discovery and optimisation of a second series of pyrimidine based antagonists is then described culminating in the identification of the clinical development candidate 13 (PF-3893787). The pre-clinical profile of 13 (PF-3893787) is presented including the development of a translatable biomarker. Our pragmatic approach to target selection, safety assessment, and testing for efficacy faced numerous challenges and we share a number of lessons which the team learned and which will assist us and others in future drug discovery projects.