Drug Target Validation of the Protein Kinase AEK1, Essential for Proliferation, Host Cell Invasion, and Intracellular Replication of the Human Pathogen Trypanosoma cruzi

Chagas disease affects 6 to 7 million people in the Americas, and its treatment has been limited to drugs with relatively high toxicity and low efficacy in the chronic phase of the infection. New validated targets are needed to combat this disease. In this work, we report the chemical and genetic validation of the protein kinase AEK1, which is essential for cytokinesis and infectivity, using a novel gene editing strategy.

Optimization of long-term human iPSC-derived spinal motor neuron culture using a dendritic polyglycerol amine-based substrate

Human induced pluripotent stem cells (h-iPSCs) derived from healthy and diseased individuals can give rise to many cell types, facilitating the study of mechanisms of development, human disease modeling, and early drug target validation. In this context, experimental model systems based on h-iPSC-derived motor neurons (MNs) have been used to study MN diseases such as spinal muscular atrophy and amyotrophic lateral sclerosis. Modeling MN disease using h-iPSC-based approaches requires culture conditions that can recapitulate in a dish the events underlying differentiation, maturation, aging, and death of MNs. Current h-iPSC-derived MN-based applications are often hampered by limitations in our ability to monitor MN morphology, survival, and other functional properties over a prolonged timeframe, underscoring the need for improved long-term culture conditions. Here we describe a cytocompatible dendritic polyglycerol amine (dPGA) substrate-based method for prolonged culture of h-iPSC-derived MNs. We provide evidence that MNs cultured on dPGA-coated dishes are more amenable to long-term study of cell viability, molecular identity, and spontaneous network electrophysiological activity. The present study has the potential to improve iPSC-based studies of human MN biology and disease.

Human rare variant and zebrafish CRISPR/Cas9-mediated mutant analyses reveal novel functions for API5, HSPB7, and LMO2 in heart failure

The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. To gain further insights into this complex pathophysiology we combined human rare variant analysis and in vivo CRISPR/Cas9-mediated mutant phenotyping in zebrafish to identify and investigate the role of 3 genes. Whole-exome sequencing of patients identified API5, HSPB7, and LMO2 as causally associated with heart failure and these genes were further investigated, alongside the positive control gata5, using CRISPR/Cas9-mediated multi-locus in vivo mutation in zebrafish. Following effective somatic mutation, we observed multiple impacts on cardiovascular development and function in F0 embryos including reductions in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2, there was also a significant impact on heart function. Our analysis suggests novel functions for API5, HSPB7, and LMO2 in human cardiovascular disease and identifies them as potential drug targets. Our data also supports in vivo CRISPR/Cas9-mediated multi-locus gene mutation analysis in F0 zebrafish as a rapid and effective primary screen for assessing gene function, as part of an integrated multi-level drug target validation strategy.

Mendelian Randomization in Stroke: A Powerful Approach to Causal Inference and Drug Target Validation

Stroke is a leading cause of death and disability worldwide. However, our understanding of its underlying biology and the number of available treatment options remain limited. Mendelian randomization (MR) offers a powerful approach to identify novel biological pathways and therapeutic targets for this disease. Around ~100 MR studies have been conducted so far to explore, confirm, and quantify causal relationships between several exposures and risk of stroke. In this review, we summarize the current evidence arising from these studies, including those investigating ischemic stroke, hemorrhagic stroke, or both. We highlight the different types of exposures that are currently under study, ranging from well-known cardiovascular risk factors to less established inflammation-related mechanisms. Finally, we provide an overview of future avenues of research and novel approaches, including drug target validation MR, which is poised to have a substantial impact on drug development and drug repurposing.

NADPH Producing Enzymes as Promising Drug Targets for Chagas Disease

Reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) is a cofactor used in different anabolic reactions, such as lipid and nucleic acid synthesis, and for oxidative stress defense. NADPH is essential for parasite growth and viability. In trypanosomatid parasites, NADPH is supplied by the oxidative branch of the pentose phosphate pathway and by enzymes associated with the citric acid cycle. The present article will review recent achievements that suggest glucose-6-phosphate dehydrogenase and the cytosolic isoform of the malic enzyme as promising drug targets for the discovery of new drugs against Trypanosoma cruzi and T. brucei. Topics involving an alternative strategy in accelerating T. cruzi drug-target validation and the concept of drug-target classification will also be revisited.

Genetic drug target validation using Mendelian randomization

Mendelian randomisation analysis has emerged as an important tool to elucidate the causal relevance of a range of environmental and biological risk factors for human disease. However, inference on cause is undermined if the genetic variants used to instrument a risk factor of interest also associate with other traits that open alternative pathways to the disease (horizontal pleiotropy). We show how the ‘no horizontal pleiotropy assumption’ in MR analysis is strengthened when proteins are the risk factors of interest. Proteins are the proximal effectors of biological processes encoded in the genome, and are becoming assayable on an-omics scale. Moreover, proteins are the targets of most medicines, so Mendelian randomization (MR) studies of drug targets are becoming a fundamental tool in drug development. To enable such studies we introduce a formal mathematical framework that contrasts MR analysis of proteins with that of risk factors located more distally in the causal chain from gene to disease. Finally, we illustrate key model decisions and introduce an analytical framework for maximizing power and elucidating the robustness of drug target MR analyses.