An explainable model of host genetic interactions linked to COVID-19 severity

We employed a multifaceted computational strategy to identify the genetic factors contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing (WES) dataset of a cohort of 2000 Italian patients. We coupled a stratified k-fold screening, to rank variants more associated with severity, with the training of multiple supervised classifiers, to predict severity based on screened features. Feature importance analysis from tree-based models allowed us to identify 16 variants with the highest support which, together with age and gender covariates, were found to be most predictive of COVID-19 severity. When tested on a follow-up cohort, our ensemble of models predicted severity with high accuracy (ACC = 81.88%; AUCROC = 96%; MCC = 61.55%). Our model recapitulated a vast literature of emerging molecular mechanisms and genetic factors linked to COVID-19 response and extends previous landmark Genome-Wide Association Studies (GWAS). It revealed a network of interplaying genetic signatures converging on established immune system and inflammatory processes linked to viral infection response. It also identified additional processes cross-talking with immune pathways, such as GPCR signaling, which might offer additional opportunities for therapeutic intervention and patient stratification. Publicly available PheWAS datasets revealed that several variants were significantly associated with phenotypic traits such as "Respiratory or thoracic disease", supporting their link with COVID-19 severity outcome.A multifaceted computational strategy identifies 16 genetic variants contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing dataset of a cohort of Italian patients

Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes

Carriers of single pathogenic variants of the CFTR (cystic fibrosis transmembrane conductance regulator) gene have a higher risk of severe COVID-19 and 14-day death. The machine learning post-Mendelian model pinpointed CFTR as a bidirectional modulator of COVID-19 outcomes. Here, we demonstrate that the rare complex allele [G576V;R668C] is associated with a milder disease via a gain-of-function mechanism. Conversely, CFTR ultra-rare alleles with reduced function are associated with disease severity either alone (dominant disorder) or with another hypomorphic allele in the second chromosome (recessive disorder) with a global residual CFTR activity between 50 to 91%. Furthermore, we characterized novel CFTR complex alleles, including [A238V;F508del], [R74W;D1270N;V201M], [I1027T;F508del], [I506V;D1168G], and simple alleles, including R347C, F1052V, Y625N, I328V, K68E, A309D, A252T, G542*, V562I, R1066H, I506V, I807M, which lead to a reduced CFTR function and thus, to more severe COVID-19. In conclusion, CFTR genetic analysis is an important tool in identifying patients at risk of severe COVID-19

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

Design and validation of bioorthogonal probes for cellular disease models

Well-validated chemical probes enable testing of biological hypotheses, investigation of target tractability and translatability to the clinical phase. Consequently, these important tool compounds play a key role in the drug discovery process. Moreover, their use for target validation may ultimately help to decrease the attrition rate encountered by new molecular entities in clinical trials. Despite the number of publications describing well-validated chemical probes, for several reasons, it remains a difficult challenge to identify and select the appropriate high quality molecule enabling the desired biological and pharmacological studies in the relevant disease model. Various reports describe sets of principles to be fulfilled by high quality chemical probes, which mainly rely on verifying that chemical probes are potent, engage their intended targets in the relevant cellular model, have sufficient exposure at the desired site of action and express functional pharmacological activities by a selective modulation of their targets. Classical chemical probes include LMW ligands, which inform on the functional consequences of interacting with a particular bio-logical target in a model system. To get information on other parameters (such as sub-cellular distribution of target or compound) different tools may need to be used, often requiring specific chemical functionalization in order to observe the molecule using the currently available technologies. Compared to classical chemical probes, bioorthogonal probes can also be small molecules that elicit a functional response but with the additional advantage of being able to undergo reaction with a variety of chemical reporters (e.g. fluorophores) in situ, in bio-logical model systems such as cells and animals. Therefore, this increases their versatility; for example their visualization using imaging technologies (bioluminescence and fluorescence imaging) can provide important insights on compound permeability, in-tracellular distribution, and potentially co-localization with its protein target in relevant cellular disease models. Hence, the use of a bespoke bioorthogonal probe may answer key biological questions, which are usually only addressed by using multiple classical probes. Herein, we describe the design and the synthesis of bioorthogonal probes to study the inhibition of the p53-Mdm2 protein-protein interaction. Furthermore, we report the development of a set of assays based on fluorescence and bioluminescence techniques enabling the validation of these molecules in a cellular osteosarcoma model. Our approach for chemical probe design and validation in cellular disease models may be applied in principle to a wide range of novel drug discovery projects to provide early mechanistic understanding

Evaluating Cellular Uptake of Drugs with Fluorescent Sensor Proteins

Here we introduce a qualitative approach to evaluate cellular uptake of inhibitors with spatiotemporal resolution in living cells. The approach is based on con-verting the protein target of a given class of inhibitors into a fluorescent biosensor. By measuring the affinity and kinetics of binding of different inhibitors to their cognate biosensor in live cells and comparing these values to those measured in vitro, the cellular uptake and concentrations of the inhibitors can be ranked. The approach is label-free and does not require the measurement of a biological read-out of the inhibition. We demonstrate the feasibility of the approach by evaluating cellular uptake of two different classes of inhibitors into the cytosol of living cells: inhibitors of the enzyme human carbonic anhydrase II and inhibitors of the protein-protein interaction between p53 and HDM2

Real-time imaging and quantification of peptide uptake in vitro and in vivo

Peptides constitute an important class of molecules for drug discovery, but many of the leads fail to advance clinically because of poor membrane and tissue permeability. Therefore, assessment of a peptide’s ability to cross cellular membrane is critical when developing novel peptide-based therapeutics. However, current methods suffer from limitations such as the necessity to introduce rather large modifications that require complex chemistry, the inability to provide kinetic information of internalization or distinguish between internalized vs membrane bound compounds, as well as requiring multiple sample manipulation steps. Herein, we report a novel “Split Luciferin Peptide” (SLP) uptake assay that provides non-invasive imaging and quantification of peptide uptake in real-time both in vitro and in vivo using a very sensitive bioluminescent readout. The method is based on a straightforward chemical modification of the peptide of interest with a D-cysteine tag retains the overall peptidic character of the original molecule. This method can in principle be adapted for screening of peptide libraries becoming an important tool for preclinical drug development both in vitro and in vivo

Real-Time Imaging and Quantification of Peptide Uptake in Vitro and in Vivo

Peptides constitute an important class of drugs for the treatment of multiple metabolic, ontological, and neurodegenerative diseases, and several hundred novel therapeutic peptides are currently in the preclinical and clinical stages of development. However, many leads fail to advance clinically because of poor cellular membrane and tissue permeability. Therefore, assessment of the ability of a peptide to cross cellular membranes is critical when developing novel peptide-based therapeutics. Current methods to assess peptide cellular permeability are limited by multiple factors, such as the need to introduce rather large modifications (e.g., fluorescent dyes) that require complex chemical reactions as well as an inability to provide kinetic information on the internalization of a compound or distinguish between internalized and membrane-bound compounds. In addition, many of these methods are based on end point assays and require multiple sample manipulation steps. Herein, we report a novel "Split Luciferin Peptide" (SLP) assay that enables the real-time noninvasive imaging and quantification of peptide uptake both in vitro and in vivo using a very sensitive bioluminescence readout. This method is based on a straightforward, stable chemical modification of the peptide of interest with a D-cysteine tag that preserves the overall peptidic character of the original molecule. This method can be easily adapted for screening peptide libraries and can thus become an important tool for preclinical peptide drug development

Bioorthogonal Probes to study MDM2-p53 inhibitors in cells and to develop high content screening assays for drug discovery

To study the behavior of MDM2-p53 inhibitors in a disease-relevant cellular model, we have developed and validated a set of bioorthogonal probes that can be fluorescently labeled in cells and used in high content screening assays. Using automated image analysis and single cell resolution, we could visualize the intracellular target binding of compounds by co-localization and quantify target upregulation upon MDM2-p53 inhibition in an osteosarcoma model. In addition, we developed a high throughput assay to quantify target occupancy of non-tagged MDM2-p53 inhibitors by competition and to identify novel chemical matter. This approach could be expanded to other targets for lead discovery applications

A Strategy to Assess the Cellular Activity of E3 Ligases against Neo-Substrates using Electrophilic Probes

Targeted protein degradation promises to enable small molecule-mediated modulation of currently undrugged proteins. While the well-characterized E3 ligases CRBN and VHL have successfully promoted the degradation of many proteins of interest, there are approximately 600 additional E3 ligase family members that may offer improved activity, substrate selectivity, or tissue distribution; however, characterizing the ability of these many ligases to promote targeted protein degradation has proven challenging. Here, we report the development of a rapid method to evaluate the ability of recombinant E3 ligase components to support the degradation of neo-substrates. Bypassing the need for hit finding to identify specific E3 ligase binders, this approach makes use of simple chemistry for Covalent Functionalization Followed by E3 Electroporation into live cells (COFFEE). We demonstrate this method using covalent E3-target binder complexes of VHL-JQ1 and VHL-dasatinib and show the degradation of Brd4 and Lyn kinase, respectively. Applying COFFEE to SPSB2, a SOCS box and SPRY-domain E3 ligase not previously shown to degrade neo-substrates, we demonstrated the ability of this method to rapidly validate an uncharacterized ligase for degradation of neo-substrates