Unveiling antiviral protein targets and novel kinases with chemical proteomics

Abstract

Chemical proteomics approaches, such as Activity-based protein profiling (ABPP), have attracted tremendous attention in recent years, which is dedicated to systematically identifying a class of proteins with specific functions in complex life systems. A probe with specific binding affinity and conserved biological activity is significant for chemical proteomics study. In the dissertation, HCQ Probe and XO44 probe are used as bio-chemical tools for mechanism exploration in different pathological aspects. Hydroxychloroquine (HCQ) and chloroquine (CQ) have attracted tremendous attention throughout the COVID-19 pandemic as potential antiviral reagents. However, their inconsistent therapeutic effects against SARS-CoV-2 under different conditions led to the debate on their antiviral activities. To date, underlying mechanisms for the varied observations of HCQ and CQ have remained a mystery, which restricts their applications to prevent future viral epidemic or ongoing clinical trials to combat other diseases. To resolve this mystery, we applied a photo-crosslinking probe and proteomics approaches and identified Cathepsin L (CTSL), a lysosomal acid protease, as the binding target of HCQ/CQ, thereby elucidating their antiviral mechanisms at a molecular level. Extensive biological, chemical and in silico analyses were conducted to validate CTSL as a major target. HCQ significantly inhibited CTSL's enzymatic activity and thus suppress coronaviruses through the CTSL-dependent endosomal entry pathway. Importantly, given its specific target, we demonstrated that HCQ was effective on viruses that heavily rely on endosomal pathway entry, but not those that adopt alternative entry pathways. The inconsistent observations of HCQ/CQ's antiviral activities were due to varied viral strain and host models chosen, underscoring the importance of test conditions that could bias the conclusions. Besides coronaviruses, the mechanism we identified was applicable to other types of viruses. This project not only provide guidance for rational usage of HCQ/CQ, but also provided CTSL as a novel therapeutic target for future drug discovery. Protein kinases are essential for regulating cellular functions and biological processes by phosphorylating serine, threonine, and tyrosine residues, thereby facilitating signal transduction and the regulation of cellular physiology. Kinase, phosphorylation regulation and kinase inhibitor exploration have always been hot topics because abnormal phosphorylation leads to adverse diseases, like cancer. To date, 536 kinases have been identified in human with different organic distributions, functions, and disease symptoms when they are abnormal. However, kinases should be deeper identified in both function and quantities as a significant protein family, which will help to illustrate the generation and regulation mechanisms of more diseases. Recording to the human genome project, we learned that only less than 2% of human genome are encoding region, while the remaining regions were considered noncoding or even "junk". In recent years, it has come to light that many non-coding RNAs have coding potential to produce polypeptides generally shorter than known proteins. Such sequences in the genome were named small open reading frames (sORFs), which located in long non-coding RNAs (IncRNAs), untranslated regions (UTRs) and intergenic frameshift, etc. In this project, by combining proteome profiling and bioinformatics approaches, we identified a microprotein from non-coding region, called SEP119, which could express and would be a novel kinase. We observed SEP119 expressed in several cell lines and the sequence is conserved in Primates. Further ATP-binding and enzymatic assay confirmed the kinase activity of the SEP119, indicating the significant function in mechanism regulation, inhibitor development as well as therapeutic potential. More importantly, the applying ABPP to explore sORF-encoded microproteins pipeline we established could be applied to scrutinize microproteins with other biological activities, which will provide methodologies and tools to a broad spectrum of the research community.