The relentless evolution of SARS-CoV-2 poses a significant threat to public health, as it adapts to immune pressure from vaccines and natural infections. Gaining insights into potential antigenic changes is critical but challenging due to the vast sequence space. Here, we introduce the Machine Learning-guided Antigenic Evolution Prediction (MLAEP), which combines structure modeling, multi-task learning, and genetic algorithms to predict the viral fitness landscape and explore antigenic evolution via in silico directed evolution. By analyzing existing SARS-CoV-2 variants, MLAEP accurately infers variant order along antigenic evolutionary trajectories, correlating with corresponding sampling time. Our approach identified novel mutations in immunocompromised COVID-19 patients and emerging variants like XBB1.5. Additionally, MLAEP predictions were validated through in vitro neutralizing antibody binding assays, demonstrating that the predicted variants exhibited enhanced immune evasion. By profiling existing variants and predicting potential antigenic changes, MLAEP aids in vaccine development and enhances preparedness against future SARS-CoV-2 variants.
© 2023. The Author(s).

Building on our previous work on ibrutinib-based reversible covalent Bruton's tyrosine kinase (BTK) PROTACs, we explored a different irreversible BTK inhibitor poseltinib as the BTK binder for PROTAC development. Different from ibrutinib, converting the irreversible cysteine reacting acrylamide group of poseltinib to a reversible covalent cyano-acrylamide group dramatically decreases the binding affinity to BTK by over 700 folds. Interestingly, one of the reversible covalent BTK PROTACs based on poseltinib with a rigid linker, dubbed as PS-RC-1, is highly potent (IC50 = ~10 nM) in Mino cells but not in other mantle cell lymphoma (MCL) cell lines, such as Jeko-1 and Rec-R cells. We showed that PS-RC-1 potently induces degradation of IKZF1 and IKZF3 but not BTK or GSPT1, accounting for its toxicity in Mino cells. We further decreased the molecular size of PS-RC-1 by shrinking the BTK binding moiety and developed PS-2 as a potent BTK and IKZF1/3 triple degrader with high specificity.

CD47 is an immune checkpoint molecule that downregulates key aspects of both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα, and it is expressed at high levels in a wide variety of tumor types. This has led to the development of biologics that inhibit SIRPα engagement including humanized CD47 antibodies and a soluble SIRPα decoy receptor that are currently undergoing clinical trials. Unfortunately, toxicological issues, including anemia related to on-target mechanisms, are barriers to their clinical advancement. Another potential issue with large biologics that bind CD47 is perturbation of CD47 signaling through its high-affinity interaction with the matricellular protein thrombospondin-1 (TSP1). One approach to avoid these shortcomings is to identify and develop small molecule molecular probes and pretherapeutic agents that would (1) selectively target SIRPα or TSP1 interactions with CD47, (2) provide a route to optimize pharmacokinetics, reduce on-target toxicity and maximize tissue penetration, and (3) allow more flexible routes of administration. As the first step toward this goal, we report the development of an automated quantitative high-throughput screening (qHTS) assay platform capable of screening large diverse drug-like chemical libraries to discover novel small molecules that inhibit CD47-SIRPα interaction. Using time-resolved Förster resonance energy transfer (TR-FRET) and bead-based luminescent oxygen channeling assay formats (AlphaScreen), we developed biochemical assays, optimized their performance, and individually tested them in small-molecule library screening. Based on performance and low false positive rate, the LANCE TR-FRET assay was employed in a ~90,000 compound library qHTS, while the AlphaScreen oxygen channeling assay served as a cross-validation orthogonal assay for follow-up characterization. With this multi-assay strategy, we successfully eliminated compounds that interfered with the assays and identified five compounds that inhibit the CD47-SIRPα interaction; these compounds will be further characterized and later disclosed. Importantly, our results validate the large library qHTS for antagonists of CD47-SIRPα interaction and suggest broad applicability of this approach to screen chemical libraries for other protein-protein interaction modulators.

The Bcl-2 family proteins are key regulators of the intrinsic apoptotic pathway and are among the validated targets for developing anticancer drugs. Protein-protein interactions between the pro- and antiapoptotic members of this family determine mitochondrial outer-membrane permeabilization. Elucidating such protein-protein interactions in a quantitative way is helpful for network pharmacology studies on the Bcl-2 family, which, in turn, will provide valuable guidance for developing new anticancer therapies. In this study, the binding affinities of the BH3 peptides derived from eight proapoptotic BH3-only proteins (i.e., Bid, Bim, Puma, Noxa, Bad, Bmf, Bik, Hrk) against five well-studied antiapoptotic proteins (i.e., Bcl-xL , Bcl-2, Mcl-1, Bcl-w, Bfl-1) in the Bcl-2 family have been measured. Three different types of binding assay (i.e., surface plasmon resonance, fluorescence polarization, and homogeneous time-resolved fluorescence) were employed for cross-validation. The results confirmed that each proapoptotic BH3 peptide exhibited a distinct binding profile against the five antiapoptotic proteins. The binding data obtained herein serve as a fresh update or correction to existing knowledge. It is expected that such binding data will be helpful for building more accurate mathematical network models for depicting the complex protein-protein interactions within the Bcl-2 family.
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

G protein-coupled receptors (GPCRs) are excellent drug targets exploited by majority of the Food and Drug Administration-approved medications, but when modulated, are often accompanied by significant adverse effects. Targeting of other elements in GPCR pathways for improved safety and efficacy is thus an unmet need. The strength of GPCR signaling is tightly regulated by regulators of G protein signaling (RGS) proteins, making them attractive drug targets. We focused on a prominent RGS complex in the brain consisting of RGS7 and its binding partners Gβ5 and R7BP. These complexes play critical roles in regulating multiple GPCRs and essential physiological processes, yet no small molecule modulators are currently available to modify its function. In this study, we report a novel high-throughput approach to screen for small molecule modulators of the intramolecular transitions in the RGS7/Gβ5/R7BP complex known to be involved in its allosteric regulation. We developed a time-resolved fluorescence energy transfer-based in vitro assay that utilizes full-length recombinant proteins and shows consistency, excellent assay statistics, and high level of sensitivity. We demonstrated the potential of this approach by screening two compound libraries (LOPAC 1280 and MicroSource Spectrum). This study confirms the feasibility of the chosen strategy for identifying small molecule modulators of RGS7/Gβ5/R7BP complex for impacting signaling downstream of the GPCRs.