Lorson Zachary C, McFadden William M, Neilsen Grace, Castaner Andres E, Slack Ryan L, Kirby Karen A, Sarafianos Stefan G
Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, 1760 Haygood Drive NE, Atlanta, GA.
Children's Healthcare of Atlanta, Atlanta, GA.
bioRxiv. 2025 Jul 24:2025.07.24.662752. doi: 10.1101/2025.07.24.662752.
Small molecule-protein interactions underpin many biological functions and play an integral role in the treatment and prevention of several human diseases. These interactions can be key to understanding the mechanism of action of these compounds. Previous methods of determining protein-protein or protein-antibody interactions have been well established; however, the use of BLI in antiviral discovery is a promising and relatively new avenue. The high-throughput nature of this method in tandem with its pM sensitivity allows for quick and seamless identification of hit compounds. Here we discuss ways to overcome common pitfalls that can occur while using BLI such as nonspecific binding (NSB) and ligand drift while offering possible solutions. Characterizing small molecule-protein interactions is not trivial and optimizing the experimental conditions is imperative. To address this gap in knowledge, we present optimized BLI protocols for the study of three cases of protein-small molecule interactions: PF74 or Lenacapavir (LEN) with HIV-1 capsid protein (CA), and Nirmatrelvir (NIR) with SARS-CoV-2 Mpro. LEN and NIR are of particular interest because they are clinically relevant, and PF74, a well-studied control, was the first compound reported to target the LEN binding site. We demonstrate that BLI can be a powerful and effective tool in calculating the binding affinities between a protein and small molecule. These newly designed methods enabled calculation of K values, the affinity between ligand and analyte, ranging from the micro to the sub-nanomolar range for CA binding events and confirmed the covalent interaction between NIR and Mpro. These protocols will facilitate efficient testing of new antivirals or derivatives in a high-throughput format.
小分子与蛋白质的相互作用是许多生物学功能的基础，在多种人类疾病的治疗和预防中发挥着不可或缺的作用。这些相互作用可能是理解这些化合物作用机制的关键。此前确定蛋白质-蛋白质或蛋白质-抗体相互作用的方法已经相当成熟；然而，生物层干涉术（BLI）在抗病毒药物研发中的应用是一个前景广阔且相对较新的途径。该方法的高通量特性及其皮摩尔级别的灵敏度，使得能够快速且无缝地鉴定出有活性的化合物。在此，我们讨论在使用BLI时可能出现的常见问题，如非特异性结合（NSB）和配体漂移，并提供可能的解决方案。表征小分子与蛋白质的相互作用并非易事，优化实验条件至关重要。为填补这一知识空白，我们针对三种蛋白质-小分子相互作用的研究，提出了优化的BLI方案：PF74或来那卡帕韦（LEN）与HIV-1衣壳蛋白（CA），以及奈玛特韦（NIR）与SARS-CoV-2 Mpro。LEN和NIR特别受关注，因为它们具有临床相关性，而PF74作为一个经过充分研究的对照，是首个被报道靶向LEN结合位点的化合物。我们证明，BLI可以成为计算蛋白质与小分子之间结合亲和力的强大而有效的工具。这些新设计的方法能够计算出K值，即配体与分析物之间的亲和力，对于CA结合事件，其范围从微摩尔到亚纳摩尔级别，并证实了NIR与Mpro之间的共价相互作用。这些方案将有助于以高通量形式高效测试新的抗病毒药物或衍生物。
