In mammals, fibroblast growth factors (FGFs) are encoded by 22 genes. FGFs bind and activate alternatively spliced forms of four tyrosine kinase FGF receptors (FGFRs 1-4). The spatial and temporal expression patterns of FGFs and FGFRs and the ability of specific ligand-receptor pairs to actively signal are important factors regulating FGF activity in a variety of biological processes. FGF signaling activity is regulated by the binding specificity of ligands and receptors and is modulated by extrinsic cofactors such as heparan sulfate proteoglycans. In previous studies, we have engineered BaF3 cell lines to express the seven principal FGFRs and used these cell lines to determine the receptor binding specificity of FGFs 1-9 by using relative mitogenic activity as the readout. Here we have extended these semiquantitative studies to assess the receptor binding specificity of the remaining FGFs 10 -23. This study completes the mitogenesis-based comparison of receptor specificity of the entire FGF family under standard conditions and should help in interpreting and predicting in vivo biological activity.

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The mammalian fibroblast growth factor (FGF) family comprises 18 polypeptides (FGF1 to FGF10 and FGF16 to FGF23) which participate in a myriad of biological processes during embryogenesis, including but not limited to gastrulation, body plan formation, somitogenesis, and morphogenesis of essentially every tissue/organ such as limb, lung, brain and kidney (3,30). FGFs execute their biological actions by binding to, dimerizing, and activating FGF receptor (FGFR) tyrosine kinases, which are encoded by four distinct genes (Fgfr1 to Fgfr4). Prototypical FGFRs consist of an extracellular domain composed of three immunoglobulin-like domains, a single-pass transmembrane domain, and an intracellular domain responsible for the tyrosine kinase activity (16). The number of principal FGFRs is increased from four to seven due to a major tissue-specific alternative splicing event in the second half of the immunoglobulin-like domain 3 of FGFR1 to FGFR3, which creates epithelial lineage-specific b and mesenchymal lineage-specific c isoforms (16, 21). Generally, the receptor-binding specificity of FGFs is divided along this major alternative splicing of receptors whereby FGFRb-interacting FGFs are produced by mesenchymal cells and FGFRc-interacting FGFs are produced by epithelial cells (21). These reciprocal expression patterns of FGFs and FGFRs result in the establishment of a paracrine epithelial-mesenchymal signaling which is essential for proper organogenesis and patterning during development as well as tissue homeostasis in the adult organism.Based on phylogeny and sequence identity, FGFs are grouped into seven subfamilies (21). The FGF core homology domain (approximately 120 amino acids long) is flanked by Nand C-terminal sequences that are highly variable in both length and primary sequence, particularly among different FGF subfamilies. The core region of FGF19 shares the highest sequence identity with FGF21 (38%) and FGF23 (36%), and therefore, these ligands are considered to form a subfamily. However, the degree of identity within the FGF19 subfamily is only 2 to 3% greater than that between FGF19 subfamily members and members of other FGF subfamilies, making this subfamily the most divergent one. FGF19 subfamily members regulate diverse physiological processes uncommon to classical FGFs, namely, energy (32) and bile acid homeostasis (FGF19) (5,8,13), glucose and lipid metabolism (FGF21) (10), and phosphate and vitamin D homeostasis (FGF23) (27). Moreover, unlike classical FGFs, FGF19 subfamily members achieve their unconventional activities in an endocrine fashion.To date, only a single structure from the endocrine-acting FGF19 subfamily has been reported (4), whereas there are crystal structures available for eight classical, paracrine-acting FGFs (2,20,22,37,38,40). The structures from the paracrine class of FGFs (FGF1, show that the core homology region folds into a globular domain composed of 12 antiparallel ␤-strands (␤1 to ␤12) known as the * Corresponding author. Mailing address:

Viral papain-like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library and performed comprehensive activity profiling of SARS-CoV-2 PLpro. On the scaffold of the best hits from positional scanning, we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro. We determined crystal structures of two of these inhibitors in complex with SARS-CoV-2 PLpro that reveals their inhibitory mechanisms and provides a molecular basis for the observed substrate specificity profiles. Last, we demonstrate that SARS-CoV-2 PLpro harbors deISGylating activity similar to SARSCoV-1 PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Together, this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repurposing.