to protein characterization and interpretation of results; Aided in manuscript preparation. Rachel A. Jones Lipinsky: Conducted chemical synthesis and characterization. Noah R. Leigh: Contributed to the automation aspect of the screen, and helped facilitate the screen. Raman G. Kutty: Contributed to the development and optimization of the pNPP assay; intellectual contribution to project design and interpretation of results. Daniel S. Sem: Key role in guiding group members in the characterization of DUSP5 enzyme kinetics and activity; aided in all aspects of project direction, compiled, prepared and edited the manuscript. Rajendra Rathore: Key role in guiding group members in the characterization of DUSP5/ERK interactions via molecular dynamic simulations. Ramani Ramchandran: Key role in guiding group members in the characterization of DUSP5 activity and purification; aided in all aspects of project direction, compiled, prepared and edited the manuscript.

B cells contribute to multiple aspects of autoimmune disorders, and B cell–targeting therapies, including B cell depletion, have been proven to be efficacious in treatment of multiple autoimmune diseases. However, the development of novel therapies targeting B cells with higher efficacy and a nondepleting mechanism of action is highly desirable. Here we describe a nondepleting, high-affinity anti–human CD19 antibody LY3541860 that exhibits potent B cell inhibitory activities. LY3541860 inhibits B cell activation, proliferation, and differentiation of primary human B cells with high potency. LY3541860 also inhibits human B cell activities in vivo in humanized mice. Similarly, our potent anti-mCD19 antibody also demonstrates improved efficacy over CD20 B cell depletion therapy in multiple B cell–dependent autoimmune disease models. Our data indicate that anti-CD19 antibody is a highly potent B cell inhibitor that may have potential to demonstrate improved efficacy over currently available B cell–targeting therapies in treatment of autoimmune conditions without causing B cell depletion.

Background: The mitogen-activated protein kinase (MAPK) pathway is functionally generic and critical in maintaining physiological homeostasis and normal tissue development. This pathway is under tight regulation, which is in part mediated by dual-specific phosphatases (DUSPs), which dephosphorylate serine, threonine, and tyrosine residues of the ERK family of proteins. DUSP5 is of high clinical interest because of mutations we identified in this protein in patients with vascular anomalies. Unlike other DUSPs, DUSP5 has unique specificity toward substrate pERK1/2. Using molecular docking and simulation strategies, we previously showed that DUSP5 has two pockets, which are utilized in a specific fashion to facilitate specificity toward catalysis of its substrate pERK1/2. Remarkably, most DUSPs share high similarity in their catalytic sites. Studying the catalytic domain of DUSP5 and identifying amino acid residues that are important for dephosphorylating pERK1/2 could be critical in developing small molecules for therapies targeting DUSP5. Results: In this study, we utilized computational modeling to identify and predict the importance of two conserved amino acid residues, H262 and S270, in the DUSP5 catalytic site. Modeling studies predicted that catalytic activity of DUSP5 would be altered if these critical conserved residues were mutated. We next generated independent Glutathione-S-Transferase (GST)-tagged full-length DUSP5 mutant proteins carrying specific mutations H262F and S270A in the phosphatase domain. Biochemical analysis was performed on these purified proteins, and consistent with our computational prediction, we observed altered enzyme activity kinetic profiles for both mutants with a synthetic small molecule substrate (pNPP) and the physiological relevant substrate (pERK) when compared to wild type GST-DUSP5 protein. Conclusion: Our molecular modeling and biochemical studies combined demonstrate that enzymatic activity of phosphatases can be manipulated by mutating specific conserved amino acid residues in the catalytic site (phosphatase domain). This strategy could facilitate generation of small molecules that will serve as agonists/antagonists of DUSP5 activity.