ABSTRACT
BACKGROUND: CD40 is a 48 kDa type I transmembrane protein that is constitutively expressed on hematopoietic cells such as dendritic cells, macrophages, and B cells. Engagement of CD40 by CD40L expressed on T cells results in the production of proinflammatory cytokines, induces T helper cell function, and promotes macrophage activation. The involvement of CD40 in chronic immune activation has resulted in CD40 being proposed as a therapeutic target for a range of chronic inflammatory diseases. CD40 antagonists are currently being explored for the treatment of autoimmune diseases and several anti-CD40 agonist mAbs have entered clinical development for oncological indications. RESULTS: To better understand the mode of action of anti-CD40 mAbs, we have determined the x-ray crystal structures of the ABBV-323 (anti-CD40 antagonist, ravagalimab) Fab alone, ABBV-323 Fab complexed to human CD40 and FAB516 (anti-CD40 agonist) complexed to human CD40. These three crystals structures 1) identify the conformational CD40 epitope for ABBV-323 recognition 2) illustrate conformational changes which occur in the CDRs of ABBV-323 Fab upon CD40 binding and 3) develop a structural hypothesis for an agonist/antagonist switch in the LCDR1 of this proprietary class of CD40 antibodies. CONCLUSIONS: The structure of ABBV-323 Fab demonstrates a unique method for antagonism by stabilizing the proposed functional antiparallel dimer for CD40 receptor via novel contacts to LCDR1, namely residue position R32 which is further supported by a closely related agonist antibody FAB516 which shows only monomeric recognition and no contacts with LCDR1 due to a mutation to L32 on LCDR1. These data provide a structural basis for the full antagonist activity of ABBV-323.
Subject(s)
Antibodies, Monoclonal/chemistry , Antigen-Antibody Complex/chemistry , CD40 Antigens/agonists , CD40 Antigens/antagonists & inhibitors , CD40 Antigens/chemistry , HEK293 Cells , Humans , Immunoglobulin Fab Fragments/chemistry , Models, Molecular , Signal Transduction , Static ElectricityABSTRACT
Inhibition of sphingosine-1-phosphate lyase has recently been proposed as a potential treatment option for inflammatory disorders such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. In this report we describe our hit-to-lead evaluation of the isoxazolecarboxamide 6, a high-throughput screening hit (in vitro IC50=1.0 µM, cell IC50=1.8 µM), as a novel S1P lyase inhibitor. We were able to establish basic structure-activity relationships around 6 and succeeded in obtaining X-ray structural information which enabled structure-based design. With the discovery of 28, enzyme activity was quickly improved to IC50=120 nM and cell potency to IC50=230 nM. The main liability in the established isoxazolecarboxamide hit series was determined to be metabolic stability. In particular we identified that future lead-optimization efforts to overcome this problem should focus on blocking the N-dealkylation on the secondary amine.
Subject(s)
Aldehyde-Lyases/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Humans , Structure-Activity RelationshipABSTRACT
We describe structure-based optimization of a series of novel 2,4-diaminopyrimidine MK2 inhibitors. Co-crystal structures (see accompanying Letter) demonstrated a unique inhibitor binding mode. Resulting inhibitors had IC(50) values as low as 19nM and moderate selectivity against a kinase panel. Compounds 15, 31a, and 31b inhibit TNFalpha production in peripheral human monocytes.
