Pharmacological activation of the STING (stimulator of interferon genes)–controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-β secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti–PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.

Proteolysis by the ubiquitin-proteasome pathway is often regulated, but the mechanisms underlying such regulation remain ill-defined. In Saccharomyces cerevisiae, cell type is controlled by the MAT transcription factors. The alpha2 repressor is a known ubiquitin pathway substrate in alpha haploid cells. We show that a1 is rapidly degraded in a haploids. In a/alpha diploids, alpha2 and a1 are stabilized by heterodimerization. Association depends on N-terminal coiled-coil interactions between a1 and alpha2. Residues in alpha2 important for these interactions overlap a critical determinant of an alpha2 degradation signal, which we delimit by extensive mutagenesis. Our data provide a detailed description of a natural ubiquitin-dependent degradation signal and point to a molecular mechanism for regulated turnover in which proteolytic signals are differentially masked in alternative multiprotein complexes.

Edited by Jeffrey E. Pessin Spleen tyrosine kinase (SYK) is a signaling node in many immune pathways and comprises two tandem Src homology (SH) 2 domains, an SH2-kinase linker, and a C-terminal tyrosine kinase domain. Two prevalent models of SYK activation exist. The "OR-gate" model contends that SYK can be fully activated by phosphorylation or binding of its SH2 domains to a dualphosphorylated immune-receptor tyrosine-based activation motif (ppITAM). An alternative model proposes that SYK activation requires ppITAM binding and phosphorylation of the SH2-kinase linker by a SRC family kinase such as LYN protooncogene, SRC family tyrosine kinase (LYN). To evaluate these two models, we generated directly comparable unphosphorylated (upSYK) and phosphorylated (pSYK) proteins with or without an N-terminal glutathione S-transferase (GST) tag, resulting in monomeric or obligatory dimeric SYK, respectively. We assessed the ability of a ppITAM peptide and LYN to activate these SYK proteins. The ppITAM peptide strongly activated GST-SYK but was less effective in activating upSYK untagged with GST. LYN alone activated untagged upSYK to a greater extent than did ppITAM, and inclusion of both proteins rapidly and fully activated upSYK. Using immunoblot and phosphoproteomic approaches, we correlated the kinetics and order of site-specific SYK phosphorylation. Our results are consistent with the alternative model, indicating that ppITAMbindingprimesSYKforrapidLYN-mediatedphosphorylation of Tyr-352 and then Tyr-348 of the SH2-kinase linker, which facilitates activation loop phosphorylation and full SYK activation. This gradual activation mechanism may also explain how SYK maintains ligand-independent tonic signaling, important for B-cell development and survival. SYK (spleen tyrosine kinase), 3 a cytoplasmic protein-tyrosine kinase (PTK), is crucial for mediating antigen-associated signals in various cell types of the innate and adaptive immune system (1, 2). This signal mediation is essential to the propagation and activation of hematopoietic cells such as B cells, mast cells, and platelets. Aggregation of IgE or ligand-binding receptors on the surface of the cells triggers the phosphorylation of All authors were or are employees of Merck & Co., Inc.

The ability of G protein-coupled receptors (GPCRs) to form homo-and heteromeric complexes has important implications for the regulation of cellular events. A no table example G protein-coupled receptors (GPCRs)1 represent the largest family of cell-surface receptors. As key controllers of diverse physiological processes, they are of considerable biological and therapeutic interest. Although this class of receptors was originally thought to act as monomers that mediate their effects through stimulation of heterotrimeric G proteins, recent studies indicate that they can also act as multimeric complexes that, besides G proteins, can directly regulate a variety of other downstream effectors, including mitogen-activated protein kinase cascades and transcription factors (1). Several early lines of evidence have suggested that certain families of GPCRs can form homo-oligomers (2, 3). More recent evidence (3-8) has confirmed this and has indicated that GPCRs can also form heteromeric complexes, either with related GPCRs or with members of distinct families of GPCRs. Moreover, some receptors appear to require interaction with additional accessory factors for proper function (9 -12). GPCRs therefore seem to function in a rather complex molecular environment. One striking example of the complexity of some GPCRs is provided by receptors for adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP), two members of the calcitonin family of peptides. In this case, a seven-transmembrane protein, the calcitonin receptor-like receptor (CRLR), has been reported to require two different types of associated proteins, receptor activity modifying proteins (RAMPs) and receptor component protein (RCP), for full activity (13,14).CGRP is a potent vasoactive neuropeptide, which has been implicated in vasodilation, migraine, and chronic pain, whereas ADM is a multifunctional regulatory peptide with a wide range of biological actions, including vasodilation, cell growth, natriuresis, and certain antimicrobial effects (15,16). Despite the physiological importance and clinical implications of these two peptides, the identification and characterization of their receptors have been difficult for some time. A seventransmembrane protein with 55% homology to the calcitonin receptor (CRLR) provided CGRP receptor function only after transfection into specific cellular backgrounds (17, 18). The lack of a CGRP response in other cell lines made positive identification of this protein as a CGRP receptor problematic and suggested that CRLR might require cell type-specific accessory factors to become CGRP-responsive. A novel accessory protein, RAMP1, was identified that generated a functional CGRP receptor upon co-transfection with CRLR into cell culture (19). Confirming the idea that cell type-specific co-factors are required for CGRP responsiveness, transfection of CRLR into cell lines only yielded a functional CGRP receptor when the cell line endogenously expresses RAMP1. Conversely, transfection with RAMP1 only resulted in CGRP receptor function...

Interleukin-1 receptor associated kinase 4 (IRAK4) is an essential signal transducer downstream of the IL-1R and TLR superfamily, and selective inhibition of the kinase activity of the protein represents an attractive target for the treatment of inflammatory diseases. A series of 5-amino-N-(1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamides was developed via sequential modifications to the 5-position of the pyrazolopyrimidine ring and the 3-position of the pyrazole ring. Replacement of substituents responsible for poor permeability and improvement of physical properties guided by cLogD led to the identification of IRAK4 inhibitors with excellent potency, kinase selectivity, and pharmacokinetic properties suitable for oral dosing.