Explore the vast range of titles available.

The baculovirus antiapoptotic protein p35 inhibited the proteolytic activity of human interleukin-1 beta converting enzyme (ICE) and three of its homologs in enzymatic assays. Coexpression of p35 prevented the autoproteolytic activation of ICE from its precursor form and blocked ICE-induced apoptosis. Inhibition of enzymatic activity correlated with the cleavage of p35 and the formation of a stable ICE-p35 complex. The ability of p35 to block apoptosis in different pathways and in distantly related organisms suggests a central and conserved role for ICE-like proteases in the induction of apoptosis

While immune checkpoint blockade leads to potent antitumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in inflammation of multiple organs, including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA4 antibody (anti-CTLA4 DVD, where CTLA4 is defined as cytotoxic T lymphocyte-associated antigen-4) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA4-binding domain. Upon reaching the tumor, the outer domain was cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA4 blockade. Anti-CTLA4 DVD markedly reduced multiorgan immune toxicity by preserving tissue-resident Tregs in Rag 1-/- mice that received naive donor CD4+ T cells from WT C57BL/6j mice. Moreover, anti-CTLA4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8+ T lymphocytes in TRAMP-C2-bearing C57BL/6j mice. Treg depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA4 without the FcγR-binding portion (anti-CTLA4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrate an approach to anti-CTLA4 blockade that depletes tumor-infiltrating, but not tissue-resident, Tregs, preserving antitumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA4 DVD provides an avenue for uncoupling antitumor efficacy from immunotherapy-induced toxicities.

Background Structure-based drug design (SBDD) can provide valuable guidance to drug discovery programs. Robust construct design and expression, protein purification and characterization, protein crystallization, and high-resolution diffraction are all needed for rapid, iterative inhibitor design. We describe here robust methods to support SBDD on an oral anti-cytokine drug target, human MAPKAP kinase 2 (MK2). Our goal was to obtain useful diffraction data with a large number of chemically diverse lead compounds. Although MK2 structures and structural methods have been reported previously, reproducibility was low and improved methods were needed. Results Our construct design strategy had four tactics: N - and C -terminal variations; entropy-reducing surface mutations; activation loop deletions; and pseudoactivation mutations. Generic, high-throughput methods for cloning and expression were coupled with automated liquid dispensing for the rapid testing of crystallization conditions with minimal sample requirements. Initial results led to development of a novel, customized robotic crystallization screen that yielded MK2/inhibitor complex crystals under many conditions in seven crystal forms. In all, 44 MK2 constructs were generated, ~500 crystals were tested for diffraction, and ~30 structures were determined, delivering high-impact structural data to support our MK2 drug design effort. Conclusion Key lessons included setting reasonable criteria for construct performance and prioritization, a willingness to design and use customized crystallization screens, and, crucially, initiation of high-throughput construct exploration very early in the drug discovery process.

The baculovirus antiapoptotic protein p35 inhibited the proteolytic activity of human interleukin-1β converting enzyme (ICE) and three of its homologs in enzymatic assays. Coexpression of p35 prevented the autoproteolytic activation of ICE from its precursor form and blocked ICE-induced apoptosis. Inhibition of enzymatic activity correlated with the cleavage of p35 and the formation of a stable ICE-p35 complex. The ability of p35 to block apoptosis in different pathways and in distantly related organisms suggests a central and conserved role for ICE-like proteases in the induction of apoptosis.

Colicin Ia and colicin Ib have three distinct functional characteristics, receptor specificity, mode of action and immunity recognition. Both colicins bind to the same receptor and kill sensitive Escherichia coli cells by depolarizing the cell membrane. Sensitive cells carrying the colicin Ib structural and immunity genes are immune to colicin Ib but sensitive to the killing effect of colicin Ia and vice versa. Analysis of the immunity specificities of hybrid proteins and the direction of transcription of the colicin Ib structural gene have led to the conclusion that the immunity recognition domain of the colicin molecule resides in the COOH-terminal half of the molecule. Hybrid proteins were constructed by ligating cloned fragments of the colicin Ib gene with fragments of the colicin Ia gene and cloning them into E. coli. Direction of transcription of the colicin Ib gene was determined by cloning the intact gene into the E. coli expression vector pKC101 which allows expression of inserted genes to be under the control of the left promotor (p(,L)) of bacteriophage Lambda. Both colicin Ia and Ib structural and immunity gene DNA sequences have been determined and a comparison of the two sequences reveal a great deal of sequence conservation. The colicin structural genes are nearly identical in what corresponds to the amino-terminal two-thirds of the molecules. The region of the molecules containing the immunity recognition domains is also the area of least homology. The DNA sequences corresponding to the immunity genes have no homology, however, the hydrophilicity profiles of the predicted proteins are very similar. Codon usage throughout the coding regions for the colicin and immunity genes does not reflect the bias of that seen in known E. coli genes. The codon choice of the colicins and their immunity genes is similar to that seen in the other membrane active colicins.

We consider the coding problem in the Stiefel manifold with chordal distance. After considering various low-dimensional instances of this problem, we use Rankin's bounds on spherical codes to prove upper bounds on the minimum distance of a Stiefel code, and then we construct several examples of codes that achieve equality in these bounds.