We have cloned two open reading frames (orf6 and orf8) from the Escherichia coli K-12 rfb region. The genes were expressed in E. coli under control of the T7lac promoter, producing large quantities of recombinant protein, most of which accumulated in insoluble inclusion bodies. Sufficient soluble protein was obtained, however, for use in a radiometric assay designed to detect UDP-galactopyranose mutase activity (the conversion of UDP-galactopyranose to UDP-galactofuranose). The assay is based upon high-pressure liquid chromatography separation of sugar phosphates released from both forms of UDP-galactose by phosphodiesterase treatment. The crude orf6 gene product converted UDP-[␣-D-U-14 C]-galactopyranose to a product which upon phosphodiesterase treatment gave a compound with a retention time identical to that of synthetic ␣-galactofuranose-1-phosphate. No mutase activity was detected in extracts from cells lacking the orf6 expression plasmid or from orf8-expressing cells. The orf6 gene product was purified by anion-exchange chromatography and hydrophobic interaction chromatography. Both the crude extract and the purified protein converted 6 to 9% of the UDP-galactopyranose to the furanose form. The enzyme was also shown to catalyze the reverse reaction; in this case an approximately 86% furanose-to-pyranose conversion was observed. These observations strongly suggest that orf6 encodes UDP-galactopyranose mutase (EC 5.4.99.9), and we propose that the gene be designated glf accordingly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified UDPgalactopyranose mutase revealed one major band, and analysis by electrospray mass spectrometry indicated a single major species with a molecular weight of 42,960 ؎ 8, in accordance with that calculated for the Glf protein. N-terminal sequencing revealed that the first 15 amino acids of the recombinant protein corresponded to those expected from the published sequence. UV-visible spectra of purified recombinant enzyme indicated that the protein contains a flavin cofactor, which we have identified as flavin adenine dinucleotide.

Summary Pancreatic ductal adenocarcinoma (PDA) is characterized by immune-tolerance and immunotherapeutic resistance. We discovered upregulation of receptor-interacting serine/threonine-protein kinase 1 (RIP1) in tumor-associated macrophages (TAMs) in PDA. To study its role in oncogenic progression, we developed a selective small molecule RIP1 inhibitor with high in vivo exposure. Targeting RIP1 reprogrammed TAMs toward an MHCIIhiTNFα+IFNγ+ immunogenic phenotype in a STAT1-dependent manner. RIP1 inhibition in TAMs resulted in cytotoxic T cell activation and T-helper cell differentiation towards a mixed Th1/Th17 phenotype, leading to tumor-immunity in mice and in organotypic models of human PDA. Targeting RIP1 synergized with PD1- and ICOS-based immunotherapies. Tumor-promoting effects of RIP1 were independent of its co-association with RIP3. Collectively, our work describes RIP1 as a checkpoint kinase governing tumor-immunity.

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RIP1 kinase regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including inflammatory and neurological diseases. Currently, RIP1 kinase inhibitors have advanced into early clinical trials for evaluation in inflammatory diseases such as psoriasis, rheumatoid arthritis, and ulcerative colitis and neurological diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. In this paper, we report on the design of potent and highly selective dihydropyrazole (DHP) RIP1 kinase inhibitors starting from a high-throughput screen and the leadoptimization of this series from a lead with minimal rat oral exposure to the identification of dihydropyrazole 77 with good pharmacokinetic profiles in multiple species. Additionally, we identified a potent murine RIP1 kinase inhibitor 76 as a valuable in vivo tool molecule suitable for evaluating the role of RIP1 kinase in chronic models of disease. DHP 76 showed efficacy in mouse models of both multiple sclerosis and human retinitis pigmentosa.

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A model system, measuring the rate of cholera-toxin-catalysed release of nicotinamide from NAD ' , has been used to identify novel compounds which may serve as substrates for toxin-directed ADP-ribosylation. In a series of guanidine-containing compounds, those in which the guanidine group was connected to a large hydrophobic domain greatly stimulated the rate of toxin-catalysed nicotinamide release. The introduction of a charge centre near the guanidine group destroyed all activity. The compounds thus identified were found to inhibit the action of cholera toxin on rat liver adenylate cyclase, and this was associated with a reduction in the amount of [32P]ADP-ribosylation of a 42-kDa protein in the membranes. Guanidine-containing compounds which did not enhance toxin-catalysed release of nicotinamide from NAD' had no effect on toxin action on adenylate cyclase, and there was a good correlation between the two activities. The results are discussed in relation to the known properties of the guanine nucleotide regulatory protein associated with adenylate cyclase sytems, which is the toxin's natural substrate.Cholera toxin activates the adenylate cyclase system of almost all eukaryotic cells (for reviews, see [l -31). In the small intestine this is thought to be the primary stimulus which ultimately results in ion and fluid secretion and hence diarrhoea [4,5]. The toxin acts by catalysing the transfer of ADPribose from intracellular NAD' to a guanine nucleotide binding protein which functions as a regulator of adenylate cyclase activity [6 -81. As a result, this protein loses the ability to hydrolyse its bound GTP and so induces persistent activation of the cyclase catalytic subunit [9].Cholera toxin also catalyses NAD' hydrolysis [lo] and the ADP-ribosylation of arginine or arginine methyl ester [ll]. It has therefore been suggested that arginine is a strong candidate for the residue(s) which is modified by cholera toxin on the guanine nucleotide binding protein. Experiments with the argmine-modifying reagent phenylglyoxal were consistent with this hypothesis [12]. Retinal transducin, which is also an effective substrate for cholera toxin, has recently been shown to be ADP-ribosylated at the guanidinium group of an arginine residue [24].Although cholera-toxin-catalysed ADP-ribosylation of arginine or its methyl ester occurs much faster than NAD' hydrolysis (ADP-ribosylation of water), this reaction is still very slow and requires high concentrations of NAD' and the acceptor guanidine. Thus, arginine itself is a rather poor substitute for the toxin's natural substrate. Recently, we outlined the discovery of much more effective toxin substrates and demonstrated that their effects on the adenylate cyclase system of rat liver membranes were those which would be expected of a compound which functions primarily as an acceptor of ADP-ribose in a toxin-catalyscd reaction [13]. This report extends these observations and describes the detailed Abbreviations. SDS, sodium dodecyl sulphate; PEI, polyethyEnzymes. Adenylate cyclas...

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A protein-0-D-mannosyltransferase (PMT) assay was optimised using a microsomal membrane preparation from Candida albicans and a peptide acceptor, YNPTSV.[14C]Mannose was transferred from dolichyl phosphate [14C]mannose to the threonine or serine residues of the peptide. During the assay, the peptide was highly susceptible to proteolysis. A blocked peptide Ac-YNPTSV-NH, was resistent to proteolysis and was apparently a better acceptor for 0-mannosylation. This peptide had a K,,, value of 4.3 mM in the assay. A number of other peptides were tested with altered sequences. Maximum incorporation of [14C]mannose was obtained with a pentapeptide YATAV (K, 2.2 mM) which was further improved by blocking both ends: Ac-YATAV-NH, (K, 0.25 mM). Finally, and unexpectedly, an improvement was noted if the acetyl group on the N terminus was replaced by a biotin residue. Biotin-YATAV-NH, had a K, of 0.075 mM. The biotin residue may be important in increasing the lipophilicity of the peptide and thus aid its adhesion to the Candida membranes. The simplest peptide that could act as an efficient mannose acceptor was Ac-ATA-NH,, whilst no incorporation was observed with Ac-GTG-NH,.Both Candida and Saccharomyces species contain carbohydrate chains which are 0-linked to the hydroxyl group of threonine and serine residues of polypeptide chains, giving rise to mannoproteins. The sequence of reactions for the formation of 0-linked mannoproteins has been described previously [l -31 and involves the transfer of mannose from a dolichyl phosphate mannose (Dol-P-Man) to the protein which is catalysed by an enzyme, protein O-mannosyltransferase (PMT), that is believed to be unique to fungi. Several attempts to purify the enzyme from Saccharomyces cerevisiae [4, 51 have been partially successful but it has proved difficult because the enzyme is membrane-bound. Little is known about the enzyme or its assay requirements, although some information is available on the use of different peptide acceptors for PMT in Saccharomyces [3, 4, 61.PMT has been even less well studied in Candida albicans; hence, we have developed an assay for the enzyme which utilises microsomal membranes as enzyme source and exogenously added peptide as the mannose acceptor. We were interested to study the peptide sequence requirements for mannosylation by the enzyme and have investigated a number of variables. In particular, we have examined the effects of peptide length, preference for a serine or threonine residue and the influence of the amino acids immediately flanking the 0-mannosylation site.Correspondence to A. Weston, Chemotherapy Department, Microbiology, Division, Glaxo Group Research Ltd, Greenford Road, Greenford, Middlesex, England, UB6 OHE Abbreviations. PMT, protein 0-D-mannosyltransferase; Dol-PMan, dolichyl phosphate mannose ; Me,SO, dimethylsulphoxide ; Abu, 2-aminobutyric acid.Enzymes. Protein 0-D-mannosyltransferase (EC 2.4.1.109). MATERIALS AND METHODSAll chemicals were analar grade unless otherwise stated. GDP-['4C]mannose (specific activity 272 Ci/mol) ...