BackgroundSAR444245 is a non-alpha IL-2 Synthorin TM molecule designed with a site-specific non-n... more BackgroundSAR444245 is a non-alpha IL-2 Synthorin TM molecule designed with a site-specific non-natural amino acid serving as a bioconjugation site for a single PEG. The non-natural amino acid is positioned to enable the PEG bioconjugation to obscure block binding to the IL-2 alpha receptor, while retaining near-native affinity with the intermediate affinity βγ IL-2 receptor. The non-alpha features of SAR444245 minimize activation of immune suppressive regulatory CD4+ T cells, while retaining activity on CD8+ T cells and NK cells expressing the IL-2 βγ receptors. NK cells exert anti-tumor activity through antibody dependent cellular cytotoxicity (ADCC) of IgG antibodies as well as antibody independent mechanisms.MethodsHere, we utilized a panel of human primary PBMC based immunoassays and transcriptomic analysis to evaluate whether SAR444245 may improve ADCC function of IgG1 anti-tumor target antibodies.ResultsWe characterized the ability of SAR444245 to enhance the cytolytic functi...
Small molecule drugs most likely exert their pharmacological effects through a multiplicity of pa... more Small molecule drugs most likely exert their pharmacological effects through a multiplicity of pathways. Profiling of metabolites produced as part of physiological responses provides a unique opportunity to explore drug pharmacology. We report on a novel platform that allows comprehensive, multiplexed analysis of eicosanoid markers and offers promise in predicting responses and toxicities in anti-inflammatory drug treatment. 122 eicosanoid lipids in human whole blood were monitored from 10 different donors upon stimulation with several inducers of immunological responses and treatment with modulators of prostaglandin and leukotriene biosynthesis, including clinical and investigational molecules. We found differentiation between drugs nominally targeting different eicosanoid biosynthetic enzymes, or even those designed to target the same enzyme. Those profiled agents affect eicosanoid biosynthesis in ways that cannot be predicted from information on their intended targets. As an exam...
The Journal of pharmacology and experimental therapeutics, 2014
Both preclinical evidence and clinical evidence suggest that α7 nicotinic acetylcholine receptor ... more Both preclinical evidence and clinical evidence suggest that α7 nicotinic acetylcholine receptor activation (α7nAChR) improves cognitive function, the decline of which is associated with conditions such as Alzheimer's disease and schizophrenia. Moreover, allosteric modulation of α7nAChR is an emerging therapeutic strategy in an attempt to avoid the rapid desensitization properties associated with the α7nAChR after orthosteric activation. We used a calcium assay to screen for positive allosteric modulators (PAMs) of α7nAChR and report on the pharmacologic characterization of the novel compound RO5126946 (5-chloro-N-[(1S,3R)-2,2-dimethyl-3-(4-sulfamoyl-phenyl)-cyclopropyl]-2-methoxy-benzamide), which allosterically modulates α7nAChR activity. RO5126946 increased acetylcholine-evoked peak current and delayed current decay but did not affect the recovery of α7nAChRs from desensitization. In addition, RO5126946's effects were absent when nicotine-evoked currents were completely b...
Proceedings of the National Academy of Sciences of the United States of America, Jan 30, 2007
Semaphorin 4D (sema4D; CD100) is an integral membrane protein and the ligand for two receptors, C... more Semaphorin 4D (sema4D; CD100) is an integral membrane protein and the ligand for two receptors, CD72 and plexin-B1. Soluble sema4D has been shown to evoke angiogenic responses from endothelial cells and impair monocyte migration, but the origin of soluble sema4D, particularly at sites of vascular injury, has been unclear. Here we show that platelets express sema4D and both of its receptors and provide evidence that these molecules promote thrombus formation. We also show that the surface expression of sema4D and CD72 increases during platelet activation, followed by the gradual shedding of the sema4D extracellular domain. Shedding is blocked by metalloprotease inhibitors and abolished in mouse platelets that lack the metalloprotease ADAM17 (TACE). Mice that lack sema4D exhibit delayed arterial occlusion after vascular injury in vivo, and their platelets show impaired collagen responses in vitro. In resting platelets, as in B lymphocytes, CD72 is associated with the protein tyrosine ...
Proceedings of the National Academy of Sciences of the United States of America, Jan 19, 2014
The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-... more The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4(+) Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7β, 27-dihydroxycholesterol (7β, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ- or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7β, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an ...
The α1A-AR is thought to couple predominantly to the Gαq/PLC pathway and lead to phosphoinositide... more The α1A-AR is thought to couple predominantly to the Gαq/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gαq coupling-defective variants of α1A-AR, as well as a combination of Ca2+ channel blockers, uncovered cross-talk between α1A-AR and β2-AR that leads to potentiation of a Gαq-independent signaling cascade in response to α1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activa...
Proceedings of the National Academy of Sciences, 1994
Sulfation of proteoglycans, secretory and membrane proteins, and glycolipids occurs in the lumen ... more Sulfation of proteoglycans, secretory and membrane proteins, and glycolipids occurs in the lumen of the Golgi apparatus. Adenosine 3'-phosphate 5'-phosphosulfate (PAPS), the sulfate donor in these reactions, must be transported from the cytosol, its site of synthesis, into the lumen of the Golgi apparatus. We have identified and purified to apparent homogeneity the rat liver Golgi membrane PAPS transporter by a combination of conventional and affinity chromatography as well as photoaffinity radiolabeling with adenosine 3',5'-bisphosphate, a competitive inhibitor of PAPS transport. The transporter, a 75-kDa protein, was purified 70,000-fold over homogenate (6% yield) and transported PAPS into phosphatidylcholine liposomes selectively and in a saturable manner (apparent Km of 1.7 microM). Radiation target-inactivation analyses of the transport activity in rat liver Golgi vesicles, together with the above described biochemical approaches, demonstrate that the PAPS trans...
Proceedings of the National Academy of Sciences, 2007
Despite their key roles in many normal and pathological processes, the molecular details by which... more Despite their key roles in many normal and pathological processes, the molecular details by which zinc-dependent proteases hydrolyze their physiological substrates remain elusive. Advanced theoretical analyses have suggested reaction models for which there is limited and controversial experimental evidence. Here we report the structure, chemistry and lifetime of transient metal–protein reaction intermediates evolving during the substrate turnover reaction of a metalloproteinase, the tumor necrosis factor-α converting enzyme (TACE). TACE controls multiple signal transduction pathways through the proteolytic release of the extracellular domain of a host of membrane-bound factors and receptors. Using stopped-flow x-ray spectroscopy methods together with transient kinetic analyses, we demonstrate that TACE's catalytic zinc ion undergoes dynamic charge transitions before substrate binding to the metal ion. This indicates previously undescribed communication pathways taking place betw...
Proceedings of the National Academy of Sciences, 1989
We have previously shown that Golgi apparatus vesicles transport nucleotide sugars and nucleotide... more We have previously shown that Golgi apparatus vesicles transport nucleotide sugars and nucleotide sulfate into their lumen. These transport activities are organelle and substrate specific and are characterized by apparent Km for nucleotide derivatives in the low micromolar range. As part of our goal of purifying and characterizing the above transport proteins, we have reconstituted a protein extract from rat liver Golgi membranes into phosphatidylcholine liposomes. The resulting proteoliposomes transport CMP-N-acetylneuraminic acid (CMP-AcNeu) and adenosine 3'-phosphate 5'-phosphosulfate with very similar affinity and inhibition characteristics as intact Golgi vesicles. Sialic acid and sodium sulfate, which are transported only very slowly into the lumen of Golgi vesicles, are transported at low rates by the reconstituted proteoliposomes. Neither rough endoplasmic reticulum-derived vesicles nor proteoliposomes made from proteins of the rough endoplasmic reticulum transport C...
The implementation of applied engineering principles to create synthetic biological systems promi... more The implementation of applied engineering principles to create synthetic biological systems promises to revolutionize medicine, but application of fundamentally redesigned organisms has thus far not impacted practical drug development. Here we utilize an engineered microbial organism with a six-letter semi-synthetic DNA code to generate a library of site-specific, click chemistry compatible amino acid substitutions in the human cytokine IL-2. Targeted covalent modification of IL-2 variants with PEG polymers and screening identifies compounds with distinct IL-2 receptor specificities and improved pharmacological properties. One variant, termed THOR-707, selectively engages the IL-2 receptor beta/gamma complex without engagement of the IL-2 receptor alpha. In mice, administration of THOR-707 results in large-scale activation and amplification of CD8+ T cells and NK cells, without Treg expansion characteristic of IL-2. In syngeneic B16-F10 tumor-bearing mice, THOR-707 enhances drug acc...
BackgroundSAR444245 is a non-alpha IL-2 Synthorin TM molecule designed with a site-specific non-n... more BackgroundSAR444245 is a non-alpha IL-2 Synthorin TM molecule designed with a site-specific non-natural amino acid serving as a bioconjugation site for a single PEG. The non-natural amino acid is positioned to enable the PEG bioconjugation to obscure block binding to the IL-2 alpha receptor, while retaining near-native affinity with the intermediate affinity βγ IL-2 receptor. The non-alpha features of SAR444245 minimize activation of immune suppressive regulatory CD4+ T cells, while retaining activity on CD8+ T cells and NK cells expressing the IL-2 βγ receptors. NK cells exert anti-tumor activity through antibody dependent cellular cytotoxicity (ADCC) of IgG antibodies as well as antibody independent mechanisms.MethodsHere, we utilized a panel of human primary PBMC based immunoassays and transcriptomic analysis to evaluate whether SAR444245 may improve ADCC function of IgG1 anti-tumor target antibodies.ResultsWe characterized the ability of SAR444245 to enhance the cytolytic functi...
Small molecule drugs most likely exert their pharmacological effects through a multiplicity of pa... more Small molecule drugs most likely exert their pharmacological effects through a multiplicity of pathways. Profiling of metabolites produced as part of physiological responses provides a unique opportunity to explore drug pharmacology. We report on a novel platform that allows comprehensive, multiplexed analysis of eicosanoid markers and offers promise in predicting responses and toxicities in anti-inflammatory drug treatment. 122 eicosanoid lipids in human whole blood were monitored from 10 different donors upon stimulation with several inducers of immunological responses and treatment with modulators of prostaglandin and leukotriene biosynthesis, including clinical and investigational molecules. We found differentiation between drugs nominally targeting different eicosanoid biosynthetic enzymes, or even those designed to target the same enzyme. Those profiled agents affect eicosanoid biosynthesis in ways that cannot be predicted from information on their intended targets. As an exam...
The Journal of pharmacology and experimental therapeutics, 2014
Both preclinical evidence and clinical evidence suggest that α7 nicotinic acetylcholine receptor ... more Both preclinical evidence and clinical evidence suggest that α7 nicotinic acetylcholine receptor activation (α7nAChR) improves cognitive function, the decline of which is associated with conditions such as Alzheimer's disease and schizophrenia. Moreover, allosteric modulation of α7nAChR is an emerging therapeutic strategy in an attempt to avoid the rapid desensitization properties associated with the α7nAChR after orthosteric activation. We used a calcium assay to screen for positive allosteric modulators (PAMs) of α7nAChR and report on the pharmacologic characterization of the novel compound RO5126946 (5-chloro-N-[(1S,3R)-2,2-dimethyl-3-(4-sulfamoyl-phenyl)-cyclopropyl]-2-methoxy-benzamide), which allosterically modulates α7nAChR activity. RO5126946 increased acetylcholine-evoked peak current and delayed current decay but did not affect the recovery of α7nAChRs from desensitization. In addition, RO5126946's effects were absent when nicotine-evoked currents were completely b...
Proceedings of the National Academy of Sciences of the United States of America, Jan 30, 2007
Semaphorin 4D (sema4D; CD100) is an integral membrane protein and the ligand for two receptors, C... more Semaphorin 4D (sema4D; CD100) is an integral membrane protein and the ligand for two receptors, CD72 and plexin-B1. Soluble sema4D has been shown to evoke angiogenic responses from endothelial cells and impair monocyte migration, but the origin of soluble sema4D, particularly at sites of vascular injury, has been unclear. Here we show that platelets express sema4D and both of its receptors and provide evidence that these molecules promote thrombus formation. We also show that the surface expression of sema4D and CD72 increases during platelet activation, followed by the gradual shedding of the sema4D extracellular domain. Shedding is blocked by metalloprotease inhibitors and abolished in mouse platelets that lack the metalloprotease ADAM17 (TACE). Mice that lack sema4D exhibit delayed arterial occlusion after vascular injury in vivo, and their platelets show impaired collagen responses in vitro. In resting platelets, as in B lymphocytes, CD72 is associated with the protein tyrosine ...
Proceedings of the National Academy of Sciences of the United States of America, Jan 19, 2014
The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-... more The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4(+) Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7β, 27-dihydroxycholesterol (7β, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ- or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7β, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an ...
The α1A-AR is thought to couple predominantly to the Gαq/PLC pathway and lead to phosphoinositide... more The α1A-AR is thought to couple predominantly to the Gαq/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gαq coupling-defective variants of α1A-AR, as well as a combination of Ca2+ channel blockers, uncovered cross-talk between α1A-AR and β2-AR that leads to potentiation of a Gαq-independent signaling cascade in response to α1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activa...
Proceedings of the National Academy of Sciences, 1994
Sulfation of proteoglycans, secretory and membrane proteins, and glycolipids occurs in the lumen ... more Sulfation of proteoglycans, secretory and membrane proteins, and glycolipids occurs in the lumen of the Golgi apparatus. Adenosine 3'-phosphate 5'-phosphosulfate (PAPS), the sulfate donor in these reactions, must be transported from the cytosol, its site of synthesis, into the lumen of the Golgi apparatus. We have identified and purified to apparent homogeneity the rat liver Golgi membrane PAPS transporter by a combination of conventional and affinity chromatography as well as photoaffinity radiolabeling with adenosine 3',5'-bisphosphate, a competitive inhibitor of PAPS transport. The transporter, a 75-kDa protein, was purified 70,000-fold over homogenate (6% yield) and transported PAPS into phosphatidylcholine liposomes selectively and in a saturable manner (apparent Km of 1.7 microM). Radiation target-inactivation analyses of the transport activity in rat liver Golgi vesicles, together with the above described biochemical approaches, demonstrate that the PAPS trans...
Proceedings of the National Academy of Sciences, 2007
Despite their key roles in many normal and pathological processes, the molecular details by which... more Despite their key roles in many normal and pathological processes, the molecular details by which zinc-dependent proteases hydrolyze their physiological substrates remain elusive. Advanced theoretical analyses have suggested reaction models for which there is limited and controversial experimental evidence. Here we report the structure, chemistry and lifetime of transient metal–protein reaction intermediates evolving during the substrate turnover reaction of a metalloproteinase, the tumor necrosis factor-α converting enzyme (TACE). TACE controls multiple signal transduction pathways through the proteolytic release of the extracellular domain of a host of membrane-bound factors and receptors. Using stopped-flow x-ray spectroscopy methods together with transient kinetic analyses, we demonstrate that TACE's catalytic zinc ion undergoes dynamic charge transitions before substrate binding to the metal ion. This indicates previously undescribed communication pathways taking place betw...
Proceedings of the National Academy of Sciences, 1989
We have previously shown that Golgi apparatus vesicles transport nucleotide sugars and nucleotide... more We have previously shown that Golgi apparatus vesicles transport nucleotide sugars and nucleotide sulfate into their lumen. These transport activities are organelle and substrate specific and are characterized by apparent Km for nucleotide derivatives in the low micromolar range. As part of our goal of purifying and characterizing the above transport proteins, we have reconstituted a protein extract from rat liver Golgi membranes into phosphatidylcholine liposomes. The resulting proteoliposomes transport CMP-N-acetylneuraminic acid (CMP-AcNeu) and adenosine 3'-phosphate 5'-phosphosulfate with very similar affinity and inhibition characteristics as intact Golgi vesicles. Sialic acid and sodium sulfate, which are transported only very slowly into the lumen of Golgi vesicles, are transported at low rates by the reconstituted proteoliposomes. Neither rough endoplasmic reticulum-derived vesicles nor proteoliposomes made from proteins of the rough endoplasmic reticulum transport C...
The implementation of applied engineering principles to create synthetic biological systems promi... more The implementation of applied engineering principles to create synthetic biological systems promises to revolutionize medicine, but application of fundamentally redesigned organisms has thus far not impacted practical drug development. Here we utilize an engineered microbial organism with a six-letter semi-synthetic DNA code to generate a library of site-specific, click chemistry compatible amino acid substitutions in the human cytokine IL-2. Targeted covalent modification of IL-2 variants with PEG polymers and screening identifies compounds with distinct IL-2 receptor specificities and improved pharmacological properties. One variant, termed THOR-707, selectively engages the IL-2 receptor beta/gamma complex without engagement of the IL-2 receptor alpha. In mice, administration of THOR-707 results in large-scale activation and amplification of CD8+ T cells and NK cells, without Treg expansion characteristic of IL-2. In syngeneic B16-F10 tumor-bearing mice, THOR-707 enhances drug acc...
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Papers by Marcos Milla