SQ3370 Activates Cytotoxic Drug via Click Chemistry at Tumor and Elicits Sustained Responses in Injected & Non-injected Lesions.

While systemic immuno-oncology therapies have shown remarkable success, only a limited subset of patients benefit from them. Our Click Activated Protodrugs Against Cancer (CAPAC™) Platform is a click chemistry-based approach that activates cancer drugs at a specific tumor with minimal systemic toxicity. CAPAC Platform is agnostic to tumor characteristics that can vary across patients and hence applicable to several types of tumors. We describe the benefits of SQ3370 (lead candidate of CAPAC) to achieve systemic anti-tumor responses in mice bearing two tumors. SQ3370 consists of a biopolymer, injected in a single lesion, followed by systemic doses of an attenuated protodrug™ of doxorubicin (Dox). SQ3370 was well-tolerated at 5.9-times the maximum dose of conventional Dox, increased survival by 63% and induced a systemic anti-tumor response against injected and non-injected lesions. The sustained anti-tumor response also correlated with immune activation measured at both lesions. SQ3370 could potentially benefit patients with micro-metastatic lesions.

Complementary Hydrogen Bonding Modulates Electronic Properties and Controls Self-Assembly of Donor/Acceptor Semiconductors.

A comprehensive investigation of the complementary H-bonding-mediated self-assembly between dipyrrolo[2,3-b:3',2'-e]pyridine (P2P) electron donors and naphthalenediimide/perylenediimide (NDI/PDI) acceptors is reported. The synthesis of parent P2P and several aryl-substituted derivatives is described, along with their optical, redox, and single-crystal packing characteristics. The dual functionality of heteroatoms in the P2P/NDI(PDI) assembly, which act as proton donors/acceptors and also contribute to π-conjugation, leads to H-bonding-induced perturbation of electronic levels. Concentration-dependent NMR and UV/Vis spectroscopic studies revealed a cooperative effect of H-bonding and π-π stacking interactions. This H-bonding-mediated co-assembly of donor (D) and acceptor (A) components leads to a new charge-transfer (CT) absorption that can be controlled throughout the visible range. The electronic interactions between D and A were further investigated by time-dependent DFT, which provided insights into the nature of the CT transition. Electropolymerization of difuryl-P2P afforded the first conjugated polymer incorporating H-bonding recognition units in its main chain.

Transformation of galena to pyromorphite produces bioavailable sulfur for neutrophilic chemoautotrophy.

The aqueous concentration of lead [Pb(II)] in geochemical environments is controlled by the solubility of Pb-bearing minerals and their weathering products. In contaminated soils, a common method for in situ stabilization of Pb(II) is the addition of phosphate to convert more redox sensitive sulfide minerals into sparingly soluble pyromorphite [Pb5 (PO4 )3 X]. In this study, we conducted experimental studies to investigate the fate of reduced sulfur during the conversion of galena [PbS] to chloropyromorphite [Pb5 (PO4 )3 Cl]. Powder X-ray diffraction analysis indicated that the reaction of phosphate with galena under oxic conditions resulted in the oxidation of sulfide and formation of elemental sulfur [S8 ]. Under oxic abiotic conditions, the S8 was retained in the solid phase, and negligible concentrations of sulfur as sulfide and thiosulfate were detected in the aqueous phase and only a small amount of sulfate. When PbS reacted in the presence of the chemoautotrophic organism Bosea sp. WAO, the S8 in the secondary mineral was oxidized to sulfate. Strain WAO produced significantly more sulfate from the secondary S8 than from the primary galena. Microscopic analysis of mineral-microbe aggregates on mineral-embedded slide cultures showed that the organism was colocalized and increased in biomass over time on the secondary mineral surface supporting a microbial role. The results of this study indicate that stimulation of sulfur-oxidizing activity may be a direct consequence of phosphate amendments to Pb(II)-contaminated soils.

Pharmacological ascorbate with gemcitabine for the control of metastatic and node-positive pancreatic cancer (PACMAN): results from a phase I clinical trial.

BACKGROUND: Treatment for pancreatic cancer with pharmacological ascorbate (ascorbic acid, vitamin C) decreases tumor progression in preclinical models. A phase I clinical trial was performed to establish safety and tolerability of pharmacological ascorbate combined with gemcitabine in patients with biopsy-proven stage IV pancreatic adenocarcinoma. DESIGN: Nine subjects received twice-weekly intravenous ascorbate (15-125 g) employing Simon's accelerated titration design to achieve a targeted post-infusion plasma level of ≥350 mg/dL (≥20 mM). Subjects received concurrent gemcitabine. Disease burden, weight, performance status, hematologic and metabolic laboratories, time to progression and overall survival were monitored. RESULTS: Mean plasma ascorbate trough levels were significantly higher than baseline (1.46 ± 0.02 vs. 0.78 ± 0.09 mg/dL, i.e., 83 vs. 44 µM, p < 0.001). Adverse events attributable to the drug combination were rare and included diarrhea (n = 4) and dry mouth (n = 6). Dose-limiting criteria were not met for this study. Mean survival of subjects completing at least two cycles (8 weeks) of therapy was 13 ± 2 months. CONCLUSIONS: Data suggest pharmacologic ascorbate administered concurrently with gemcitabine is well tolerated. Initial data from this small sampling suggest some efficacy. Further studies powered to determine efficacy should be conducted.

Mechanistic studies on the chiral recognition of polysaccharide-based chiral stationary phases using liquid chromatography and vibrational circular dichroism: reversal of elution order of N-substituted alpha-methyl phenylalanine esters.

Enantiomeric separation of two aromatic alpha-substituted alanine esters was achieved on two commercially available polysaccharide-based chiral stationary phases (CSPs): amylose tris(3,5-dimethylphenylcarbamate) (ADMPC) and cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC). The interactions between enantiomeric analytes and the CSPs were investigated using chromatographic methods and vibration circular dichroism (VCD). The two analytes differ on the aromatic portion of the molecules where one analyte has a pi-acceptor aromatic ring (1) while the other has a pi-donor aromatic ring (2). When an ADMPC CSP was employed, an increase in the polarity of the mobile phase leads to a reversal of the elution order for the two enantiomers of 1. The elution order of compound 2 was not affected by the polarity of the mobile phase. In order to gain an understanding of these phenomena, the enantiomeric separation of 1 and 2 was also performed on the CDMPC CSP. Interestingly, no reversal of elution order was observed upon the chromatographic separation of both pairs of enantiomers of compounds 1 and 2 upon increasing the solvent polarity when a CDMPC CSP was utilized. To understand the underlying mechanism governing these chiral separations, VCD was applied to study the structure of the ADMPC and CDMPC polymers and their conformational behaviors under chromatographic conditions. For the first time the conformations of the side chains of both polymers were revealed based on the VCD spectra along with DFT calculations. Furthermore, the interactions between the two analytes and the two CSPs were directly probed by VCD. By comparing the spectral differences of the two CSPs in the presence of the two analytes, the detailed interactions involving different functional groups associated with the chiral recognition were elucidated and thus explained the unusual reversal of elution order associated with increasing solvent polarity.

Se(VI) reduction and the precipitation of Se(0) by the facultative bacterium Enterobacter cloacae SLD1a-1 are regulated by FNR.

The fate of selenium in the environment is controlled, in part, by microbial selenium oxyanion reduction and Se(0) precipitation. In this study, we identified a genetic regulator that controls selenate reductase activity in the Se-reducing bacterium Enterobacter cloacae SLD1a-1. Heterologous expression of the global anaerobic regulatory gene fnr (fumarate nitrate reduction regulator) from E. cloacae in the non-Se-reducing strain Escherichia coli S17-1 activated the ability to reduce Se(VI) and precipitate insoluble Se(0) particles. Se(VI) reduction by E. coli S17-1 containing the fnr gene occurred at rates similar to those for E. cloacae, with first-order reaction constants of k = 2.07 x 10(-2) h(-1) and k = 3.36 x 10(-2) h(-1), respectively, and produced elemental selenium particles with identical morphologies and short-range atomic orders. Mutation of the fnr gene in E. cloacae SLD1a-1 resulted in derivative strains that were deficient in selenate reductase activity and unable to precipitate elemental selenium. Complementation by the wild-type fnr sequence restored the ability of mutant strains to reduce Se(VI). Our findings suggest that Se(VI) reduction and the precipitation of Se(0) by facultative anaerobes are regulated by oxygen-sensing transcription factors and occur under suboxic conditions.

XAFS determination of the bacterial cell wall functional groups responsible for complexation of Cd and U as a function of pH.

Bacteria, which are ubiquitous in near-surface geologic systems, can affect the distribution and fate of metals in these systems through adsorption reactions between the metals and bacterial cell walls. Recently, Fein et al. (1997) developed a chemical equilibrium approach to quantify metal adsorption onto cell walls, treating the sorption as a surface complexation phenomenon. However, such models are based on circumstantial bulk adsorption evidence only, and the nature and mechanism of metal binding to cell walls for each metal system have not been determined spectroscopically. The results of XAFS measurements at the Cd K-edge and U L3-edge on Bacillus subtilis exposed to these elements show that, at low pH, U binds to phosphoryl groups while Cd binds to carboxyl functional groups.

A comparison of myocardial beta-adrenoreceptor density and ligand binding affinity among selected teleost fishes.

This study quantified the cell surface beta-adrenoreceptor density and ligand binding affinity in the ventricular tissue of seven teleost species; skipjack tuna (Katsowonus pelamis), yellowfin tuna (Thunnus albacares), Pacific mackerel (Scomber japonicus), mahimahi (dolphin fish; Coryphaena hippurus), sockeye salmon (Oncorhynchus nerka), rainbow trout (Oncorhynchus mykiss) and an Antarctic nototheniid (Trematomus bernacchii). Beta-Adrenoreceptor density varied by almost fourfold among these species, being highest for the athletic fish: sockeye salmon among the salmonids and skipjack tuna among the scombrids. Beta-Adrenoreceptor density was lowest for the Antarctic icefish. Beta-Adrenoreceptor binding affinity varied by almost threefold. We conclude that there is a significant species-specific variability in myocardial beta-adrenoreceptor density and binding affinity and these interspecific differences cannot be attributed to temperature even though intraspecifically cold temperature can stimulate an increase in myocardial beta-adrenoreceptor density. Instead, we suggest that interspecifically myocardial beta-adrenoreceptor density is highest in fish that inhabit tropical water.

Self-regeneration of stereocenters: a practical enantiospecific synthesis of LFA-1 antagonist BIRT-377.

[reaction: see text] An efficient enantiospecific synthesis of the LFA-1 antagonist BIRT-377 has been achieved in 43% overall yield in eight steps. The key transformations involve the stereospecific formation of the trans imidazolidinone 7, subsequent alkylation, and the efficient hydrolysis of disubstituted imidazolidinone 9. The process is practical, robust, and cost-effective; it has been successfully implemented in the pilot plant to produce multikilogram quantities of the drug BIRT-377.

A novel synthesis of 2-aryl-2H-indazoles via a palladium-catalyzed intramolecular amination reaction.

[reaction: see text] A variety of 2-aryl-2H-indazoles were synthesized by the palladium-catalyzed intramolecular amination of the corresponding N-aryl-N(o-bromobenzyl)hydrazines. Of several sets of reaction conditions surveyed, the combination of Pd(OAc)2/dppf/tBuONa gave the best results. This method applies to a wide scope of substrates containing electron-donating and electron-withdrawing substituents.

c-kit receptor signaling through its phosphatidylinositide-3'-kinase-binding site and protein kinase C: role in mast cell enhancement of degranulation, adhesion, and membrane ruffling.

In bone marrow-derived mast cells (BMMCs), the Kit receptor tyrosine kinase mediates diverse responses including proliferation, survival, chemotaxis, migration, differentiation, and adhesion to extracellular matrix. In connective tissue mast cells, a role for Kit in the secretion of inflammatory mediators has been demonstrated as well. We recently demonstrated a role for phosphatidylinositide-3' (PI 3)-kinase in Kit-ligand (KL)-induced adhesion of BMMCs to fibronectin. Herein, we investigated the mechanism by which Kit mediates enhancement of Fc epsilon RI-mediated degranulation, cytoskeletal rearrangements, and adhesion in BMMCs. Wsh/Wsh BMMCs lacking endogenous Kit expression, were transduced to express normal and mutant Kit receptors containing Tyr-->Phe substitution at residues 719 and 821. Although the normal Kit receptor fully restored KL-induced responses in Wsh/Wsh BMMCs, Kit gamma 719F, which fails to bind and activate PI 3-kinase, failed to potentiate degranulation and is impaired in mediating membrane ruffling and actin assembly. Inhibition of PI 3-kinase with wortmannin or LY294002 also inhibited secretory enhancement and cytoskeletal rearrangements mediated by Kit. In contrast, secretory enhancement and adhesion stimulated directly through protein kinase C (PKC) do not require PI 3-kinase. Calphostin C, an inhibitor of PKC, blocked Kit-mediated adhesion to fibronectin, secretory enhancement, membrane ruffling, and filamentous actin assembly. Although cytochalasin D inhibited Kit-mediated filamentous actin assembly and membrane ruffling, secretory enhancement and adhesion to fibronectin were not affected by this drug. Therefore, Kit-mediated cytoskeletal rearrangements that are dependent on actin polymerization can be uncoupled from the Kit-mediated secretory and adhesive responses. Our results implicate receptor-proximal PI 3-kinase activation and activation of a PKC isoform in Kit-mediated secretory enhancement, adhesion, and cytoskeletal reorganization.

Biosynthesis of cyclic diterpene hydrocarbons in rice cell suspensions: conversion of 9,10-syn-labda-8(17),13-dienyl diphosphate to 9beta-pimara-7,15-diene and stemar-13-ene.

The biosynthesis of diterpene hydrocarbons with enzyme extracts from rice cell suspension cultures was investigated to verify proposed pathways and intermediates in the production of the momilactone and oryzalexin phytoalexins. Diterpene synthase activity in cells treated with chitin to elicit the phytoalexin response was compared with the activity in untreated cells using the acyclic substrates [1-3H](E,E,E)- and [1-3H] (E,Z,E)-geranylgeranyl diphosphates (GGPPs 4-OPP and 11-OPP) as well as the bicyclic substrates [15-3H]ent-copalyl and [15-3H] syn-copalyl diphosphates (CPPs, 5-OPP, and 6-OPP). ent-kaurene (7), ent-sanda, racopimaradiene (8), 9 beta H-pimara-7,15-diene (9), and stemar-13-ene (10) were identified as major products by comparisons with authentic standards. Marked increases in diterpene synthase activities were observed with enzyme from chitin-treated cells: (E,E,E)-GGPP (approximately 100 fold), ent-CPP (approximately 3 fold), and syn-CPP (approximately 60 fold). The very low conversions of (E,Z,E)-GGPP to hydrocarbon products excludes its role in the biosynthesis of 9,10-syn-diterpenes in rice cells. ent-Kaurene was the major diterpene formed from ent-CPP with enzyme from unelicited cells. In contrast the enzyme from chitin-treated cells converted ent-CPP to a mixture of ent-kaurene, ent-sandaracopimaradiene, and a third unidentified diterpene. With syn-CPP as substrate the induced syntheses afforded a mixture of 9 beta-pimaradiene, stemarene, and a third, unidentified syn-diterpene. Overall the results are consistent with the hypothesis that rice cells respond to treatment with chitin fragments by producing new diterpene synthases not present in the untreated cells. These induced cyclases initiate phytoalexin biosynthesis by diverting (E,E,E)-GGPP into new cyclization modes that produce ent-sandaracopimaradiene, stemarene, and 9 beta-pimaradiene, the presumed precursors to oryzalexins A-F, oryzalexin S, and momilactones A-C, respectively. The intermediate role of 9,10-syn-CPP in syn diterpene biosynthesis is verified.

Differential roles of PI3-kinase and Kit tyrosine 821 in Kit receptor-mediated proliferation, survival and cell adhesion in mast cells.

The pleiotropic effects of the Kit receptor system are mediated by Kit-Ligand (KL) induced receptor autophosphorylation and its association with and activation of distinct second messengers, including phosphatidylinositol 3'-kinase (PI3-kinase), p21ras and mitogen-activated protein kinase (MAPK). To define the role of PI3-kinase, p21ras and MAPK in Kit-mediated cell proliferation, survival and adhesion in bone marrow-derived mast cells (BMMC), mutant Kit receptors were expressed in Wsh/Wsh BMMC lacking endogenous c-kit expression. The introduction of both murine Kit(S) and KitL (isoform containing a four amino acid insert) into Wsh/Wsh BMMC restored KL-induced proliferation, survival and adhesion to fibronectin, as well as activation of PI3-kinase, p21ras and MAPK, and induced expression of c-fos, junB, c-myc and c-myb mRNA. Substitution of tyrosine 719 in the kinase insert with phenylalanine (Y719F) abolished PI3-kinase activation, diminished c-fos and junB induction, and impaired KL-induced adhesion of BMMC to fibronectin. In addition, the Y719F mutation had partial effects on p21ras activation, cell proliferation and survival, while MAP kinase activation was not affected. On the other hand, Y821F substitution impaired proliferation and survival without affecting PI3-kinase, p21ras and MAPK activation, and induction of c-myc, c-myb, c-fos and c-jun mRNA, while KL-induced cell adhesion to fibronectin remained intact. In agreement with a role for PI3-kinase in Kit-mediated cell adhesion, wortmannin blocked Kit-mediated cell adhesion at concentrations known to specifically inhibit PI3-kinase. We conclude, that association of Kit with p85PI3-K, and thus with PI3-kinase activity, is necessary for a full mitogenic as well as adhesive response in mast cells. In contrast, tyrosine 821 is essential for Kit-mediated mitogenesis and survival, but not cell adhesion.

Long-term generation of human mast cells in serum-free cultures of CD34+ cord blood cells stimulated with stem cell factor and interleukin-3.

The generation of murine mast cells is supported by several cytokines, and mast cell lines are frequently established in long-term cultures of normal murine marrow cells. In contrast, growth of human mast cells was initially dependent on coculture with murine fibroblasts. The growth factor produced by murine fibroblasts and required to observe differentiation of human mast cells is attributable in part to stem cell factor (SCF). However, other factors are likely involved. We have previously shown that the combination of SCF and interleukin-3 (IL-3) efficiently sustains proliferation and differentiation of colony-forming cells (CFCs) from pre-CFC enriched from human umbilical cord blood by CD34+ selection. With periodic medium changes and the addition of fresh growth factors, five consecutive cultures of different cord blood samples gave rise to differentiated cells and CFCs for more than 2 months. Although differentiated cells continued to be generated for more than 5 months, CFCs were no longer detectable by day 50 of culture. The cells have the morphology of immature mast cells, are Toluidine blue positive, are karyotypically normal, are CD33+, CD34-, CD45+, c-kit-, and c-fms-, and die in the absence of either SCF or IL-3. These cells do not form colonies in semisolid culture and are propagated in liquid culture stimulated with SCF and IL-3 at a seeding concentration of no less than 10(4) cells/mL. At refeedings, the cultures contain a high number (> 50%) of dead cells and have a doubling time ranging from 5 to 12 days. This suggests that subsets of the cell population die because of a requirement for a growth factor other than SCF or IL-3. These results indicate that the combination of cord blood progenitor and stem cells, plus a cocktail of growth factors including SCF and IL-3, is capable with high efficiency of giving rise in serum-deprived culture to human mast cells that behave like factor-dependent cell lines. These cells may represent a useful tool for studies of human mast cell differentiation and leukemia.