Search

Endothelial cell (EC) dysfunction is the underlying cause for the development of several pathologies, and the interdependency between the pancreatic β-cells and ECs has been established in the pathophysiology of diabetes. ECs release several factors that govern the expression of genes involved in the proliferation, physiology, and survival of the β-cells. Of the known factors that collapse this intricately balanced system, endothelial dysfunction is the crucial condition that manifests as the causative factor for micro and macrovascular diseases. Our earlier studies demonstrated that activation of nuclear factor erythroid-related factor (Nrf2) renders protection to the ECs experiencing ER stress. In this study, using a co-culture system, the crosstalk between pancreatic cells under ER stress and ECs and the effect of a novel Nrf2 activator Rosolic Acid (RA), on the crosstalk was investigated. ECs pre-treated with different concentrations RA and co-cultured with thapsigargin-induced ER stressed pancreatic β-cells showed increased levels of Nrf2 and its downstream targets such as heme oxygenase-1 (HO-1) and NADPH-quinone oxidoreductase-1 (NQO-1), and reduction of ER stress evinced by the decreased levels of glucose-regulated protein (GRP) 78 and C/ERB homologous protein (CHOP). The sensitization of ECs using RA, offered protection to pancreatic cells against ER stress as displayed by increased intracellular insulin and upregulated expression of cell survival and proliferative genes BCl2 and PDX-1. In addition, RA treatment resulted in elevated levels of various angiogenic factors, while inflammatory (TNF-α and IL-1β) and apoptotic markers (CXCL10 and CCL2) decreased. RA treatment normalized the levels of 115 proteins of the 277, which were differentially regulated as revealed by proteomic studies of ER stressed pancreatic β-cells in co-culture conditions. These findings clearly indicate the role of small molecule activators of Nrf2 not only in restoring the functioning of pancreatic cells but also in increasing the cell mass. Further, the study impinges on the strategies that can be developed to balance the pancreatic microenvironment, leading to the restoration of β-cell mass and their normophysiology in diabetic patients.

Endoplasmic reticulum (ER) plays a key role in the folding, modification, and trafficking of proteins. When the homeostasis of the ER is disturbed, un/misfolded proteins accumulate in the ER which leads to ER stress. Sustained ER stress results in apoptosis, which is associated with various diseases. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a major transcription factor in redox homeostasis by regulating various genes associated with detoxification and cell-protective mechanisms. We found that Rosolic acid (RA) treatment dose-dependently activates Nrf2 in endothelial cells using the enzyme fragment complementation assay. The cytoprotective role of RA against ER stress-induced endothelial apoptosis and its molecular mechanism was explored in the present study. The Nrf2 and its target genes, as well as ER stress marker expressions, were measured by qPCR in ER stress-exposed endothelial cells. The contribution of Nrf2 in RA-mediated defense mechanism in endothelial cells was established by knockout studies using Nrf2-CRISPR/Cas9. The treatment with RA to ER stress-induced endothelial cells exhibited activation of Nrf2, as demonstrated by Nrf2 translocation and reduction of ER stress markers. We found that the Nrf2 knockout sensitized the endothelial cells against ER stress, and further, RA failed to mediate its cytoprotective effect. Proteomic studies using LC-MS/MS revealed that among the 1370 proteins detected, we found 296 differentially regulated proteins in ER stress-induced endothelial cells, and RA administration ameliorated 71 proteins towards the control levels. Of note, the ER stress in endothelial cells was attenuated by the treatment with the RA, suggesting the role of the Nrf2 activator in the pathological conditions of ER stress-associated diseases., Competing Interests: We declare that we have no competing financial interests and personal relationships with other people or organizations that can inappropriately influence our work. There is no professional or other personal interests of any nature or kind in any product, service, and/or company that could be construed as influencing the position presented in, or the review of, the manuscript., (Copyright © 2021 Karan Naresh Amin et al.)

Despite recent advances in therapy, allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative option for a range of high-risk hematologic malignancies. However, acute graft-versus-host disease (aGVHD) continues to limit the long-term success of HSCT, and new therapies are still needed. We previously demonstrated that aGVHD depends on the ability of donor conventional T cells (T con s ) to express the lymph node trafficking receptor, CC-Chemokine Receptor 7 (CCR7). Consequently, we examined the ability of cosalane, a recently identified CCR7 small-molecule antagonist, to attenuate aGVHD in mouse HSCT model systems. Here we show that the systemic administration of cosalane to transplant recipients after allogeneic HSCT did not prevent aGVHD. However, we were able to significantly reduce aGVHD by briefly incubating donor T cons with cosalane ex vivo before transplantation. Cosalane did not result in T con toxicity and did not affect their activation or expansion. Instead, cosalane prevented donor T con trafficking into host secondary lymphoid tissues very early after transplantation and limited their subsequent accumulation within the liver and colon. Cosalane did not appear to impair the intrinsic ability of donor T con s to produce inflammatory cytokines. Furthermore, cosalane-treated T con s retained their graft-versus-leukemia (GVL) potential and rejected a murine P815 inoculum after transplantation. Collectively, our data indicate that a brief application of cosalane to donor T con s before HSCT significantly reduces aGVHD in relevant preclinical models while generally sparing beneficial GVL effects, and that cosalane might represent a viable new approach for aGVHD prophylaxis., (Copyright © 2019 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

CC-chemokine receptor 7 (CCR7) is a G protein-coupled receptor expressed on a variety of immune cells. CCR7 plays a critical role in the migration of lymphocytes into secondary lymphoid tissues. CCR7 expression, however, has been linked to numerous disease states. Due to its therapeutic relevance and absence of available CCR7 inhibitors, we undertook a high-throughput screen (HTS) to identify small-molecule antagonists of the receptor. Here, we describe a robust HTS approach using a commercially available β-galactosidase enzyme fragment complementation system and confirmatory transwell chemotaxis assays. This work resulted in the identification of several compounds with activity against CCR7. The most potent of these was subsequently determined to be cosalane, a cholesterol derivative previously designed as a therapeutic for human immunodeficiency virus. Cosalane inhibited both human and murine CCR7 in response to both CCL19 and CCL21 agonists at physiologic concentrations. Furthermore, cosalane produced durable inhibition of the receptor following a cellular incubation period with subsequent washout. Overall, our work describes the development of an HTS-compatible assay, completion of a large HTS campaign, and demonstration for the first time that cosalane is a validated CCR7 antagonist. These efforts could pave the way for new approaches to address CCR7-associated disease processes.

Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) are sensitive techniques used for detecting nucleic acids and proteins in cultured cells. However, these techniques are rarely applied together, and standard protocols are not readily compatible for sequential application on the same specimen. Here, we provide a user-friendly step-by-step protocol to perform multicolor RNA-FISH in combination with IF to simultaneously detect the subcellular localization of distinct RNAs and proteins in cultured cells. We demonstrate the use of our protocol by analyzing changes in the subcellular distribution of RNAs and proteins in cells exposed to a variety of stress conditions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Yersinia sp. bacteria owe their viability and pathogenic virulence to the YopH factor, which is a highly active bacterial protein tyrosine phosphatase. Inhibition of YopH phosphatase results in the lack of Yersinia sp. pathogenicity. We have previously described that aurintricarboxylic acid inhibits the activity of YopH at nanomolar concentrations and represents a unique mechanism of YopH inactivation due to a redox process. This work is a continuation of our previous studies. Here we show that modifications of the structure of aurintricarboxylic acid reduce the ability to inactivate YopH and lead to higher cytotoxicity. In the present paper we examine the inhibitory properties of aurintricarboxylic acid analogues, such as eriochrome cyanine R (ECR) and pararosaniline. Computational docking studies we report here indicate that ATA analogues are not precluded to bind in the YopH active site and in all obtained binding conformations ECR and pararosaniline bind to YopH active site. The free binding energy calculations show that ECR has a stronger binding affinity to YopH than pararosaniline, which was confirmed by experimental YopH enzymatic activity studies. We found that ATA analogues can reversibly reduce the enzymatic activity of YopH, but possess weaker inhibitory properties than ATA. The ATA analogues induced inactivation of YopH is probably due to oxidative mechanism, as pretreatment with catalase prevents from inhibition. We also found that ATA analogues significantly decrease the viability of macrophage cells, especially pararosaniline, while ATA reveals only slight effect on cell viability., Competing Interests: The authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Pharmacological induction of proteotoxic stress is rapidly emerging as a promising strategy for cancer cell-directed chemotherapeutic intervention. Here, we describe the identification of a novel drug-like heat shock response inducer for the therapeutic induction of proteotoxic stress targeting malignant human melanoma cells. Screening a focused library of compounds containing redox-directed electrophilic pharmacophores employing the Stress & Toxicity PathwayFinder(TM) PCR Array technology as a discovery tool, a drug-like triphenylmethane-derivative (aurin; 4-[bis(p-hydroxyphenyl)methylene]-2,5-cyclohexadien-1-one) was identified as an experimental cell stress modulator that causes (i) heat shock factor transcriptional activation, (ii) up-regulation of heat shock response gene expression (HSPA6, HSPA1A, DNAJB4, HMOX1), (iii) early unfolded protein response signaling (phospho-PERK, phospho-eIF2α, CHOP (CCAAT/enhancer-binding protein homologous protein)), (iv) proteasome impairment with increased protein-ubiquitination, and (v) oxidative stress with glutathione depletion. Fluorescence polarization-based experiments revealed that aurin displays activity as a geldanamycin-competitive Hsp90α-antagonist, a finding further substantiated by molecular docking and ATPase inhibition analysis. Aurin exposure caused caspase-dependent cell death in a panel of human malignant melanoma cells (A375, G361, LOX-IMVI) but not in non-malignant human skin cells (Hs27 fibroblasts, HaCaT keratinocytes, primary melanocytes) undergoing the aurin-induced heat shock response without impairment of viability. Aurin-induced melanoma cell apoptosis depends on Noxa up-regulation as confirmed by siRNA rescue experiments demonstrating that siPMAIP1-based target down-regulation suppresses aurin-induced cell death. Taken together, our data suggest feasibility of apoptotic elimination of malignant melanoma cells using the quinone methide-derived heat shock response inducer aurin., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Complement plays a vital role in both the innate and adaptive immune systems. It recognizes a target, opsonizes it, generates anaphylatoxins, and directly kills cells through the membrane attack complex (MAC). This final function, which assembles C5b-9(n) on viable cell surfaces, can kill host cells through bystander lysis. Here we identify for the first time compounds that can inhibit bystander lysis while not interfering with the other essential functions of complement. We show that aurin tricarboxylic acid (ATA), aurin quadracarboxylic acid (AQA), and aurin hexacarboxylic acid (AHA), block the addition of C9 to C5b-8 so that the MAC cannot form. These molecules inhibit hemolysis of human, rat, and mouse red cells with a half maximal inhibitory concentration (IC(50)) in the nanomolar range. When given orally to Alzheimer disease type B6SJL-Tg mice, they inhibit MAC formation in serum and improve memory retention. On autopsy, they show no evidence of harm to any organ. Aurin tricarboxylic acid, aurin quadracarboxylic acid, and aurin hexacarboxylic acid may be effective therapeutic agents in Alzheimer disease and other degenerative disorders where self damage from the MAC occurs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

DNA methyltransferase 1 (DNMT1) is an emerging target for the treatment of cancer, brain disorders, and other diseases. Currently, there are only a few DNMT1 inhibitors with potential application as therapeutic agents or research tools. 5,5-Methylenedisalicylic acid is a novel scaffold previously identified by virtual screening with detectable although weak inhibitory activity of DNMT1 in biochemical assays. Herein, we report enzyme inhibition of a structurally related compound, trimethylaurintricarboxylic acid (NSC97317) that showed a low micromolar inhibition of DNMT1 (IC(50) = 4.79 μM). Docking studies of the new inhibitor with the catalytic domain of DNMT1 suggest that NSC97317 can bind into the catalytic site. Interactions with amino acid residues that participate in the mechanism of DNA methylation contribute to the binding recognition. In addition, NSC97317 had a good match with a structure-based pharmacophore model recently developed for inhibitors of DNMT1. Trimethylaurintricarboxylic acid can be a valuable biochemical tool to study DNMT1 inhibition in cancer and other diseases related to DNA methylation.

Cosalane and related compounds are a peculiar group of anti-HIV agents with activities against a broad range of viral targets, such as viral entry and reverse transcriptase (RT). Cosalane and its analogues having anionic pharmacophore inhibit the binding of gp120 to CD4 as well as the fusion of the viral envelope with the cell membrane. The alkenyldiarylmethanes (ADAMs), characterized by the lack of the steroidal moiety of cosalane, are a unique class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that have potential value in the treatment of HIV infection. In this article, the structural modifications, structure-activity relationship (SAR) studies and/or crystallographic studies of cosalane related derivatives as potent antiviral agents were reviewed, which will be beneficial to the discovery of next generation cosalane derivatives with improved antiviral potency, metabolic stability and bioavailability.

Glucocorticoids are widely used to suppress inflammation and treat various immune-mediated diseases. Some glucocorticoid receptor (GR)-regulated genes mediate the therapeutic response, whereas others cause debilitating side effects. To discover selective modulators of the GR response, we developed a high-throughput, multiplexed system to monitor regulation of 4 promoters simultaneously. An initial screen of 1,040 natural products and Food and Drug Administration-approved drugs identified modulators that caused GR to regulate only a subset of its target promoters. Some compounds selectively inhibited GR-mediated gene activation without altering the repression of cytokine expression by GR. This approach will facilitate identification of genes and small molecules that augment beneficial effects of GR and diminish deleterious ones. Our results have important implications for the development of GR modulators and the identification of cross-talk pathways that control selective GR gene regulation.

Previously, we have reported that aurintricarboxylic acid (ATA) is one of the most potent inhibitors of the DNA binding of transcription factor NF-kappaB. We now report the NF-kappaB-DNA binding inhibitory activity of ATA analogues. An electrophoretic mobility shift assay has shown that bromopyrogallol red (BPR) is the most effective inhibitor of NF-kappaB-DNA binding among the studied analogues. The molecular modeling studies showed that BPR makes a strong network of hydrogen bonds with the DNA-binding region of the p50 subunit of NF-kappaB and has electronegative potential on its peripheral surface. Because zinc has been reported to influence the DNA binding of NF-kappaB, the interaction of these analogues with zinc was studied. Chemical speciation and formation-constant studies showed that BPR forms the most stable 1:1 complex with zinc. BPR has also been found to be the most potent antioxidant among the studied analogues.

The induction of heme oxygenase-1 (HO-1) is widely recognized as an effective cellular strategy to counteract a variety of stressful events. We have shown that curcumin and caffeic acid phenethyl ester, two naturally occurring phytochemicals that possess antioxidant, anti-inflammatory, and anticarcinogenic activities, induce HO-1 in many cell types. This suggests that stimulation of HO-1 could partly underlie the beneficial effects exerted by these plant-derived constituents. Here we examined the ability of additional plant constituents to up-regulate heme oxygenase activity and HO-1 in aortic endothelial cells. Incubation of endothelial cells with a series of polyphenolic chalcones (5-50 microM) resulted in increased heme oxygenase activity; interestingly, the chemical structure dictated the pattern of heme oxygenase induction, which was unique to each particular compound employed. We also found that rosolic acid, a constituent isolated from the rhizome of Plantago asiatica L. dramatically increased HO-1 in a concentration- and time-dependent manner. Severe cytotoxicity was observed after prolonged exposure (24 or 48 h) of cells to curcumin and caffeic acid phenethyl ester, whereas 2'-hydroxychalcone and rosolic acid did not affect cell viability. By using different mitogen-activated protein kinase inhibitors, we determined that the extracellular signal-regulated kinase, p38, and c-Jun NH(2)-terminal protein kinase pathways play only a minor role in the induction of HO-1 by rosolic acid and 2'-hydroxychalcone. On the other hand, increased intra- and extracellular thiols markedly reduced the rise in heme oxygenase activity elicited by rosolic acid. Thus, this study identified novel plant constituents that highly induce HO-1 in endothelial cells and investigated some of the mechanisms involved in this effect.

Purpose: Cosalane is a potent inhibitor of HIV replication with activity against a broad range of viral targets. However, oral bioavailability of this highly lipophilic compound is extremely poor (<1%). The purpose of this study is to screen a variety of permeation enhancers (cyclodextrin derivatives, cremophor EL, bile salts and mixed micelles) for their ability to enhance the transport of cosalane and its analogs/prodrugs across Caco-2 cell monolayers., Methods: Cosalane and its different analogs/prodrugs were synthesized and their physicochemical properties were determined. Caco-2 cells were cultured at a density of 66,000 cells/cm(2) either on collagen coated clear polyester membranes or Transwell inserts. Side-bi-side diffusion cells and Transwell inserts were employed to study for the transport of cosalane and its analogs/prodrugs with various permeation enhancers across Caco-2 cell monolayers., Results: Permeabilities of EH-3-39, EH-3-55 and EH-3-57 significantly improved compared to that of cosalane in the presence of bile salt, sodium desoxycholate. Among the various cyclodextrins studied, hydroxypropyl beta cyclodextrin (HP-beta-CD) and dimethyl beta cyclodextrin (DM-beta-CD) exhibited 22.3-fold and 19-fold permeability enhancement of cosalane respectively across Caco-2 cell monolayers. Sodium desoxycholate (10 mM) also showed a remarkable (105-fold) enhancement on the permeability of cosalane (P(app) 11.72+/-3.31 x 10(-6) cm/s) without causing any measurable cellular damage. Cremophor EL resulted in higher transport of 14C mannitol. The mechanism of enhancement effect can be mainly attributed to the alteration of membrane fluidity by cyclodextrin and opening of tight junctions by cremophor EL., Conclusions: Among the enhancers tested, 10 mM sodium desoxycholate and HP-beta-CD appear to be viable candidates for further development of an oral formulation of cosalane and its congeners.

Cosalane and its amino acid conjugates are potent inhibitors of HIV replication. The purpose of this study was to investigate: (1) the pharmacokinetic disposition of the diglycine (GC) and the diaspartic acid (ASPC) conjugates of cosalane in male Sprague-Dawley rats; (2) intestinal absorption of cosalane and its amino acid conjugates using in vitro (small intestinal segments), in situ (closed loop); and (3) biodistribution of GC and its absolute oral bioavailability in rat. Cosalane and its conjugates exhibited biexponential disposition with very long half-lives upon intravenous dosing. However, these compounds failed to permeate the small intestine unless sodium desoxycholate (5-20 mM) was used as an intestinal permeation enhancer. A rank order correlation in terms of permeation enhancement in a descending order is as follows: GC>Cosalane>ASPC. In situ studies revealed that although the bile salt enhanced the permeation of cosalane across the enterocyte, its hepatic uptake was extensive. However, 66% of the absorbed dose of GC escaped uptake by the reticuloendothelial system (RES) and its biodistribution studies showed that the uptake by the RES was significantly lower compared to the parent compound. GC had an absolute oral bioavailability of 5.10+/-1.51%. Therefore, GC appears to be a favorable candidate for further development.

A new series of cosalane analogues incorporating two fragments of the dichlorodisalicylmethane pharmacophore has been synthesized. In order to identify the position for the attachment of the pharmacophore fragments to the steroid ring that results in the most potent analogues, two types of compounds were designed. In the first type, the two pharmacophore fragments were attached at C-3 and C-17 of the steroid ring by using appropriate linker units. In the second type, both pharmacophore groups were connected to C-3 of the steroid through an alkenyl chain containing an amide moiety. All of the new compounds displayed antiviral activity versus HIV-1(RF), HIV-1(IIIB), and HIV-2(ROD) in cell culture. The relative potencies of the compounds resulting from the two attachment strategies were found to depend on the viral strain as well as the cell type. Overall, the attachment of the second pharmacophore did not result in either a large gain or a large loss in anti-HIV activity, and the results are therefore consistent with the hypothesis that the two pharmacophores act independently, and one at a time, with positively charged amino acid side chains present on the surface of gp120 and CD4.

Cosalane is a potent inhibitor of HIV replication with multiple sites of action. The purposes of this study were to (a) determine the extent and nature of cosalane binding to mucin, alpha(1)-acid glycoprotein (AAG), plasma, and human (HSA) and bovine serum (BSA) albumin, and (b) determine the primary site(s) of cosalane binding to HSA. Plasma protein binding of cosalane was studied by a gel filtration technique. Cosalane binding to HSA was also determined in the presence of salicylic acid. Competitive inhibition studies were conducted using warfarin, digitoxin, and diazepam to determine the primary HSA binding site(s) of cosalane. The drug was bound extensively to HSA and BSA and required 500-550 moles to saturate 1 mole of protein. Stoichiometries of cosalane binding to alpha(1)-acid glycoprotein (AAG) and mucin were between 30 and 50 mol/mol of either glycoprotein. The binding isotherm deviated from a rectangular hyperbola, suggesting self-association of the ligand. Salicylic acid decreased cosalane binding to HSA by one order of magnitude. Inhibition studies of cosalane to HSA revealed that the compound binds primarily to warfarin site with a K(i) of 1.24 +/- 0.24 nM. In summary, cosalane binds extensively to serum albumins and to a lesser extent to both AAG and mucin., (Copyright 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association.)

Cosalane and its synthetic derivatives inhibit the binding of gp120 to CD4 as well as the fusion of the viral envelope with the cell membrane. The binding of the cosalanes to CD4 is proposed to involve ionic interactions of the negatively charged carboxylates of the ligands with positively charged arginine and lysine amino acid side chains of the protein. To investigate the effect of anion spacing on anti-HIV activity in the cosalane system, a series of cosalane tetracarboxylates was synthesized in which the two proximal and two distal carboxylates are separated by 6--12 atoms. Maximum activity was observed when the proximal and distal carboxylates are separated by 8 atoms. In a series of cosalane amino acid derivatives containing glutamic acid, glycine, aspartic acid, beta-alanine, leucine, and phenylalanine residues, maximum activity was displayed by the di(glutamic acid) analogue. A hypothetical model has been devised for the binding of the cosalane di(glutamic acid) conjugate to CD4. In general, the compounds in this series are more potent against HIV-1(RF) in CEM-SS cells than they are vs HIV-1(IIIB) in MT-4 cells, and they are least potent vs HIV-2(ROD) in MT-4 cells.

The anti-HIV agent cosalane and several of its analogues inhibited RANTES-induced migration of human monocytes, but they did not inhibit migration induced by MIP1alpha or MIP1beta. RANTES-induced migration of single receptor CCRI-HEK transfectants was also inhibited by the cosalanes. Acetylation of the reactive amino groups of RANTES abrogated the inhibitory activity of cosalane. The data suggest that cosalane and its structural analogues may interfere with the RANTES/CCR1 interaction by binding to RANTES.

The binding of the anti-HIV agent cosalane to CD4 is thought to involve ionic interactions of negatively charged carboxylates of the ligand with positively charged residues on the surface of the protein. The purpose of the present study was to examine the hypothesis that the two carboxyl groups of cosalane could be sacrificed through conjugation to amino acids, and the anti-HIV activity still be retained, provided that at least two new carboxyl groups are contributed by the amino acid residues.

The binding of the anti-HIV agent cosalane to CD4 is thought to involve ionic interactions of negatively charged carboxylates of the ligand with positively charged residues on the surface of the protein. An investigation of the optimal anion distances for anti-HIV activity in a series of cosalane tetracarboxylate analogues has been completed, and maximal activity results when the two proximal and the two distal carboxylates are separated by eight atoms.

Cosalane is a potent inhibitor of HIV replication with activity against a broad range of viral targets. However, the oral bioavailability of this highly lipophilic compound is extremely poor (<1%). Also, cosalane accumulates in high concentration in the liver after intravenous administration, with clear resistance to hepatic metabolism. In the present study, the transcellular permeability of cosalane was examined using Transwell(R) filter as well as plastic-grown confluent Caco-2 cell monolayers. A cell-culture-based biophysical model was adopted to understand the interactions of protein binding, membrane partitioning, and aqueous solubility of cosalane in limiting transcellular flux of cosalane across Caco-2 cell monolayers. The transcellular permeability (P(app)) of cosalane was extremely low (4.494 x 10(-8) cm/s) and the effect of p-glycoprotein on the efflux of cosalane was negligible. A characteristic disparity exists between the kinetics of cosalane uptake from apical (AP) donor solution and efflux into basolateral (BL) receiver side. The AP uptake of cosalane was rapid, exhibiting exponential kinetics, and reached equilibrium within 60 min, whereas the concomitant appearance of the compound into the BL receiver side was slow but linear over time. Furthermore, the uptake of cosalane was significantly reduced in the presence of bovine serum albumin (BSA). In unidirectional efflux studies, AP efflux of cosalane was limited in the absence of BSA. Also, no detectable metabolites were found in Caco-2 cell incubations. In conclusion, the present study demonstrates that diffusion of cosalane across Caco-2 cell monolayers is extremely limited and kinetically regulated essentially by the equilibrium between protein-bound and free drug partitioning into cell membrane., (Copyright 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89:826-833, 2000.)

Cosalane and dihydrocosalane are potent inhibitors of HIV replication with a broad range of activity. The purpose of this study was to investigate: 1) the pharmacokinetic disposition of both cosalane and dihydrocosalane in male Sprague-Dawley rats, and 2) biliary excretion, enterohepatic circulation, and tissue distribution of cosalane after i.v. and/or oral administration. Animals were administered i.v. (10 mg/kg) cosalane or dihydrocosalane through a jugular vein to obtain plasma profiles. Dose dependence of cosalane was studied over a dose range of 1.0 to 10 mg/kg. The extent of enterohepatic recycling, biliary excretion, and tissue distribution were studied after i.v. administration. Both cosalane and dihydrocosalane exhibited a biexponential disposition with very long half-lives of 749 +/- 216 and 1016 +/- 407 min, along with very large volumes of distribution 23.1 +/- 4.4 and 24.4 +/- 2. 5 liter/kg, respectively. Both cosalane (nondetectable) and dihydrocosalane (<1%) showed very poor oral bioavailability. The biliary and renal excretions of cosalane were found to be negligible with no detectable metabolites either in urine or bile. After oral administration, more than 87% of the cosalane dose was excreted in the feces as the parent compound. Also, cosalane was sequestered significantly in liver with quantifiable levels in all tissues tested, even 48 h after the dose was administered. Therefore it was concluded that the poor oral bioavailability of cosalane may be due to its poor enterocytic transport coupled with sequestration in liver parenchymal cell membrane layers.

Introduction of an amido group or an amino moiety into the alkenyl linker chain of cosalane (1) provided a new series of analogues 3-8. The new compounds were evaluated as inhibitors of the cytopathic effect of HIV-1 and HIV-2 in cell culture. The replacement of the 1' and 2' carbons in the linker chain of I by an amido group was generally tolerated. The length of the linker chain and the stereochemistry of the substituent at C-3 of the steroidal ring had significant effects on the antiviral activity and potency. Incorporation of an amino moiety into the linker completely abolished the anti-HIV activity. There are several steps in the HIV replication cycle that have been proposed as targets for the development of therapeutic agents (De Clercq, E. J. Med. Chem. 1995, 38, 2491; De Clercq, E. Pure Appl. Chem. 1998, 70, 567). However, currently approved anti-HIV drugs are only directed against the viral enzymes reverse transcriptase or protease (Carpenter. C. C. J.; Fischl, M. A.; Hammer, S. M.; Hirsch, M. S.; Jacobsen, D. M.; Katzenstein, D. A.; Montaner, J. S. G.; Richman, D. D.; Saag, M. S.; Schooley, R. T.; Thompson, M. A.; Vella, S.; Yeni, P. G.; Volberding, P. A. JAMA 1998, 280, 78). Drugs capable of interfering with other steps of the virus life cycle will be highly valuable in the antiretroviral therapy of AIDS, as they will have different patterns of resistance mutations than the drugs currently used clinically. In addition, their utilization in combination with other therapeutic agents could provide more potent drug 'cocktails' capable of completely suppressing virus replication. Consequently, there is an urgent need for the discovery of clinically useful anti-HIV agents possessing novel mechanisms of action.

Cosalane is a potent inhibitor of HIV replication with a broad range of activity. In this study, the hepatic disposition of cosalane was investigated with a noncirculating isolated perfused rat liver technique. When 6 microM cosalane was infused into livers from untreated rats, the drug was highly extracted by the liver (only 2. 5% of influent cosalane concentration appeared in the effluent perfusate). Pretreatment of rats with various inducers of cytochrome P-450 before perfusion neither altered the effluent cosalane concentration nor resulted in the appearance of detectable metabolites in the effluent perfusate or liver homogenates. Hepatic uptake of cosalane was negligible when the drug was infused in the presence of BSA, and infusion of albumin after cosalane resulted in a significant displacement of the drug into the effluent perfusate. Furthermore, permeabilization of perfused livers with digitonin significantly diminished effluent cosalane concentration while enhancing cosalane uptake by the liver. Based on our data, it appears that a significant proportion of cosalane does not penetrate the hepatocyte membrane and may accumulate in the lipid bilayer of the cell membrane. This finding supports the proposed mechanism explaining the antiviral effect of cosalane which stipulates that this compound appears to imbed perpendicularly in the lipid bilayer of the cell membrane and the viral envelope. Also, cosalane does not seem to be metabolized by the liver as evidenced by the lack of detectable metabolites in the effluent perfusate, liver homogenates, and liver microsomal incubations.

The anti-HIV agent cosalane inhibits both the binding of gp120 to CD4 as well as an undefined postattachment event prior to reverse transcription. Several cosalane analogues having an extended polyanionic "pharmacophore" were designed based on a hypothetical model of the binding of cosalane to CD4. The analogues were synthesized, and a number of them displayed anti-HIV activity. One of the new analogues was found to possess enhanced potency as an anti-HIV agent relative to cosalane itself. Although the new analogues inhibited both HIV-1 and HIV-2, they were more potent as inhibitors of HIV-1 than HIV-2. Mechanism of action studies indicated that the most potent of the new analogues inhibited fusion of the viral envelope with the cell membrane at lower concentrations than it inhibited attachment, suggesting inhibition of fusion as the primary mechanism of action.

A novel cosalane analog having an extended polyanionic pharmacophore was synthesized in order to target specific cationic residues on the surface of CD4. The design rationale is based on a hypothetical binding model of cosalane to the surface of the protein. The new analog displayed an EC50 of 0.55 microM as an inhibitor of the cytopathic effect of HIV-1RF in CEM-SS cells, which represents a significant increase in potency over cosalane itself (EC50 5.1 microM). Both cosalane and the new analog are inhibitors of viral entry into target cells.

Purpose: The selection of cosalane (NSC 658586) by the National Cancer Institute for further development as a potential drug candidate for the treatment of AIDS led to the exploration of the solubility behavior of this extremely hydrophobic drug, which has an intrinsic solubility (S0 approaching 1 ng/ml. This study describes attempts to reliably measure the intrinsic solubility of cosalane and examine its pH-solubility behavior., Methods: S0 was estimated by 5 different strategies: (a) direct determination in an aqueous suspension: (b) facilitated dissolution; (c) estimation from the octanol/water partition coefficient and octanol solubility (d) application of an empirical equation based on melting point and partition coefficient; and (e) estimation from the hydrocarbon solubility and functional group contributions for transfer from hydrocarbon to water., Results: S0 estimates using these five methods varied over a 5 x 107-fold range Method (a) yielded the highest values, two-orders of magnitude greater than those obtained by method (b) (facilitated dissolution. 1.4 +/- 0.5 ng/ml). Method (c) gave a value 20-fold higher while that from method (d) was in fair agreement with that from facilitated dissolution. Method (e) yielded a value several orders-of-magnitude lower than other methods. A molecular dynamics simulation suggests that folded conformations not accounted for by group contributions may reduce cosalane's effective hydrophobicity. Ionic equilibria calculations for this weak diprotic acid suggested a 100-fold increase in solubility per pH unit increase. The pH-solubility profile of cosalane at 25 degrees C agreed closely with theory., Conclusions: These studies highlight the difficulty in determining solubility of very poorly soluble compounds and the possible advantage of the facilitated dissolution method. The diprotic nature of cosalane enabled a solubility enhancement of > 107-fold by simple pH adjustment.

Inhibition of binding of HIV via gp120 to its principle cellular receptor, CD4, remains an attractive site for intervention in the viral replicative cycle despite the poor clinical trial results demonstrated to date for sCD4. Based on a model structure we recently proposed for gp120, we have examined the predicted binding sites for several synthetic and natural products which selectively bind to gp120 and inhibit binding to CD4. Correlation between the decrease in internal energies of the ligand/gp120 complexes versus the reported inhibitory constants of the known ligands suggests that the derived gp120 structure is sufficiently accurate to perform computer simulations to guide the design of improved ligands for the CD4 binding site on gp120.

The effects of linker chain modifications were investigated in a series of cosalane analogues. The modifications investigated included: (1) shortening the three-carbon linker chain between the dichlorodisalicylmethane and the cholestane moiety by one carbon atom; (2) lengthening the linker chain by one carbon; (3) hydrogenation of the double bond in the linker chain; (4) changing the point of attachment of the linker chain from C-3 to C-6; (5) insertion of a phosphate between the steroid and the linker chain. With the exception of the phosphate modification, which abolished anti-HIV activity and increased cytotoxicity, the linker chain modifications produced relatively minor changes in anti-HIV activity and increased cytotoxicity, the linker chain modifications produced relatively minor changes in anti-HIV potency. The steroid and attached linker chain of cosalane therefore appear only to provide a general lipophilic appendage for the dichlorodisalicylmethane pharmacophore.

Several novel alkenyldiarylmethane (ADAM) non-nucleoside HIV-1 reverse transcriptase inhibitors were synthesized. The most potent of these proved to be 3',3"-dibromo-4',4"-dimethoxy-5'5"-bis(methoxycarbonyl)-1,1-diphenyl-1-+ ++heptene (8) ADAM 8 inhibited the cytopathic effect of HIV-1 in CEM cell culture with an EC50 value of 7.1 microM and was active against an array of laboratory strains of HIV-1 in CEM-SS and MT-4 cells, but was inactive as an inhibitor of HIV-2. In common with the other known non-nucleoside reverse transcriptase inhibitors, ADAM 8 was an effective inhibitor of HIV-1 reverse transcriptase (IC50 1 microM) with poly(rC).oligo(dG), but not with poly(rA).oligo(dT), as the template/primer. ADAM 8 was inactive against HIV-1 reverse transcriptases containing non-nucleoside reverse transcriptase inhibitor resistance mutations at residues 101, 106, 108, 139, 181, 188, and 236, while it remained active against enzymes with mutations at residues 74, 98, 100, 103, and at 103/181. An AZT-resistant virus having four mutations in reverse transcriptase was more sensitive to inhibition by ADAM 8 than the wild-type HIV-1. In addition, ADAM 8 displayed synergistic activity with AZT, but lacked synergy with ddI. ADAM 8 or a structurally related analog may therefore be useful as an antiviral agent in combination with AZT or with other NNRTIs that are made ineffective by mutations at residues which do not confer resistance to ADAM 8.

In order to define the role of the cholestane moiety in the anti-HIV agent cosalane, a series of cosalane analogs was synthesized in which the cholestane ring system was replaced by normal alkenyl and phosphodiester substituents having varied chain lengths and lipophilicities. The compounds containing simple alkenyl substituents were found to be more potent as inhibitors of the cytopathic effect of HIV-1 in cell culture than the phosphodiesters. In addition, the potencies of the alkene congeners correlated positively with chain length and lipophilicity of the alkene. The results indicate that the cholestane moiety of cosalane functions as a lipophilic accessory appendage to escort the dichlorodisalicylmethane pharmacophore to a lipid environment.

Several new analogues of the novel anti-HIV agent cosalane have been synthesized and evaluated as inhibitors of HIV-1 integrase and protease, HIV-1 replication, HIV-1 and HIV-2 cytopathicity, HIV-1- and HIV-2-mediated syncytium formation, and cytopathicity of a variety of human pathogenic viruses. The congeners displayed enhanced potencies relative to cosalane itself as inhibitors of HIV-1 integrase and protease. The two most potent analogues against HIV-1 integrase displayed IC50 values of 2.2 microM, while the three most potent compounds against HIV-1 protease had IC50 values in the 0.35-0.39 microM range. In addition to its activity against HIV-1 and HIV-2 cytopathicity, cosalane inhibited the cytopathic effects of herpes simplex virus-1, herpes simplex virus-2, and human cytomegalovirus at concentrations that were well below the cytotoxic concentrations. Potentially useful antiviral activities were also revealed for some of the new cosalane congeners against influenza virus, Junin virus, and Tacaribe virus.

Cosalane (3), a novel anti-HIV agent having a disalicylmethane unit linked to C-3 of cholestane by a three-carbon linker, was synthesized from commercially available starting materials by a convergent route. Cosalane proved to be a potent inhibitor of HIV with a broad range of activity against a variety of laboratory, drug-resistant, and clinical HIV-1 isolates, HIV-2, and Rauscher murine leukemia virus. The cytotoxicity of cosalane is relatively low as reflected by an in vitro therapeutic index of > 100. Although cosalane inhibits HIV-1 reverse transcriptase and protease, time of addition experiments indicate that it prevents the cytopathic effect of HIV by acting earlier than reverse transcription in the viral replication cycle. The available evidence indicates that the primary mechanism of action of cosalane involves inhibition of gp120-CD4 binding as well as inhibition of a postattachment event prior to reverse transcription.

The activity of 45 compounds against bloodstream forms of Trypanosoma cruzi was investigated. The aim was to consider new agents which might subsequently be assayed for chemoprophylaxis in donated blood. In a preliminary screening the drugs were assayed (50 to 1,000 microM at 29 degrees C) and those active against bloodstream forms at concentrations below 600 microM were selected for further assays under blood-bank conditions (4 degrees C/24 h). Three compounds isolated from natural sources and six synthetic agents were selected. The active compounds of plant origin included purpurin, a member of the trihydroxylated anthraquinone group, which is known to exhibit trypanocidal activity. Among the active synthetic compounds, five displayed a common structural feature in that they were potentially one-electron acceptors, via reductive functional groups. All five compounds form tricentered C or N intermediates, joined in a hypothetical 'Y' radical pattern. It is possible that the trypanocidal mechanisms initiated by these compounds are similar to those found with crystal violet, since this dye, which is already used in endemic areas for the treatment of banked blood, also conforms to this general Y structural pattern.

Several aurintricarboxylic acid (ATA) monomers, monomer analogs, and polymer fractions have been tested as inhibitors of HIV-1 integration protein (IN). Both of the ATA monomers and all of the ATA polymer fractions inhibited a selective DNA cleavage reaction catalyzed by IN. The ATA monomer analogs were inactive or had low activity. The activities of the substances as inhibitors of HIV IN correlated in a positive way with their activities as inhibitors of the cytopathic effect of HIV-1 in CEM and HIV-2 in MT4 cells. These results suggest that inhibition of HIV IN may contribute to the antiviral activity of the ATA monomers and monomer analogs in cell culture.

To determine whether heparin potentiation of platelet aggregation is related to platelet GP IIb/IIIa and GP Ib receptors, four series of experiments were performed on blood from normal volunteers. In the first experiment pretreatment with the monoclonal antibody 7E3 (MAb 7E3), which antagonizes at the GP IIb/IIIa receptor, potently inhibited the collagen-induced platelet aggregation (p less than 0.001). With heparin added to blood pretreated with MAb 7E3, the aggregation increased (p less than 0.005) to an extent similar to that when only saline was used for pretreatment. In the second experiment, monoclonal antibody 10E5 (MAb 10E5) and peptide RGDS, substances which also antagonize at the GP IIb/IIIa receptor, decreased collagen-induced platelet aggregation to an extent similar to that after pretreatment with MAb 7E3. Following pretreatment with RGDS, heparin increased platelet aggregation (p less than 0.03), while after pretreatment with antibody MAb 10E5 heparin did not enhance platelet aggregation. In the third experiment aurin, an inhibitor of von Willebrand factor and its interaction with the platelet GPIb receptor, decreased platelet aggregation dose-dependently. In the fourth experiment heparin enhanced platelet aggregation to a similar extent (p less than 0.005), regardless of pretreatment of the blood with saline, aurin or monoclonal antibody 6D1 (MAb 6D1), the latter an antagonist at the GP Ib receptor. In conclusion, the potentiation of collagen-induced platelet aggregation by heparin was not inhibited by MAb 7E3, RGDS, aurin or MAb 6D1, but was abolished by MAb 10E5, implying that the heparin effect is related to activation of the platelet GP IIb/IIIa receptor complex.

The X-ray crystal structure of recombinant wild-type azurin from Pseudomonas aeruginosa was determined by difference Fourier techniques using phases derived from the structure of the mutant His35Leu. Two data sets were collected from a single crystal of oxidized azurin soaked in mother liquor buffered at pH 5.5 and pH 9.0, respectively. Both data sets extend to 1.93 A resolution. The two pH forms were refined independently to crystallographic R-factors of 17.6% (pH 5.5) and 17.5% (pH 9.0). The conformational transition previously attributed to the protonation/deprotonation of residue His35 (pKa(red) = 7.3, pKa(ox) = 6.2), which lies in a crevice of the protein close to the copper binding site, involves a concomitant Pro36-Gly37 main-chain peptide bond flip. At the lower pH, the protonated imidazole N delta 1 of His35 forms a strong hydrogen bond with the carbonyl oxygen from Pro36, while at alkaline pH the deprotonated N delta 1 acts as an acceptor of a weak hydrogen bond from HN Gly37. The structure of the remainder of the azurin molecule, including the copper binding site, is not significantly affected by this transition.

Aurintricarboxylic acid (ATA) was fractionated by a combination of dialysis, ultrafiltration, and gel permeation chromatography. The number average and weight average molecular weights of the ATA fractions were determined by the universal calibration method. The sulfonic acid analogue of ATA was prepared and separated in high and low molecular weight fractions. The phosphonic acid analogue of ATA was also synthesized. All of the ATA fractions were tested for prevention of the cytopathic effect of HIV-1 and HIV-2 in MT-4 cell culture as well as against HIV-1 in CEM cell culture. The abilities of the fractions and analogues to inhibit syncytium formation between HIV-1- and HIV-2-infected HUT-78 cells and uninfected MOLT-4 cells were evaluated. In addition, the fractions and analogues were tested for cytotoxicity in mock-infected MT-4 cells, prevention of the binding of the OKT4A monoclonal antibody to the CD4 receptor, inhibition of the binding of anti-gp120 monoclonal antibody to gp120, inhibition of attachment of HIV-1 virions to MT-4 cells, and inhibition of HIV-1 reverse transcriptase. In all of these assays except cytotoxicity, there was a correlation of potency with molecular weight. The higher the molecular weight, the higher the activity. Several of the lower molecular weight fractions of ATA, which bound to gp120 but not to CD4, prevented HIV-1 and HIV-2 cytopathicity. A similar profile was observed for the phosphonic acid analogue of ATA and the lower molecular weight fraction of the sulfonic acid analogue. The results on the ATA fractions indicate that the binding of ATA to gp120 in the absence of CD4 binding is sufficient for anti-HIV activity. The active compounds bind more avidly to gp120 than to CD4. The anti-HIV activity of the ATA fractions is due to inhibition of virus binding due to an interference with the gp120-CD4 interaction.

Several compounds corresponding to fragments of the schematic representation of the polymeric structure of aurintricarboxylic acid (ATA) have been prepared and tested for prevention of the cytopathic effect of HIV-1 and HIV-2 in MT-4 cell culture and HIV-1 in CEM cell culture. Both the triphenylcarbinol 3 as well as the triphenylmethane 5 were found to afford protection against the cytopathogenicity of HIV-2 in MT-4 cells and HIV-1 in CEM cells, but they were inactive against HIV-1 in MT-4 cells. Both substances were also found to inhibit syncytium formation when MOLT-4 cells were cocultured with HIV-2-infected HUT-78 cells, but were inactive in this assay against HIV-1-infected cells. When observed, the activity is generally moderate in degree of protection and requires concentrations in the 10(-4) molar range. In contrast to ATA, both of these substances were inactive when tested for prevention of the binding of the OKT4A monoclonal antibody to the CD4 receptor and also for inhibition of HIV-1 reverse transcriptase. These substances therefore appear act by a mechanism that is distinct from that of polymeric ATA. Several active and inactive structural analogues of 3 and 5 were also synthesized. The anti-HIV activity in this series seems to depend on the presence of anionic carboxylate groups, since the methyl esters 4, 6, and 12 were uniformly inactive. The diphenylmethanes 8, 14, 18, and 19 also reproducibly inhibited the cytopathic effect of HIV-1 in CEM cell culture.

Eliminating rosolic acid from M-FC medium improves the MFC procedure by allowing higher fecal coliform colony recoveries with greater ease in counting. Samples of unchlorinated and chlorinated domestic sewage, creek, lake, and river water were analyzed for fecal coliforms by standard procedures. Results of 200 comparisons of fecal coliform counts on M-FC medium without and with rosolic acid showed that higher counts were obtained 71% of the time when rosolic acid was excluded without an overgrowth of background colonies. Results from analyzing chlorinated sewage showed that eliminating rosolic acid improved the recovery of fecal coliform bacteria by 49%. A total of 1,675 blue colonies and 766 nonblue colonies were verified. Of the 1,675 blue colonies, 1,566 were confirmed as fecal coliform bacteria, for a verification of 93.5%. The percent verification of nonblue colonies as noncoliform bacteria was 84.2% (644/766).

MacConkey agar, standard M-FC agar, M-FC agar without rosolic acid, M-FC agar with a resuscitation top layer, Teepol agar, and pads saturated with Teepol broth, were evaluated as growth media for membrane filtration counting of fecal coliform bacteria in water. In comparative tests on 312 samples of water from a wide variety of sources, including chlorinated effluents, M-FC agar without rosolic acid proved the medium of choice because it generally yielded the highest counts, was readily obtainable, easy to prepare and handle, and yielded clearly recognizable fecal coliform colonies. Identification of 1,139 fecal coliform isolates showed that fecal coliform tests cannot be used to enumerate Escherichia coli because the incidence of E. coli among fecal coliforms varied from an average of 51% for river water to 93% for an activated sludge effluent after chlorination. The incidence of Klebsiella pneumoniae among fecal coliforms varied from an average of 4% for the activated sludge effluent after chlorination to 32% for the river water. The advantages of a standard membrane filtration procedure for routine counting of fecal coliforms in water using M-FC agar without rosolic acid as growth medium, in the absence of preincubation or resuscitation steps, are outlined.

Crude aurintricarboxylic acid synthesized by the conventional method was fractionated into 8 components (A1-A8) by silica gel thin layer chromatography. Some of the components were isolated and their effects were tested on nuclear swelling and on in vitro RNA synthesis and DNA synthesis using isolated polymerases. Only two components with low RF (A7 and A8) induced strong nuclear swelling. The other components were almost inactive. Formaurin-dicarboxylic acid, a contaminating polyanion of crude aurintricarboxylic acid, was synthesized separately. It was as effective as the two active components in nuclear swelling and inhibited RNA and DNA polymerases on naked DNA template. However, it stimulated RNA synthesis on chromatin template probably by dissociating histones from DNA. Unfractionated aurintricarboxylic acid showed the same effects at higher concentrations. A preliminary analysis indicated one of the inactive components (A4) is genuine aurintricarboxylic acid. The results suggest that the observed activity of crude aurintricarboxylic acid is due to some contaminating substances, one of which is formaurindicarboxylic acid.

Aurintricarboxylic acid is a potent inhibitor of cell-free protein synthesis by the post-mitochondrial supernatant of chick brain and the translation of mRNA by wheat germ lysate. Comparison of commercially available and chemically synthesized analogues of aurintricarboxylic acid indicates that the unique aurin triphenyl methane ring system and the carboxylic acid groups are both necessary for inhibition of cell-free protein synthesis in both systems.