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Pneumocystis jirovecii pneumonia (PCP) incidence is increasing in kidney transplant recipients (KTR), but risk factors remain poorly defined. CD4+ T lymphopenia and mannose-binding lectin (MBL) deficiency are common immunodeficiencies in KTR. Here, we investigated whether CD4+ T lymphopenia and/or MBL deficiency would be risk factors for PCP in KTR. Furthermore, the role of thymic function in CD4+ T lymphopenia and outcome was studied by assessing CD45RA+CD31+CD4+ T cell numbers (RTE, recent thymus emigrants). In 321 de novo KTR serial determinations of peripheral T lymphocyte subsets (n=281, mean 4.2 times between days 0-365) and/or MBL levels (n=112, mean 1.8 times between days 30-180) were performed. 22/321 patients developed a PCP episode on average at day 199 (107-398) post-Tx. Age correlated inversely with RTE, CD4+ and CD8+ T-cell counts until day 180 post-Tx. RTE correlated with CD4+ T-cell counts at all time-points pre- and post-Tx. PCP patients had more CMV infections (p=0.045) within the first 3 months compared to controls. Importantly, PCP patients were older (p=0.008), and had lower RTE (p=0.046) pretransplant, and lower CD4+ T-cell counts pretransplant (p=0.017), at day 60 (p=0.032) and for the average of all post-Tx values (p=0.027) compared to controls. Though treatment with T-cell depleting antibodies was associated with consecutive CD4+ T lymphopenia in the whole cohort, the number of patients who received T-cell depleting antibodies was comparable between PCP and control patients (p=0.754). A multivariate stepwise logistic regression model identified only pretransplant CD4+ T-cell counts (OR 0.011, p=0.010) and acute rejection (OR 4.66, p=0.023) as predictors of PCP. In contrast, MBL levels and incidence of MBL deficiency (

There is significant clinical interest in primers which are specific for fungi and do not hybridize to DNA of other eukaryotes or prokaryotes. Such primers would allow specific amplification of fungal DNA from human tissue samples containing fungi. Fungal identification to the species level could follow by direct sequencing or restriction analysis. Several previously described primer systems cross-react with DNA of plants and animals. We have designed a primer system that amplifies a fragment of the gene coding for the small ribosomal subunit 18S rRNA. Database searches and sequence analyses were performed using the HUSAR (Heidelberg Unix Sequence Analysis Resources) computer system at the German Cancer Research Centre, Heidelberg, Germany. Primers TR1 (5'-GTTTCTAGGACCGCCGTA) and TR2 (5'-CTCAAACTTCCATCGACTTG) bind to sequences which are homologous within the fungi, but differ from corresponding DNA fragments of plants and animals. The amplified fragment is 581 base pairs in length and contains variable, and therefore species-specific, regions. The DNA of Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton terrestre, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum and of several yeast species was amplified by the primers, but not the DNA from 42 normal human skin samples. Furthermore, other DNA preparations from plants and animals, including those from radish, cabbage, wheat and mouse, did not show amplification reactions.

BACKGROUND: Cytomegalovirus (CMV) infection has been associated with allograft rejection in solid organ transplantation. However, the immunologic mechanisms behind this observation have not been elucidated. One proposed mechanism is direct cross-reactivity of antiviral T-cells with allogeneic MHC/peptide complexes, a process termed heterologous immunity. Another model favours indirect stimulation of alloimmunity by CMV-induced proinflammatory cytokines and upregulation of MHC class II and adhesion molecules. Recently, we found that protection from CMV disease was correlated with high levels of CMV-immediate early-1 (IE-1) specific IFN-gamma-producing T-cell responses in heart and lung transplant recipients. The aim of this study was to define the relation of CMV-specific T-cell responses to acute rejection, donor-reactive memory T cells, and allograft function after kidney transplantation. METHODS: To address this issue, IFN-gamma-producing T-cell responses following ex-vivo stimulation with pools of overlapping peptides representing the CMV pp65 and IE-1 proteins, as well as donor-reactive IFN-gamma-producing T-cells were determined at multiple time points before (pre-Tx) and during the first 6 months posttransplant (post-Tx) in 36 kidney transplant recipients using an enzyme linked immunoabsorbent spot assay (ELISPOT). RESULTS: CMV-specific T cells were not exclusively detectable in CMV seropositive patients, as 3/12 seronegative patients had significant pre- and post-Tx pp65/IE-1-specific T-cell responses. In patients with detectable anti-CMV antibody or T-cell responses, no difference in CMV-specific T-cell frequencies was found between patients with versus without acute rejection. However, early (week 1, r=0.457, p=0.037) and average IE-1-specific T-cell responses (r=-0.415, p=0.032) during 6 months post-Tx showed a significant inverse correlation with average post-Tx donor-reactive T-cell responses. Furthermore, average post-Tx IE-1-specific T-cell responses correlated significantly with 6 and 12 months glomerular filtration rate (GFR). In contrast, pp65-specific T-cell responses did not correlate with donor-reactive T cells or graft function. Only 2/36 patients developed CMV disease, both showing very weak IE-1-specific T-cell responses during the whole monitoring period. CONCLUSION: No evidence for heterologous immunity could be found in patients with high levels of CMV-specific T cells. On the contrary, less alloreactivity and improved graft function were found in patients with strong IE-1-specific T-cell responses. These results emphasize the importance of immediate early antigens (IE) as targets for T-cell immunity to CMV. We hypothesize that IE-1-specific T cells might effectively suppress IE-1-induced indirect effects such as inflammation and upregulation of MHC class II and adhesion molecules.

Donor-reactive cellular sensitization does not routinely suggest humoral sensitization and vice versa, but both predict poor kidney transplant outcome. Irrespective of donor reactivity, panel-reactive antibody (PRA) screening identifies patients who are at enhanced risk. Therefore, it was hypothesized that panel-reactive memory T cell reactivity (PRT) might be an additional risk assessment factor of dialysis patients who are on the transplant waiting list. IFN-gamma-enzyme-linked immunosorbent spot memory T cell frequencies were determined in 10 healthy volunteers and 41 hemodialysis patients using for stimulation an allogeneic cell bank (ACB) from 17 healthy individuals who represented the most frequent white HLA antigens. Positive responses to ACB were analogous to PRA defined as percentage of positive assays of the ACB sets. Hemodialysis patients expressed higher PRT levels compared with healthy volunteers. Five of 10 PRT++ patients were PRA negative, and only four of 10 PRA++ patients exhibited PRT reactivity, suggesting independence of humoral and cellular sensitization. Pretransplantation PRT testing of recipients might improve individual risk assessment to make individualized therapy decisions.

P2X receptors are cation channels gated by extracellular ATP and related nucleotides. Because of the widespread distribution of P2X receptors and the high subtype diversity, potent and selective antagonists are needed to dissect their roles in intact tissues. Based on suramin as a lead compound, several derivates have been described that block recombinant P2X receptors with orders of magnitude higher potency than suramin. Here we characterized the suramin analogue 4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) with respect to its potency to antagonize ATP or alphabeta-methyleneadenosine 5'-trisphosphate-induced inward currents of homomeric rat P2X(1)-P2X(4) receptors or heteromeric P2X(1 + 5) and P2X(2+3) receptors, respectively. NF449 most potently blocked P2X(1) and P2X(1 + 5) receptors with IC(50) values of 0.3 nM and 0.7 nM, respectively. Three to four orders of magnitude higher NF449 concentrations were required to block homomeric P2X(3) or heteromeric P2X(2 + 3) receptors (IC(50) 1.8 and 0.3 microM, respectively). NF449 was least potent at homomeric P2X(2) receptors (IC(50) 47 microM) and homomeric P2X(4) receptors (IC(50) > 300 microM). Altogether, these results characterize NF449 as the so far most potent and selective antagonist of receptors incorporating the P2X(1) subunit such as the P2X(1) homomer and the P2X(1 + 5) heteromer.

Protein-tyrosine phosphatases (PTPs) are important signaling enzymes that have emerged within the last decade as a new class of drug targets. It has previously been shown that suramin is a potent, reversible, and competitive inhibitor of PTP1B and Yersinia PTP (YopH). We therefore screened 45 suramin analogs against a panel of seven PTPs, including PTP1B, YopH, CD45, Cdc25A, VHR, PTPalpha, and LAR, to identify compounds with improved potency and specificity. Of the 45 compounds, we found 11 to have inhibitory potency comparable or significantly improved relative to suramin. We also found suramin to be a potent inhibitor (IC(50) = 1.5 microm) of Cdc25A, a phosphatase that mediates cell cycle progression and a potential target for cancer therapy. In addition we also found three other compounds, NF201, NF336, and NF339, to be potent (IC(50) < 5 microm) and specific (at least 20-30-fold specificity with respect to the other human PTPs tested) inhibitors of Cdc25A. Significantly, we found two potent and specific inhibitors, NF250 and NF290, for YopH, the phosphatase that is an essential virulence factor for bubonic plague. Two of the compounds tested, NF504 and NF506, had significantly improved potency as PTP inhibitors for all phosphatases tested except for LAR and PTPalpha. Surprisingly, we found that a significant number of these compounds activated the receptor-like phosphatases, PTPalpha and LAR. In further characterizing this activation phenomenon, we reveal a novel role for the membrane-distal cytoplasmic PTP domain (D2) of PTPalpha: the direct intramolecular regulation of the activity of the membrane-proximal cytoplasmic PTP domain (D1). Binding of certain of these compounds to PTPalpha disrupts D1-D2 basal state contacts and allows new contacts to occur between D1 and D2, which activates D1 by as much as 12-14-fold when these contacts are optimized.

Extracellular adenine and uracil 5'-nucleotides are important signalling molecules that exert a great variety of effects in numerous tissues and cell types through the activation of P2 receptors. In the past eight years, an extended series of P2 receptors (P2X(17), ionotropic subunits; P2Y(1,2,4,6,11,12), metabotropic receptors) has been cloned from vertebrate tissues. In this rapidly expanding field, one of the main current challenges is to relate the cloned P2 receptor subtypes to the diverse physiological responses mediated by the pharmacological phenotypes of native P2 receptors. Unfortunately, subtype-selective P2 ligands, especially potent and selective antagonists, have been only slowly forthcoming, and this acts as a considerable impediment to progress. However, a number of new P2 receptor antagonists have recently been described which to some degree are more potent and more selective than earlier antagonists like suramin or pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). This work moves us closer to the ideal goal of classifying the recombinant and native P2 receptor subtypes on the basis of antagonist profiles. This review begins with a brief account of the current status of P2 receptors and their ligands. It then focuses on structure-activity relationships of PPADS and suramin analogues and will finish with a brief discussion of some related therapeutic possibilities.

Intracellular microelectrodes were used to record the transmembrane potential and excitatory junction potentials (e.j.p.s) produced by sympathetic nerve stimulation (1 Hz) in smooth muscle cells of the guinea-pig isolated vas deferens. The symmetrical 3′-urea of 8-(benzamido)naphthalene-1,3,5-trisulphonic acid (NF023) produced a concentration-dependent inhibition of e.j.p. magnitude (IC50=4.8×10−6 M), but had no effect on the resting membrane potential of the smooth muscle cells. Pyridoxal-5-phosphate (P-5-P) also depressed e.j.p. magnitude in a concentration-dependent manner, but was less potent than NF023 (IC50=2.2×10−5 M). At 10−4 M and above P-5-P significantly depolarized the smooth muscle cells. The nucleoside triphosphatase inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156) (5×10−5 M) significantly increased e.j.p. amplitude. ARL 67156 (10−4 M) further increased e.j.p. amplitude such that they often reached threshold for initiation of action potentials, causing muscle contraction and expulsion of the recording electrode. After reduction of e.j.p.s by NF023 or P-5-P (both 10−5 M), subsequent co-addition of ARL 67156 (10−4 M) significantly increased their magnitude. The overflow of endogenous ATP evoked by field stimulation of sympathetic nerves (8 Hz, 1 min) was measured by HPLC and flurometric detection. ARL 67156 (10−4 M) enhanced ATP overflow by almost 700% compared to control. We conclude that for electrophysiological studies NF023 is preferable to other P2X receptor antagonists such as pyridoxalphosphate -6-azophenyl-2′,4′-disulphonic acid (PPADS), suramin or P-5-P. Furthermore, breakdown of endogenous ATP by nucleoside triphosphatases is an important modulator of purinergic neurotransmission in the guinea-pig vas deferens. British Journal of Pharmacology (2000) 129, 1089–1094; doi:10.1038/sj.bjp.0703163

The effect of the suramin analogue 8,8'-(carbonylbis(imino-4, 1-phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(1,3 , 5-naphthalenetrisulfonic acid) (NF279) was analyzed on human P2X(1) and P2X(7) receptor subtypes (human P2X(1) and human P2X(7)) heterologously expressed in Xenopus oocytes using the two-microelectrode voltage-clamp technique. At activating ATP concentrations of 1 microM (human P2X(1)) and 10 microM ATP (human P2X(7)), IC(50) values of 0.05 microM and 2.8 microM were found for human P2X(1) and human P2X(7) receptors, respectively. An increase in the activating [ATP] shifted the NF279 concentration-inhibition curve rightwards for both receptors. NF279 slowed the activation of both human P2X(1) and human P2X(7) as well as the desensitization of human P2X(1). The data support a model in which desensitization of P2X(1) is dependent on preceding activation of these P2X receptors. It is concluded that NF279 acts as a competitive antagonist with much higher potency at human P2X(1) than at P2X(7) receptors. NF279 may hence be suited to discriminate between both receptors in native tissues.

1. The effects of NF023, the symmetrical 3'-urea of 8-(benzamido)naphthalene-1,3,5-trisulphonic acid), and its parent compound suramin were investigated on vasoconstrictor responses to alpha, beta-methylene ATP in rabbit isolated saphenous artery and vasodilator responses to ATP in noradrenaline-precontracted rabbit isolated thoracic aorta. 2. In rabbit isolated saphenous artery, alpha, beta-methylene ATP-induced vasoconstrictor responses via P2X-receptors were concentration-dependently and competitively antagonised by NF023 (30-300 microM; pA2 = 5.69 +/- 0.04). Suramin (100-1000 microM) also competitively blocked vasoconstrictor responses to alpha, beta-methylene ATP, albeit with lower potency (pA2 = 4.79 +/- 0.05). In contrast, NF023 (100 microM) did not significantly affect contractile responses to noradrenaline or histamine in the saphenous artery. 3. In noradrenaline-precontracted rabbit isolated thoracic aorta preparations, ATP (3-3000 microM) concentration-dependently induced relaxations via endothelium-dependent or smooth muscle P2Y-receptor subtypes. NF023 (30-300 microM) failed to block relaxant responses to ATP at endothelium-dependent P2Y-receptors, whereas suramin (100-1000 microM) did antagonise endothelium-dependent vasodilator responses to ATP. Neither NF023 (100 microM) nor suramin (300 microM) influenced vasorelaxant responses to ATP via endothelium-independent P2Y-receptors. 4. In conclusion, this study outlines the selectivity of NF023 as an effective P2X-receptor antagonist in rabbit isolated blood vessels without affecting endothelium-dependent or endothelium-independent P2Y-receptor subtypes, adrenoceptors or histamine receptors.

Clonotype analysis is essential for complete characterization of antigen-specific T cells. Moreover, knowledge on clonal identity allows tracking of antigen-specific T cells in whole blood and tissue infiltrates and can provide information on antigenic specificity. Here, we developed a next generation sequencing (NGS)-based platform for the highly quantitative clonotype characterization of T cells and determined requirements for the unbiased characterization of the input material (DNA, RNA, ex vivo derived or cell culture expanded T cells). Thereafter we performed T cell receptor (TCR) repertoire analysis of various specimens in clinical settings including cytomegalovirus (CMV), polyomavirus BK (BKV) reactivation and acute cellular allograft rejection. Our results revealed dynamic nature of virus-specific T cell clonotypes; CMV reactivation was linked to appearance of new highly abundant antigen-specific clonalities. Moreover, analysis of clonotype overlap between BKV-, alloantigen-specific T cell-, kidney allograft- and urine-derived lymphocytes provided hints for the differential diagnosis of allograft dysfunction and enabled appropriate therapy adjustment. We believe that the established approach will provide insights into the regulation of virus-specific/anti-tumor immunity and has high diagnostic potential in the clinical routine.

BK virus (BKV) infection represents a serious complication in renal transplant patients resulting in BKV-associated nephropathy and subsequent allograft loss. Natural killer cells are crucial in the antiviral immune response; however, an understanding of the role of natural killer cells in protection against BKV is limited. To elucidate whether killer-cell immunoglobulin-like receptors and their interaction between donor-/recipient-related ligands have a role in BKV infection, we performed genotyping analysis in 48 kidney transplant recipients with a history of severe BKV infection/BKV-associated nephropathy and 110 recipients with stable renal function and no BKV reactivation. Of interest, we found that telomeric gene content motif was significantly associated with severe course of BKV infection/BKV-associated nephropathy and detected significantly higher percentage of patients with BKV-associated nephropathy carrying low numbers of activating receptors compared with the control group. Detailed analysis of each single receptor revealed significantly lower frequencies of the activating receptor KIR3DS1 in patients with BKV infection/nephropathy as compared with the controls. Thus, our study supports protective effects of activating receptors in BKV infection and suggest natural killer-cell–related genetic predisposition to the development of BKV-associated nephropathy.

This report demonstrates the ability of the anti-cancer drug suramin to interfere with the binding of interleukin (IL)-1 to its receptor and to inhibit IL-1-induced biological activities. In a radioreceptor cell based assay, suramin inhibits the binding of IL-1 alpha to several murine cell lines expressing predominantly type I and type II IL-1 receptors. Affinity cross-linking experiments using IL-1 alpha and EL-4.6.1 cells confirms that suramin inhibits the binding of the ligand to the 80 kDa IL-1 type I receptor. In contrast, suramin fails to displace significantly prebound IL-1. In a cell-free system, suramin prevents the binding of IL-1 alpha and IL-1 beta to murine and human recombinant soluble type I IL-1 receptors. For example, the IC50 for suramin inhibiting IL-1 alpha and IL-1 beta binding to soluble human IL-1 receptor were 204 microM and 186 microM, respectively. The suramin analogues, NF-058 and NF-103 (which bear the same number of sulfate groups as suramin), are between three- and ten-fold less active than suramin in inhibiting IL-1 binding to EL-4.6.1 cells, and to recombinant soluble IL-1 receptor. Furthermore, in a dose-dependent manner suramin prevents several IL-1 mediated biological responses, including thymocyte proliferation, PGE-2 synthesis and IL-6 production. The inhibitory effect of the drug can be significantly reversed by the addition of excess cytokine. Taken together, the results indicate that suramin is a competitive IL-1 receptor antagonist. Because IL-1 participates in a broad range of immunological and inflammatory functions, the data suggest that suramin administration may influence important activities beyond those associated strictly with tumor inhibition.

The infection of human colonic epithelial cells HT-29 by human immunodeficiency virus type 1 (HIV-1) occurs independently of CD4, the main HIV-1 receptor expressed on lymphocytes and macrophages. Recent studies from our group have shown that HT-29 cells express the glycosphingolipid galactosylceramide (GalCer), a potential alternative receptor for the HIV-1 envelope glycoprotein gp120. The binding of recombinant gp120 to GalCer was blocked by monoclonal antibodies directed against the third variable region (V3) of gp120, suggesting that the V3 domain was implicated in GalCer recognition. In the present report, we show that suramin, a polysulfonyl naphtylurea known to inhibit retroviral reverse transcriptases in vitro, blocks HIV-1 infection in HT-29 cells. The effect is dose dependent, with a half-maximal inhibition (IC50) achieved for a suramin concentration of 54 micrograms/ml. Since [3H]suramin was not significantly internalized into HT-29 cells during our infection assay (i.e. 2 h), we have considered the possibility that the drug could act at an extracellular step of the HIV-1 cycle. Using a high performance thin layer chromatography binding assay, we show that suramin inhibits binding of HIV-1 gp120 to purified GalCer with an IC50 of 25 micrograms/ml. Suramin does not bind to GalCer, since preincubation of GalCer with suramin did not prevent the subsequent attachment of gp120. Using a solid-phase assay, we show that [3H]suramin specifically binds to recombinant gp120 and that this binding could be blocked by a monoclonal antibody specific for the conserved GPGRAF motif of the V3 domain of gp120. We also demonstrate that [3H]suramin binds to multibranched synthetic GPGRAF peptides that block HIV-1 infection in HT-29 cells. Binding of [3H]suramin to V3 peptides is specific and inhibited by unlabeled suramin (IC50 of 28 micrograms/ml). In contrast, the suramin derivative NF036, that is unable to block HIV-1 infection in HT-29 cells, does not inhibit the binding of [3H]suramin to V3 peptides. Taken together, these results suggest that suramin blocks HIV-1 infection in HT-29 cells because it binds to the V3 domain of gp120 and hence prevents the interaction between gp120 and the GalCer receptor.

Polyomavirus associated nephropathy (PVAN) affects up to 10% of kidney transplant recipients and is a major risk factor for graft loss. Mannose-binding lectin (MBL) is an important recognition molecule of the innate immune system, and its deficiency has been associated with susceptibility to various infections. In transplantation, on the other hand, high MBL levels have been associated with increased tissue damage in ischemia-reperfusion models and poorer graft and patient survival in solid organ transplant patients. To investigate the relation between MBL and BK virus infection, post-transplant (post-Tx) MBL levels were determined in a cohort of de novo kidney transplant patients with and without BK viremia.41 de novo kidney transplant patients with high (n=16, group 1) or low level BK viremia (n=25, group 2) and 64 patients without BK viremia (group 3) were included. In every patient, functional MBL levels were determined at 1-3 time points (days 30, 90 or 180) post-Tx using an MBL oligomer ELISA.MBL levels remained unchanged between days 30 and 180 post-Tx independent of BKV viremia. Frequencies of MBL deficiency (500 ng/mL) and MBL levels were not significantly different between the 3 groups. However, group 2 patients showed a trend towards lower MBL serum levels compared to group 1 patients, notably in patients without acute rejection (p=0.076).MBL deficiency was not associated with higher risk for BK viremia. In contrast, we hypothesize that BK virus replication in patients with low MBL levels might imply lower risk for progression towards PVAN compared to patients with high MBL levels. This view is supported by recent data demonstrating local complement activation in BK nephropathy.

The reverse transcriptase (RT) inhibition and the specificity of 15 aminonaphthalenesulfonic acid derivatives were examined with RT of a simian immunodeficiency virus derived from an African green monkey (SIVagmTYO-7). The two compounds with the strongest RT inhibition (NF415) or the highest specificity (NF345), together with suramin, were evaluated against polymerase alpha-primase complex from calf thymus. We have also compared the kinetics of inhibition of the viral and the cellular polymerase by these three compounds. While RT inhibition followed a mixed competitive and non-competitive mechanism, inhibition of the DNA polymerase alpha was competitive for suramin and non-competitive for NF415 and NF345. Certain structural characteristics appeared to be common for specific RT inhibitors.

Suramin is a polyanionic compound currently used under evaluation for antineoplastic activity. One of the main problems encountered during clinical trials was an adverse neurotoxic effect, probably due to a direct cytotoxic effect on neural cells. Suramin is also known to trigger differentiation of human colon cancer cells, yet a chronic treatment induces a lysosomal storage disorder. The aim of this study was to evaluate suramin analogs for their effect: (i) on the lysosomal system of the human colon cancer cell clone HT29-D4; and (ii) on C6 glioma cell growth and morphology. One of the derivatives tested, NF036, induced terminal differentiation of HT29-D4 cells without any impairment of the lysosomal system. Furthermore, in contrast to suramin, NF036 did not alter C6 cell growth and morphology. We conclude that there is a relationship between the ability of a suramin derivative to induce a lysosomal storage disorder in human colon cancer cells and its neurotoxic effect. A double screening of suramin analogs on HT29-D4 and C6 cells allowed us to identify a new candidate antineoplastic drug: NF036.

The interface between receptors and G proteins can be considered as a drug target. Various classes of low molecular weight inhibitors have been identified that block the ability of receptors to interact with G proteins (e.g. peptides, suramin analogues and amphiphilic cations). Here we have tested if there are compounds that differentially affect the interaction of one receptor with two different (related) G protein alpha-subunits. Fusion proteins comprising the human A1-adenosine receptor and Galphai-1 (A1/Galphai-1) or Galphao (A1/Galphao) were expressed in HEK293 cells. Suramin analogues were screened for their ability to differentially affect high affinity binding of the agonist (-) N6-3-[125I](iodo-4-hydroxyphenylisopropyl) adenosine (IHPIA). One compound [NF326 = 8,8'-(carbonylbis-(imino-3,1-phenylenecarbonylimino))bis-(1-naphthol-3,6-disulfonic acid, disodium salt)] was identified that inhibited high affinity agonist binding to the fusion protein A1/Galphai-1 but modestly enhanced binding of IHPIA to A1/Galphao. This action was specific because NF326 did not affect antagonist binding to either fusion protein. In addition, it was unrelated to a difference in affinity of the receptor for the G protein fusion moiety because the stability of ternary complexes formed by IHPIA + A1/Galphai-1 and IHPIA + A1/Galphao) is comparable and because lowering the affinity of the receptor for the G protein (by introducing point mutations at cys351 of Galphai-1) enhanced the uncoupling effect of NF326. Finally, NF326 did not discriminate between a fusion protein comprising the alpha2A-adrenoceptor and Galphai-1 (alpha2A)/Galphai-1) or Galphao-1 (alpha2A)/Galphao-1); binding of the agonist [3H]UK14304 (bromoxidine) to both fusion proteins was inhibited over a comparable concentration range while binding of the antagonist [3H]yohimbine was unaffected. These observations are consistent with the interpretation that the contact sites that are formed between individual receptors and G proteins differ. These differences suffice to allow for selective disruption by G protein inhibitors of different classes. Using NF326 we show that the bulk of the A1-adenosine receptors in human cerebrocortical membranes interacts with Galphao rather than Galphai.

Antagonistic effects of the novel suramin analogue 4,4',4",4"'-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) were studied on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alphabetameATP; mediated by P2X1 receptors), contractions of the guinea-pig ileal longitudinal smooth muscle elicited by alphabetameATP (mediated by P2X3 receptors) or adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; mediated by P2Y1 receptors), ATP-induced increases of [Ca2+]i in human embryonic kidney (HEK) 293 cells (mediated by P2Y2 receptors), inward currents evoked by ATP in follicle cell-free Xenopus laevis oocytes expressing rP2X1 or rP2X3 receptors and degradation of ATP by ecto-nucleotidases in folliculated Xenopus laevis oocytes. In addition, NF449 was examined for its P2 receptor specificity in rat vas deferens (alpha1A-adrenoceptors) and guinea-pig ileum (histamine H1 and muscarinic M3 receptors). At native (pIC50=7.15) and recombinant (pIC50=9.54) P2X1 receptors, NF449 was a highly potent antagonist. The P2X3 receptors present in guinea-pig ileum (pIC50=5.04) or expressed in oocytes (pIC50 approximately 5.6) were much less sensitive for NF449. It also was a very weak antagonist at P2Y1 receptors in guinea-pig ileum (pIC50=4.85) and P2Y2 receptors in HEK 293 cells (pIC50=3.86), and showed very low inhibitory potency on ecto-nucleotidases (pIC503.5). NF449 (100 microM) did not interact with alpha1A-adrenoceptors or histamine H1 and muscarinic M3 receptors. Thus, the antagonism by NF449 is highly specific for P2 receptors. In conclusion, the subnanomolar potency at rP2X1 receptors and the rank order of potency, P2X1P2X3P2Y1P2Y2ecto-nucleotidases, make NF449 unique among the P2 receptor antagonists reported to date. NF449 may fill the long-standing need for a P2X1-selective radioligand.

The suramin analogue 8,8′-(carbonylbis(imino-3,1-phenylene carbonylimino)bis(1,3,5-naphthalenetrisulfonic acid) (NF023) antagonizes in a competitive fashion P2X receptor-mediated responses in certain vascular and visceral smooth muscles. In the present study, the effect of NF023 on voltage-clamped Xenopus oocytes heterologously expressing homomultimeric P2X 1 -P2X 4 as well as heteromultimeric P2X 2 /P2X 3 receptors has been characterized. P2X 1 receptors were most sensitive to inhibition by NF023 with IC 50 values of 0.24 and 0.21 μM for the rat and human homologue, respectively. P2X 3 receptors have an intermediate sensitivity with IC 50 values of 8.5 and 28.9 μM for rat and human subtypes, respectively and P2X 2 was the least sensitive subtype (IC 50 >50 μM). P2X 4 receptors were insensitive to NF023 at concentrations up to 100 μM. Coexpression of rat P2X 3 with rat P2X 2 resulted in receptors whose sensitivity to NF023 was identical to that obtained for homomultimeric rat P2X 3 receptors ( αβ meATP as agonist; IC 50 =1.4 and 1.6 μM, respectively). NF023 inhibited P2X 1 receptors in a voltage-insensitive manner. In addition, NF023 (5 and 30 μM) caused a shift of the concentration-response curve to the right without affecting the maximal response to ATP ( K B =1.1±0.2 μM). Our results indicate that NF023 is a subtype-selective and surmountable antagonist at P2X 1 receptors heterologously expressed in Xenopus oocytes.

African trypanosomes contain a cytosolic serine oligopeptidase, called OP-Tb, that is reversibly inhibited by the active principles of three of the five most commonly used trypanocidal drugs: pentamidine, diminazene and suramin. OP-Tb was inhibited by pentamidine in a competitive manner, and by suramin in a partial, non-competitive manner. The inhibition of OP-Tb by a variety of suramin analogues correlated with the trypanocidal efficacy of these analogues (P = 0.03; by paired Student's t-test). Since intracellular (therapeutic) concentrations of pentamidine and suramin are reported to reach approximately 206Ki and 15Ki respectively, we suggest that these drugs may exert part of their trypanocidal activity through the inhibition of OP-Tb.

8,8'-(Carbonylbis(imino-4, 1 -phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(1,3, 5-naphthalenetrisulfonic acid) (NF279) antagonized P2X receptor-mediated contractions in rat vas deferens, evoked by alpha,beta-methylene ATP (10 microM; pIC50=5.71) without affecting responses mediated via alpha1A-adrenoceptors, adenosine A1 and A2B receptors, histamine H1, muscarinic M3 and nicotinic receptors. The low inhibitory potency of NF279 on P2Y receptors in guinea-pig taenia coli (pA2=4.10) and at ecto-nucleotidases in folliculated Xenopus laevis oocytes (IC50 > 100 microM) indicates that NF279 is a novel specific and selective P2X receptor antagonist.

Progress in developing new contraceptive agents, particularly for the male, is extremely slow. Here, we report on a novel property of the anti-trypanosomal drug suramin: the ability to act at the sperm-egg interface and prevent fertilization. Suramin is a polysulphonated compound that inhibits binding of capacitated mouse spermatozoa to the zona pellucida in vitro with an IC50 of 12.4 microM. The drug is only effective at the time of fertilization and is not inhibitory if gametes are pre-treated separately. Autoradiographic localization of suramin binding sites on mouse, boar and human spermatozoa shows that they are intracellular, principally in the head region. The sperm protein recognized by suramin has been identified as proacrosin which is known to interact with sulphate groups on zona glycoproteins. Zona pellucida glycoproteins do not bind suramin, suggesting that the drug blocks the ability of proacrosin/acrosin, exposed during the acrosome reaction, to mediate the secondary binding phase of spermatozoa to the zona during fertilization. Structure-based design studies have the potential to generate safe suramin mimetics that would form the basis for a new generation of non-steroidal contraceptive agents.

The reverse transcriptase (RT) was partially purified by a newly developed procedure from the simian immunodeficiency virus TYO-7 isolated from an African green monkey (SIVagmTYO-7). The method comprised lysis of the virus with nonionic detergent followed by two centrifugations in isopycnic sucrose density gradients and one velocity sedimentation in a glycerol gradient. The enzyme exhibited a purity of 70-80% and showed an exceptional high specific activity of 135 nmol incorporation of dTMP per milligram of protein in 1 h with poly(rA).oligo(dT) as template-primer (TP). The molecular weight of the native enzyme was estimated by velocity sedimentation analysis as 120K-130K. Investigation of the RT by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the active enzyme is a heterodimer composed of a 64- and a 50-kDa subunit. The two subunits were identified to be RT specific by Western blot analysis. In activity gels, both subunits exhibited enzymatic activity, whereby the 64-kDa subunit showed the predominant activity. The RT preferred the TP poly(rA).oligo(dT) over poly(rC).oligo(dG). With poly(rCm).oligo(dG), only marginal activity was detected, and no activity was measured with poly(dA).oligo(dT). The TP specificity was influenced by the reaction temperature. The highest activity was measured around the melting temperature of the TP used. Furthermore, the enzyme activity was more thermolabile when measured with poly(rA).oligo(dT) than with poly(rC).oligo(dG). To compare the specificity of RT inhibitors, their inhibition efficiency (IE) was defined as the ratio of the 50% inhibiting concentration (ID50) obtained with the RT in viral lysates to the ID50 of purified RT.

Zwei mono-Chlorderivate, 7a und 7b, und zwei di-Chlor-derivate, 7c und 7d, von 6-(4-Diathylamino-1-methyl-butylamino)-5,8-dimethoxychinolin (1a) wurden synthetisiert, um den Einflus von Chlorsubstituenten auf Malaria-Wirksamkeit und Toxizitat von 1a zu untersuchen. 7a–d besitzen nur eine geringe Wirksamkeit gegen Plasmodium vinckei (in der Maus). Antimalarial 6-Aminoquinolines, VI: 2-, 3- and 4-Chloro Derivatives of 6-(4-Diethylamino-1-methylbutylamino)-5.8-dimethoxyquinoline Two monochloro derivatives, 7a and 7b, and two dichloro derivatives, 7c and 7d, of 6-(4-diethylamino-1-methylbutylamino)-5.8-dimethoxyquinoline (1a) were synthesized with the aim of studying the influence of chloro substituents on the antimalarial activity and toxicity of 1a. 7a–d showed only slight activity against Plasmodium vinckei (in mice).

Ninety analogues of suramin have been examined for their ability to inhibit the exogenous reverse transcriptase (RT) of human immunodeficiency virus type I (HIV-I). Of these compounds, 57 inhibited the poly(rC).oligo(dG)-dependent RT activity. Three classes of dose-response curves could be discriminated. Allocation of a compound to one class did not correspond with obvious structural features. Twenty-four substances were superior to suramin in our RT inhibition assay. The RT-inhibitory activity of these compounds did not correlate with their effect against filariae or trypanosomes. Preliminary antiviral evaluation in susceptible human T cells inoculated with HIV-I demonstrated in vitro therapeutic efficacy for some compounds with lower drug-related cellular toxicity than suramin. Certain structural features relevant for the RT-inhibitory effect of these compounds were recognized. Predictions are made for the design of more effective RT inhibitors. Such compounds will help to understand the molecular mechanism of reverse transcription and might be useful in the therapy of retroviral infections.

The chromosome of N. gonorrhoeae contains several complete expression genes coding for variant opacity proteins. DNA sequence analysis of two opacity genes derived from the same locus ( opa E1) of two isogenic gonococcal variants reveals common and variable regions in these genes. Genomic blotting experiments using synthetic probes suggest gene conversion as a principle for the assembly of variant sequence information in opacity genes. The 5′ region of opacity genes is composed of identical pentameric pyrimidine units (CTCTT) encoding the hydrophobic portion of the opacity leader peptide. This coding repeat is variable in a given locus with respect of the number of pentameric units. While all expression loci in a single cell are constitutively transcribed, the production of opacity proteins is determined by the coding repeat sequence on the translational level.

In N. gonorrhoeae, the expression of pilus and opacity (Op) proteins can be switched on and off and a single cell apparently has a whole repertoire of genes to express many serologically distinguishable protein types. We describe the isolation of several different Op genes and of nonexpressing gene equivalents, all derived from isogenic gonococcal variants. In the E. coli host, Op proteins identical with those made in the respective N. gonorrhoeae strain are produced. The Op genes map near the pilus expression locus. Genomic blotting experiments with an Op gene probe reveal complex hybridization patterns but little heterogeneity among the genes of Op variants. It appears that colonial variation involving the Op protein of N. gonorrhoeae is based on minor sequence alterations, in contrast to the pilus variation system, in which changes in the expression can be evoked by substantial genomic rearrangements.

Gonococcal virulence is thought to rely on multiple phenomena to which antigenic variation of surface proteins as well as the production of an extracellular protease specific for human IgA1 can be accounted. The expression of pilin and opacity (Op) protein in Neisseria gonorrhoeae can be switched on and off and a single cell apparently has a whole repertoire of genes to express many serologically distinguishable protein types. In order to investigate the molecular mechanisms associated with this surface variability, we have cloned the genes for pilin and Op expression in Escherichia coli. Using these genes as probes in DNA filter hybridizations, we revealed two complex gene systems which undergo genomic reorganizations and/or gene conversions which occur concomitant to changes in the expression of these surface proteins. To study the role of IgA proteases in bacterial pathogenesis, we cloned the IgA protease gene of N. gonorrhoeae MS11. This clone promotes expression and extracellular secretion of active enzyme in E. coli and other gram-negative bacterial species.