Suramin Inhibits Chikungunya Virus Replication by Interacting with Virions and Blocking the Early Steps of Infection.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that can cause a debilitating disease that is primarily characterized by persistent joint pain. CHIKV has been emerging globally, while neither a vaccine nor antiviral medication is available. The anti-parasitic drug suramin was previously shown to inhibit CHIKV replication. In this study we aimed to obtain more detailed insight into its mechanism of action. We found that suramin interacts with virions and can inhibit virus binding to cells. It also appeared to inhibit post-attachment steps of the infection process, likely by preventing conformational changes of the envelope glycoproteins required for fusion and the progression of infection. Suramin-resistant CHIKV strains were selected and genotyping and reverse genetics experiments indicated that mutations in E2 were responsible for resistance. The substitutions N5R and H18Q were reverse engineered in the E2 glycoprotein in order to understand their role in resistance. The binding of suramin-resistant viruses with these two E2 mutations was inhibited by suramin like that of wild-type virus, but they appeared to be able to overcome the post-attachment inhibitory effect of suramin. Conversely, a virus with a G82R mutation in E2 (implicated in attenuation of vaccine strain 181/25), which renders it dependent on the interaction with heparan sulfate for entry, was more sensitive to suramin than wild-type virus. Using molecular modelling studies, we predicted the potential suramin binding sites on the mature spikes of the chikungunya virion. We conclude that suramin interferes with CHIKV entry by interacting with the E2 envelope protein, which inhibits attachment and also interferes with conformational changes required for fusion.

Suramin inhibits Zika virus replication by interfering with virus attachment and release of infectious particles.

Zika virus (ZIKV) is a mosquito-borne flavivirus that mostly causes asymptomatic infections or mild disease characterized by low-grade fever, rash, conjunctivitis, and malaise. However, the recent massive ZIKV epidemics in the Americas have also linked ZIKV infection to fetal malformations like microcephaly and Guillain-Barré syndrome in adults, and have uncovered previously unrecognized routes of vertical and sexual transmission. Here we describe inhibition of ZIKV replication by suramin, originally an anti-parasitic drug, which was more recently shown to inhibit multiple viruses. In cell culture-based assays, using reduction of cytopathic effect as read-out, suramin had an EC50 of ∼40 µM and a selectivity index of 48. In single replication cycle experiments, suramin treatment also caused a strong dose-dependent decrease in intracellular ZIKV RNA levels and a >3-log reduction in infectious progeny titers. Time-of-addition experiments revealed that suramin inhibits a very early step of the replication cycle as well as the release of infectious progeny. Only during the first 2 h of infection suramin treatment strongly reduced the fraction of cells that became infected with ZIKV, suggesting the drug affects virus binding/entry. Binding experiments at 4 °C using 35S-labeled ZIKV demonstrated that suramin interferes with attachment to host cells. When suramin treatment was initiated post-entry, viral RNA synthesis was unaffected, while both the release of genomes and the infectivity of ZIKV were reduced. This suggests the compound also affects virion biogenesis, possibly by interfering with glycosylation and the maturation of ZIKV during its traffic through the secretory pathway. The inhibitory effect of suramin on ZIKV attachment and virion biogenesis and its broad-spectrum activity warrant further evaluation of this compound as a potential therapeutic.

Polysulfonate suramin inhibits Zika virus infection.

Zika virus (ZIKV) is an arthropod-borne flavivirus that causes newborn microcephaly and Guillian-Barré syndrome in adults. No therapeutics are available to treat ZIKV infection or other flaviviruses. In this study, we explored the inhibitory effect of glycosaminoglycans and analogues against ZIKV infection. Highly sulfated heparin, dextran sulfate and suramin significantly inhibited ZIKV infection in Vero cells. De-sulfated heparin analogues lose inhibitory effect, implying that sulfonate groups are critical for viral inhibition. Suramin, an FDA-approved anti-parasitic drug, inhibits ZIKV infection with 3-5 log10 PFU viral reduction with IC50 value of ∼2.5-5 µg/ml (1.93 µM-3.85 µM). A time-of-drug-addition study revealed that suramin remains potent even when administrated at 1-24 hpi. Suramin inhibits ZIKV infection by preventing viral adsorption, entry and replication. Molecular dynamics simulation revealed stronger interaction of suramin with ZIKV NS3 helicase than with the envelope protein. Suramin warrants further investigation as a potential antiviral candidate for ZIKV infection. Heparan sulfate (HS) is a cellular attachment receptor for multiple flaviviruses. However, no direct ZIKV-heparin interaction was observed in heparin-binding analysis, and downregulate or removal of cellular HS with sodium chlorate or heparinase I/III did not inhibit ZIKV infection. This indicates that cell surface HS is not utilized by ZIKV as an attachment receptor.

Homing of bone marrow mesenchymal stem cells mediated by sphingosine 1-phosphate contributes to liver fibrosis.

BACKGROUND/AIMS: Myofibroblasts play a central role in the pathogenesis of liver fibrosis. Myofibroblasts of bone marrow (BM) origin have recently been identified in fibrotic liver. However, little is known about the mechanism that controls their mobilization in vivo. Here we confirmed that BM mesenchymal stem cells (BMSCs) can migrate to the damaged liver and differentiate into myofibroblasts. We also investigated the mechanism underlying the homing of BMSCs after liver injury. METHODS: ICR mice were lethally irradiated and received BM transplants from enhanced green fluorescent protein transgenic mice. Carbon tetrachloride or bile duct ligation was used to induce liver fibrosis. The fibrotic liver tissue was examined by immunofluorescent staining to identify BM-derived myofibroblasts. RESULTS: BMSCs contributed significantly to myofibroblast population in fibrotic liver. Moreover, analysis in vivo and in vitro suggested that homing of BMSCs to the damaged liver was in response to sphingosine 1-phosphate (S1P) gradient between liver and BM. Furthermore, S1P receptor type 3 (S1P3) was required for migration of BMSCs triggered by S1P. CONCLUSIONS: S1P mediates liver fibrogenesis through homing of BMSCs via S1P3 receptor, which may represent a novel therapeutic target in liver fibrosis through inhibiting S1P formation and/or receptor activation.

IGF-1 receptor mediates differentiation of primary cultures of mouse skeletal myoblasts.

Studies involving immortalized myoblasts suggested that insulin-like growth factors (IGFs) promote differentiation of skeletal muscle, but gene targeting experiments in mice did not provide support for this hypothesis. To address this discrepancy, we examined differentiation of primary cultures of mouse myoblasts. Differentiation was normally unaffected by addition of IGFs to the differentiation medium. However, when we interrupted IGF-mediated signaling, by incubating myoblasts with suramin or with a monoclonal antibody to the IGF-I receptor, differentiation was inhibited. Inhibition was reversed by exogenous IGF-I or IGF-II, but not by insulin. Differentiation was enhanced in myoblasts that were incubated with an inhibitor of the mitogen-activated protein kinase signaling pathway (PD098059) and such cells were responsive to exogenous IGF-I. Our results demonstrate that IGF action contributes to the differentiation of non-immortalized mouse myoblasts and that these cells represent a model system that can be experimentally manipulated to study the molecular events involved in skeletal muscle differentiation.

Ectonucleotide diphosphohydrolase activity in Crithidia deanei.

In this work we describe the ability of living Crithidia deanei to hydrolyze extracellular ATP. In intact cells at pH 7.2, a low level of ATP hydrolysis was observed in the absence of any divalent metal (0.41+/-0.13 nmol P(i) h(-1) 10(7) cells(-1)). The ATP hydrolysis was stimulated by MgCl(2) and the Mg(2+)-dependent ecto-ATPase activity was 4.05+/-0.17 nmol P(i) h(-1) 10(7) cells(-1). Mg(2+)-dependent ecto-ATPase activity increased linearly with cell density and with time for at least 60 min. The addition of MgCl(2) to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.93+/-0.26 mM MgCl(2). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2), but not CaCl(2) or SrCl(2). The apparent K(m) for Mg-ATP(2-) was 0.26+/-0.03 mM. ATP was the best substrate for this enzyme; other nucleotides, such as ITP, GTP, UTP and CTP, produced lower reaction rates. In the pH range from 6.6 to 8.4, in which the cells were viable, the acid phosphatase activity also present in this cell decreased, while the Mg(2+)-dependent ATPase activity did not change. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, vanadate, molybdate, sodium fluoride and tartrate. To confirm that this Mg(2+)-dependent ATPase was an ecto-ATPase, we used the impermeant inhibitor 4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. The cell surface location of the ATP-hydrolyzing site was also confirmed by cytochemical analysis.

Inhibition of human chymase by suramin.

Chymase is a chymotrypsin-like protease localized in mast cells in complexes with heparin. In the present study, we demonstrated that suramin, a hexasulfonated naphthylurea used as an anti-cancer drug, inhibits the activity of purified human chymase in vitro. The inhibition was ionic-strength-dependent. It was observed that suramin competed with heparin-Sepharose gel for binding to chymase and the inhibition of chymase activity by suramin was partially impaired by heparin. Our results show that suramin may become a prototype of a new type of chymase inhibitor because of its unique character.

Reversal by transferrin of growth-inhibitory effect of suramin on hormone-refractory human prostate cancer cells.

BACKGROUND: The second leading cause of cancer-related deaths in American men is metastatic hormone-refractory adenocarcinoma of the prostate, for which there is currently no effective treatment. Transferrin is abundant in bone stroma and has been found to stimulate models of hormone-refractory metastatic prostate cancer. Suramin, a compound that has been used to treat metastatic prostate cancer, has been demonstrated to antagonize the binding of transferrin to the transferrin receptor and to suppress uptake of iron by hematopoietic cells. PURPOSE: The purpose of our study was to determine whether transferrin may reverse the inhibitory action of suramin on metastatic prostate-derived cell lines. METHODS: Five human prostate cell lines (PC-3, PC-3M, DU-145, TSU-Pr1, and LNCaP) derived from metastatic deposits were examined for response to growth stimulation by apotransferrin, for the presence of transferrin receptors by binding of 125I-labeled transferrin, and for relative transferrin receptor messenger RNA (mRNA) content by ribonuclease protection assays. We measured the amount of growth inhibition by suramin in low serum assays to demonstrate maximal inhibition over the apotransferrin to reverse the inhibition of suramin in these tumors. RESULTS: The results clearly demonstrate that the androgen-insensitive metastatic cell lines (PC-3, PC-3M, DU-145, and TSU-Pr1) demonstrate increased cell numbers when exposed to holotransferrin or apotransferrin, while the androgen-sensitive cell line (LNCaP) did not show any increase. All cell lines demonstrated a similar number of transferrin receptors and transferrin receptor mRNA. We used these maximally inhibitory, but clinically relevant, concentrations of suramin to determine whether transferrin could reverse the inhibition, and it did, but only in the androgen-insensitive metastatic lines. Indeed, in the PC-3 cells, inhibition turned to stimulation with the addition of transferrin, and even at the highest concentration of suramin tested, 400 microM, a concentration that would be toxic to patients, the amount of inhibition by suramin was still reduced by more than 50% by transferrin in TSU-Pr1 cells. In the androgen-sensitive LNCaP cells, however, transferrin had limited ability to block the inhibitory activity of suramin. CONCLUSIONS: Concentrations of tumor-stimulating factors, such as transferrin, in the metastatic microenvironment need to be taken into consideration in the use of suramin and suramin-like derivatives. Novel strategies need to be identified that will negate the action of transferrin on androgen-insensitive cells.

Suramin interference with transforming growth factor-beta inhibition of human renal cell carcinoma in culture.

Suramin is a polyanionic compound used clinically for the treatment of trypanosomiasis, which is known to inhibit the action of many protein factors in vitro. Transforming growth factor-beta (TGF-beta) is a multifunctional regulatory protein which inhibits the growth of renal cell carcinoma in culture. While suramin at 50-500 micrograms/ml had no significant effect on the growth of renal cell carcinoma in culture in our experiments, it did partially reverse the growth inhibition induced by TGF-beta in the two cell lines tested. This effect apparently is caused by suramin's direct interference with 125I-labeled TGF-beta's ability to bind to the cell, and not by any effect of suramin on the TGF-beta receptor. Furthermore, suramin dissociates TGF-beta bound to the cell with a t1/2 of less than 30 min. These results are consistent with those previously reported regarding suramin's interaction with other protein growth factors, and suggest that suramin may interact with the TGF-beta protein itself to inactivate it.

The ability of suramin to block CD4-gp120 binding is reversed in the presence of albumin.

We have shown that suramin can directly inhibit the binding of the human immunodeficiency virus type 1 gp120 envelope protein to immobilized CD4, thus helping to explain the previously described antiviral properties of suramin. However, physiological concentrations of serum albumin significantly attenuated suramin's antiviral effects, suggesting that only free suramin has antiviral properties. Reported discrepancies between in vitro and clinical activities of suramin may be due to differences between free suramin levels in experimental assays and those achievable clinically.

The effect of thymopoietin 32-34 (TP3) on suramin-induced inhibition on delayed type hypersensitivity in guinea pigs.

Suramin administration inhibits virus replication and produces impaired immunoregulation. Delayed type hypersensitivity (DTH)-reaction was inhibited by Suramin (200 mg/kg, i.p.) injection in guinea-pigs immunized with BCG vaccine. Suramin-inhibited DTH-reaction was restored to normal level after 4 days of treatment with thymopoietin 32-34 (TP 3) and TP5 (1 mg/kg, i.p.), TP3 greater than TP5. TP3 produced the strongest restoration when applied together with Suramin, but a single treatment with TP3 or TP5 oligopeptide inhibited the development of DTH-reaction. TP3 with Suramin treatment might be advantageous to eliminate defective side effects of Suramin on immune system and TP3 with indirect inducing action on IL-2, IFN-gamma production may be useful in therapy of AIDS patients.

[Trial prevention of the embryolethal properties of suramin in the mouse by adjuvant progesterone]. / Essai de prévention du pouvoir embryoléthal de la suramine chez la souris par adjonction de progestérone

Suramine, a trypanocidal drug, has an embryo-lethal and teratogenic action in Rodents. Like Triton W.R. 1339 suramine modifies the lysosomes of the visceral yolk sac. In the mouse the attempts to correct the embryolethal action by a simultaneous progesterone treatment failed while the hormonal treatment is capable to suppress the embryolethality in Triton W.R. 1339 treated mice.