Drug Repurposing and De Novo Drug Discovery of Protein Kinase Inhibitors as New Drugs against Schistosomiasis.

Schistosomiasis is a neglected tropical disease affecting more than 200 million people worldwide. Chemotherapy relies on one single drug, praziquantel, which is safe but ineffective at killing larval stages of this parasite. Furthermore, concerns have been expressed about the rise in resistance against this drug. In the absence of an antischistosomal vaccine, it is, therefore, necessary to develop new drugs against the different species of schistosomes. Protein kinases are important molecules involved in key cellular processes such as signaling, growth, and differentiation. The kinome of schistosomes has been studied and the suitability of schistosomal protein kinases as targets demonstrated by RNA interference studies. Although protein kinase inhibitors are mostly used in cancer therapy, e.g., for the treatment of chronic myeloid leukemia or melanoma, they are now being increasingly explored for the treatment of non-oncological conditions, including schistosomiasis. Here, we discuss the various approaches including screening of natural and synthetic compounds, de novo drug development, and drug repurposing in the context of the search for protein kinase inhibitors against schistosomiasis. We discuss the status quo of the development of kinase inhibitors against schistosomal serine/threonine kinases such as polo-like kinases (PLKs) and mitogen-activated protein kinases (MAP kinases), as well as protein tyrosine kinases (PTKs).

JQ-1 ameliorates schistosomiasis liver fibrosis by suppressing JAK2 and STAT3 activation.

Schistosomiasis is a serious parasitic infection caused by Schistosoma. The parasite deposits eggs in the host liver, causing inflammation that activates hepatic stellate cells (HSCs), which leads to liver fibrosis. Currently, there is no effective therapy for liver fibrosis; thus, treatments are urgently needed. Therefore, in the present study, mice infected with Schistosoma japonicum were treated with JQ-1, a small-molecule bromodomain inhibitor with reliable anti-tumor and anti-inflammatory activities. The fibrotic area of the liver measured by computer-assisted morphometric analysis and the expression levels of the cytoskeletal protein alpha smooth muscle actin (α-SMA) and of collagen assessed by quantitative PCR, Western blot and immunohistochemistry were significantly decreased in the liver following JQ-1 treatment compared with vehicle-treated controls. Total RNA was extracted from the liver of JQ-1-treated Schistosoma-infected mice for RNA-sequencing analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that JQ-1 affected biological processes and the expression of cellular components known to play key roles in the transdifferentiation of HSCs to myofibroblasts. In vitro treatment with JQ-1 of JS-1 cells, a mouse HSC line, indicated that JQ-1 significantly inhibited JS-1 proliferation but had no effect on JS-1 activity, senescence, or apoptosis. Western blot results showed that JQ-1 inhibited the expression levels of phosphorylated JAK2 and phosphorylated STAT3 without altering expression levels of these non-phosphorylated proteins. Taken together, these findings suggested that JQ-1 treatment ameliorated S. japonicum egg-induced liver fibrosis, at least in part, by suppressing HSC activation and proliferation through the inhibition of JAK2/STAT3 signaling. These results lay a foundation for the development of novel approaches to treat and control liver fibrosis caused by S. japonicum.

Increased expression of NTPDases 2 and 3 in mesenteric endothelial cells during schistosomiasis favors leukocyte adhesion through P2Y1 receptors.

Schistosomiasis is caused by an intravascular parasite and linked to phenotypic changes in endothelial cells that favor inflammation. Endothelial cells express P2Y1 receptors (P2Y1R), and their activation by ADP favors leukocyte adhesion to the endothelial monolayer. We aimed to evaluate the influence of schistosomiasis upon endothelial purinergic signaling-mediated leukocyte adhesion. Mesenteric endothelial cells and mononuclear cells from control and Schistosoma mansoni-infected mice were used in co-culture. P2Y1R levels were similar in both groups. Basal leukocyte adhesion was higher in the infected than in the control group; leukocyte adhesion increased after treatment with the P2Y1R agonist 2-MeSATP in both groups, though it only marginally increased in the infected group. Pre-incubation with the selective P2Y1R antagonist MRS2179 (0.3µM) prevented the agonist effect. However, in the infected group it also reduced the basal leukocyte adhesion, suggesting endothelial cell pre-activation. The endothelial expressions of NTPDases 2 and 3 were significantly increased in the infected group, increasing extracellular ATP hydrolysis and ADP formation by endothelial cells. Therefore, mesenteric endothelial cells are primed by schistosomiasis to a pro-inflammatory phenotype characterized by an increased expression of NTPDases 2 and 3, favoring ADP accumulation and mononuclear cell adhesion, possibly contributing to mesenteric inflammation and schistosomiasis morbidity.

[Observation on serum γ-glutamyl transpeptidase levels of patients with subclinical schistosomiasis before and after pathogen treatment].

OBJECTIVE: To observe the changes of serum γ-glutamyl transpeptidase (GGT) levels before and after the pathogen treatment in patients with subclinical schistosomiasis, and explore its clinical value in the diagnosis and treatment of subclinical schistosomiasis. METHODS: Totally 109 patients with subclinical schistosomiasis, who were found in the endemic investigation of schistosomiasis in Ezhou City, were selected as the investigation subjects, and then they were treated with praziquantel. The serum GGT levels of the subjects before and after the treatment were detected and compared. RESULTS: Before the treatment, the average value of the GGT levels of the 109 patients was (48.1 ± 45.9) IU/L, among which, the GGT levels of 69 cases (63.3%) were normal, and the levels of 40 cases (36.7%) were increased. After the treatment, the average GGT level of the patients was (32.1 ± 23.4) IU/L, which decreased by 33.3% comparing with that before the treatment, and the difference had a statistical significance (U＝2.17, P = 0.01). The GGT levels of 65 patients decreased in different degrees. Among the 40 patients whose GGT levels had increased before the treatment, the GGT levels of 31 ones returned to the normal. CONCLUSIONS: The GGT level detection can accurately reflect the liver function in the patients with subclinical schistosomiasis, and also it has certain clinical application value to judge the liver function damage and recovery of the patients before and after the pathogen treatment.

Targeting kinases in Plasmodium and Schistosoma: Same goals, different challenges.

With respect to parasite-induced infectious diseases of worldwide importance, members of the genera Plasmodium and Schistosoma are top pathogens. Nearly half a billion people suffer from malaria caused by Plasmodium spp. and schistosomiasis (bilharzia) induced by Schistosoma spp. Resistance against essentially all drugs used for malaria treatment has been reported. For schistosomiasis justified fear of upcoming resistance is discussed against the background of only one widely used drug for treatment. Research of the recent decade has demonstrated that essential steps of the biology of these and other parasites are controlled by kinases, which represent attractive targets for new-generation antiparasitic compounds. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

[Clinical efficacy of sodium tanshinone IIA sulfonate in treatment of advanced schistosomiasis].

OBJECTIVE: To evaluate the clinical efficacy of sodium tanshinone IIA sulfonate (STS) in the treatment of advanced schistosomiasis. METHODS: Fifty cases with advanced schistosomiasis admitted to the Touzao Township Hospital of Dongtai City during the period from November 2012 to November 2013 were treated with STS for 10 days, and the internal diameter of the portal vein, levels of ALT, AST, gamma-GT, PIIIP, CIV, HA and LN were measured and compared before and after the administration of STS. RESULTS: The mean levels of all serological parameters except HA were within the normal range before STS treatment, while the highest positive rate was detected in gamma-GT (26.0%) and HA (54.0%). Following the STS treatment, the mean levels of all parameters and the positive rates reduced, with the greatest reduction observed in gamma-GT (36.7%) and HA (37.8%); however, the mean HA level was still higher than the normal range. The mean internal diameter of the portal vein reduced from (10.5 +/- 1.7) mm before the STS treatment to (9.8 +/- 1.3) mm after the STS administration, with a significant diffrtence (P < 0.05). CONCLUSION: STS appears effective in the treatment of advanced schistosomiasis.

Hepatic cytochrome P450s, phase II enzymes and nuclear receptors are downregulated in a Th2 environment during Schistosoma mansoni infection.

Inflammation and infection downregulate the activity and expression of cytochrome P450s (P450s) and other drug metabolizing enzymes (DMEs) involved in hepatic drug clearance. Schistosoma mansoni infection was reported to cause a downregulation of hepatic P450-dependent activities in mouse liver, but little is known about the specific enzymes affected or whether phase II DMEs are also affected. Here we describe the effect of murine schistosomiasis on the expression of hepatic P450s, NADPH-cytochrome P450 reductase (Cpr), phase II drug metabolizing enzymes, and nuclear receptors at 30 and 45 days postinfection (dpi). Although the hepatic expression of some of these genes was altered at 30 dpi, we observed substantial changes in the expression of the majority of P450 mRNAs and proteins measured, Cpr protein, as well as many of the UDP-glucuronosyltransferases and sulfotransferases at 45 dpi. S. mansoni infection also altered nuclear receptor expression, inducing mRNA levels at 30 dpi and depressing levels at 45 dpi. S. mansoni evoked a T helper 2 (Th2) inflammatory response at 45 dpi, as indicated by the induction of hepatic Th2 cytokine mRNAs [interleukins 4, 5, and 13], whereas the hepatic proinflammatory response was relatively weak. Thus, chronic schistosomiasis markedly and selectively alters the expression of multiple DMEs, which may be associated with Th2 cytokine release. This would represent a novel mechanism of DME regulation in disease states. These findings have important implications for drug testing in infected mice, whereas the relevance to humans with schistosomiasis needs to be determined.

[Relationship between advanced schistosomiasis and HBV infection].

OBJECTIVE: To discuss the relationship between advanced schistosomiasis and HBV infection. METHODS: A total of 250 advanced schistosomiasis patients were examined with ultrasound, and their serum samples were detected for liver function, HBsAg, etc. The correlations between HBV infection and advanced types, abnormal liver function, liver cancer and mortality were analyzed, respectively. RESULTS: The positive rate of HBsAg was 58.4% (146/250). The rates of abnormal liver function (46.6%), jaundice (23.3%), cancer (17.8%) and mortality (23.3%) were significantly higher in the advanced schistosomiasis patients with HBsAg than in the advanced schistosomiasis patients without HBsAg (P values < 0.05 or 0.01). CONCLUSION: Advanced schistosomiasis combined with HBV infection aggravates the liver damage.

Increased endothelial cell-leukocyte interaction in murine schistosomiasis: possible priming of endothelial cells by the disease.

BACKGROUND AND AIMS: Schistosomiasis is an intravascular parasitic disease associated with inflammation. Endothelial cells control leukocyte transmigration and vascular permeability being modulated by pro-inflammatory mediators. Recent data have shown that endothelial cells primed in vivo in the course of a disease keep the information in culture. Herein, we evaluated the impact of schistosomiasis on endothelial cell-regulated events in vivo and in vitro. METHODOLOGY AND PRINCIPAL FINDINGS: The experimental groups consisted of Schistosoma mansoni-infected and age-matched control mice. In vivo infection caused a marked influx of leukocytes and an increased protein leakage in the peritoneal cavity, characterizing an inflamed vascular and cellular profile. In vitro leukocyte-mesenteric endothelial cell adhesion was higher in cultured cells from infected mice as compared to controls, either in the basal condition or after treatment with the pro-inflammatory cytokine tumor necrosis factor (TNF). Nitric oxide (NO) donation reduced leukocyte adhesion to endothelial cells from control and infected groups; however, in the later group the effect was more pronounced, probably due to a reduced NO production. Inhibition of control endothelial NO synthase (eNOS) increased leukocyte adhesion to a level similar to the one observed in the infected group. Besides, the adhesion of control leukocytes to endothelial cells from infected animals is similar to the result of infected animals, confirming that schistosomiasis alters endothelial cells function. Furthermore, NO production as well as the expression of eNOS were reduced in cultured endothelial cells from infected animals. On the other hand, the expression of its repressor protein, namely caveolin-1, was similar in both control and infected groups. CONCLUSION/SIGNIFICANCE: Schistosomiasis increases vascular permeability and endothelial cell-leukocyte interaction in vivo and in vitro. These effects are partially explained by a reduced eNOS expression. In addition, our data show that the disease primes endothelial cells in vivo, which keep the acquired phenotype in culture.

Piggy-backing the concept of cancer drugs for schistosomiasis treatment: a tangible perspective?

The fear that schistosomes will become resistant to praziquantel (PZQ) motivates the search for alternatives to treat schistosomiasis. Recent studies of signaling proteins in schistosomes uncovered a way of achieving this goal relatively quickly. It was shown that protein kinases (PKs) control important biological processes in schistosomes. Concurrently, the involvement of mutant forms of PKs was demonstrated in the etiology of cancer. Therefore, different anticancer drugs have been developed to inhibit deregulated PKs. These can also inhibit schistosome PKs, thus blocking parasite development. Recent studies characterizing schistosome PKs are summarized and we discuss the concept of PK inhibitors, including approved cancer drugs, as novel candidate anti-schistosome agents. This is also likely to be of significance for other worm infections.

Succinate cytochrome c reductase in schistosomiasis: in vitro inhibition by some schistosomicidal drugs.

Enzymes in mitochondria play an important role in biological oxidation and energy production. To understand the effect of schistosomiasis on these important processes, succinate cytochrome c reductase (SCR) from control and Schistosoma-infected mice was subjected for investigation. In this article, we report that SCR from Schistosoma-infected mouse showed a significant decrease in its Vmax and Km compared to control using both cytochrome c and 2,6-dichlorophenolindophenol as substrates. Furthermore, the kinetic studies of the purified SCR in the absence and presence of the schistosomicidal drugs praziquantel and Commiphora extract reveal that both drugs have an inhibitory action on the enzyme from the control and Schistosoma-infected mice and praziquantel changes the type of inhibition of SCR towards cytochrome c from mixed type in control to a competitive one in the case of the infection.

Structure mechanism insights and the role of nitric oxide donation guide the development of oxadiazole-2-oxides as therapeutic agents against schistosomiasis.

Schistosomiasis is a chronic parasitic disease affecting hundreds of millions of individuals worldwide. Current treatment depends on a single agent, praziquantel, raising concerns of emergence of resistant parasites. Here, we continue our explorations of an oxadiazole-2-oxide class of compounds we recently identified as inhibitors of thioredoxin glutathione reductase (TGR), a selenocysteine-containing flavoenzyme required by the parasite to maintain proper cellular redox balance. Through systematic evaluation of the core molecular structure of this chemotype, we define the essential pharmacophore, establish a link between the nitric oxide donation and TGR inhibition, determine the selectivity for this chemotype versus related reductase enzymes, and present evidence that these agents can be modified to possess appropriate drug metabolism and pharmacokinetic properties. The mechanistic link between exogenous NO donation and parasite injury is expanded and better defined. The results of these studies verify the utility of oxadiazole-2-oxides as novel inhibitors of TGR and as efficacious antischistosomal agents.

CD10 expression in tumour and stromal cells of bladder carcinoma: an association with bilharziasis.

CD10 is a cell surface zinc metalloprotease expressed in a variety of normal cell types, including lymphoid precursor cells, germinal centre B lymphocytes and some epithelial cells. The aim of this study was to assess the prognostic value of CD10 in bladder carcinoma. The expression of CD10 was immunohistochemically assessed in 49 cases of primary bladder carcinoma in comparison with 10 non-neoplastic normal bladder mucosa specimens. 27 cases (55%) were tumour CD10(+) and tumour CD10 positivity was significantly correlated with advanced stage, larger tumor size, and shorter mean survival time. Extensive tumoral staining assessed by H score further documented the positive correlation of CD10 with worse prognostic factors in the whole group and its subdivisions (SCC and TCC), in addition to its significant association with bilharziasis in SCC. Only CD10-tumour positivity in the whole group proved to be an independent prognostic factor for overall survival by multivariate analysis. No significant value of stromal CD10 expression in the investigated bladder carcinoma cases was found. This study points to the prognostic value of neoplastic CD10 expression in bladder carcinoma and its possible importance in facilitating tumour invasion and metastasis. Bilharziasis could have a role in upregulation of CD10 expression in SCC.

Protein tyrosine kinases as new potential targets against human schistosomiasis.

In spite of the numerous efforts made to control their transmission, parasite schistosomes still represent a serious public health concern and a major economic problem in many developing countries. Praziquantel (PZQ) is the drug of choice for the treatment of schistosomiasis and the only one that is available for mass chemotherapy. However, its widespread use and its inefficacy on juvenile parasites raise fears that schistosomes will develop drug resistance, and make the development of alternative drugs highly desirable. Protein tyrosine kinases (PTKs) are key molecules that control cell differentiation and proliferation and they already represent important targets for molecular cancer therapy. The recent characterization in Schistosoma mansoni of several cytosolic and receptor PTKs, with properties similar but also divergent from their vertebrate counterparts, opens new perspectives for the development of novel strategies in chemotherapy of schistosomiasis, which could be based on the use of parasite-specific tyrosine phosphorylation inhibitors.

In vitro effect of schistosomicidal drugs on hepatic arylsulfatase B from the schistosoma-infected mouse.

Arylsulfatase B (ASB) hydrolyzes the desulfation of N-acetylgalactosamine-4-sulfate at the non-reducing terminal of glycosaminoglycans. This enzyme activity was found to be elevated in mice schistosomiasis. In the present study, the catalytic and immunological properties of purified ASB from the liver of Schistosoma-infected mouse was investigated in the presence and absence of the schistosomicidal drugs praziquantel and Commiphora extract. The in vitro effect of praziquantel was found to be inhibitory with a Ki value of 5.5 x 10(-4) M while that of commiphora extract was as an activator. Furthermore, these drugs did not have an observed effect on the immunological properties of ASB with regard to its binding to its polyclonal rabbit antibody. These results indicate that some schistosomicidal drugs may reverse the alteration of the catalytic properties of the enzyme in schistosomiasis.

Development of the rapid and simple elisa (whole blood-ELISA) using coconut water-tween as a wash solution for whole blood sample from Schistosoma japonicum-infected rabbit and human.

PBS-Tween as a wash solution, prepared with distilled water, is used in ELISA. In areas where schistosomiasis is endemic, however, distilled water is hard to come by. We have modified a WHOLE BLOOD-ELISA test to use coconut water-Tween as a wash solution, because coconut water is easy to come by and cheap in the tropics. We applied the test to whole blood samples from rabbits and humans infected with Schistosoma japonicum. This modified WHOLE BLOOD-ELISA was confirmed to be a rapid, simple, and cost-effective method.

Activity of some hepatic enzymes in schistosomiasis and concomitant alteration of arylsulfatase B.

The levels of arylsulfatases A and B, alpha-amylase, aspartate transcarbamylase, and gamma-glutamyl transpeptidase were investigated during the infection of mice with schistosoma mansoni. This infection caused a significant (p < 0.001) increase in the activity of hepatic arylsulfatase B (ASB), aspartate transcarbamylases and gamma-glutamyl transpeptidase. A non-significant difference occurred for alpha-amylase (p < 0.3) and arylsulfatase A (p > 0.5) when compared to the control. The specific activity of hepatic ASB was progressively increased with the progression of the Schistosoma-infection. Moreover, the kinetic studies of hepatic ASB in Schistosoma-infection showed that a slight decrease in the value of K(m) and about a 40% increase in V(max) when compared to the control. In addition, the pH optimum of hepatic ASB was altered from 6 to 7 as a result of schistosomiasis. These observations suggest that there are schistosomiasis-associated changes of the catalytic and kinetic properties of hepatic ASB.

Detection of inducible nitric oxide synthase in Schistosoma japonicum and S. mansoni.

Schistosoma japonicum and S. mansoni were tested for reactivity with an anti-inducible nitric oxide (iNOS) antibody and the distribution of iNOS was studied by immunofluorescent tests in different stages of the parasites. Reactivity was associated with the tegument in both larval schistosomes (sporocysts and cercariae) and eggs. With adult worms, the majority of the immunofluorescence was predominantly subtegumental in S. japonicum and parenchymal in S. mansoni. Fluorescence was also observed in host tissues (snails and mouse liver). In Western blots, the enzyme of S. japonicum had an apparent molecular weight of about 210 kDa. The possible role of worm and host iNOS in the parasite-host interrelation remains to be clarified.

Evaluation of nitric oxide (NO) levels in hepatitis C virus (HCV) infection: relationship to schistosomiasis and liver cirrhosis among Egyptian patients.

Nitric oxide (NO), a recently discovered free radical, is overproduced in liver cirrhosis. Hepatitis C virus (HCV) might increase NO levels via increased inducible NO synthase (iNOS). This work was carried out to study the effect of HCV-induced liver cirrhosis on NO levels among Egyptian patients. The study included 46 patients with liver cirrhosis, and 30 healthy individuals of matched age and sex. NO levels determined as the stable endproduct nitrate, showed a statistically significant increase among patients compared to the control group (P < 0.001). Furthermore, NO levels increased proportionally with the severity of liver cirrhosis as assessed by Child's classification (P < 0.05). Moreover, schistosomial infection enhanced NO levels in cirrhotic patients with HCV infection compared to non-bilharzial patients (P < 0.001). Polymerase chain reaction (PCR) and branched DNA assays were used for detection of HCV RNA positivity, and measurement of the virus load, respectively. Both showed a positive correlation with the NO levels (P < 0.001). At a nitrate cutoff value of 70 micromol/L, the sensitivity and specificity were 83.0% and 73.0%, respectively. Chi square analysis showed a significant correlation between ALT levels and both HCV RNA positivity by polymerase chain reaction (PCR) (P < 0.02), and virus load (P<0.05). Interestingly enough, there was a significant positive correlation between HCV RNA and schistosomal antibody titer as measured by hemaglutination inhibition assay (HAI) (P < 0.05). The data presented in this report indicated an association between NO levels and the development and progression of liver cirrhosis. Furthermore, the findings obtained from this study demonstrated that schistomiasis is an important risk factor involved in enhancement of NO levels and virus replication. The latter may aggravate liver cell injury and hence the development of cirrhosis.

Nitric oxide synthase immunoreactivity in human bladder carcinoma.

AIMS: To study the expression of the endothelial and inducible isoforms of nitric oxide synthase (eNOS and iNOS, respectively) in human bladder carcinoma and schistosomal bladder disease, and to compare it with normal adult and fetal urothelium. Nitric oxide is thought to play a complex role in human carcinogenesis, but has only recently been investigated in bladder cancer. METHODS: Immunohistochemistry was performed on paraffin wax embedded sections of 33 human bladder carcinomas and five bladder carcinoma cell lines; in addition, seven schistosomal bladder cases and normal and fetal urothelium were investigated. In the cell lines enzymatic activity was examined by the NADPH diaphorase reaction. RESULTS: Immunoreactivity for eNOS was present in most cells of all 31 cases examined. Immunoreactivity for iNOS was less abundant and was seen in 23 of 25 cases. Similar findings were noted in schistosomal bladder cancer. In the normal bladder mucosa, eNOS immunoreactivity was found only in the superficial cell layer and iNOS was not expressed, whereas in the fetal urothelium immunoreactivity for both isoforms was seen in all cell layers. Enzymatic activity and immunoreactivity for eNOS and iNOS were evident in the five bladder carcinoma cell lines. CONCLUSIONS: It is possible that NOS plays a role in the differentiation of the transitional epithelium in fetal life, has a biological function in the adult bladder mucosa, and is involved in bladder carcinogenesis. eNOS and iNOS immunoreactivity do not differ in schistosomal and non-schistosomal bladder carcinoma, but resemble the pattern of expression typical of fetal urothelium.