Human schistosomiasis, one of the most important neglected tropical diseases caused by parasitic worms of the genus Schistosoma. It is accompanied by severe clinical symptoms as well as socioeconomic problems, and more than 200,000 deaths per year. According to the WHO, some 229 million people in 78 countries are affected. Although disease transmission is more common in tropical and subtropical areas, a potential shift of schistosomiasis into new habitat like south Europe is anticipated due to climate change. No vaccine is available, only the drug Praziquantel (PZQ) is used for treatment. However, PZQ still having some basic problems as, constant selection pressure that caused a serious concern for the spread of drug-resistance to this monotherapy and the cure rate for PZQ is usually 60-90% but never 100%. In addition, PZQ is active against mature parasites only and not active against juvenile parasites which leads to re-activation since the patient is still harboring juvenile worms. Due to PZQ treatment limitations and constant selection pressure, there is an urgency of developing new therapies for schistosomiasis. Our starting point in drug development was a previous treatment of Schistosoma, Oxamniquine (OXA). OXA is a prodrug that requires activation by an endogenous schistosome sulfotransferase. However, OXA has fallen out of use due to drug resistance and limited efficacy. To overcome these treatment limitations, we have successfully derivatized OXA using structure-function-guided approaches to design a new series of compounds with major advancements over both PZQ and OXA. Guided by data from X-ray crystallography and Schistosoma killing assays, we designed, synthesized, and tested 350 compounds. We have successfully identified CIDD-015610 that has major advancement. In vivo studies demonstrated that CIDD-0150610 can reduced the infection up to 80%. CIDD-015610 is effective against the main clinically important Schistosoma species. and, importantly, they are also effective against juvenile parasite and PZQ-resistant strains. We conclude that CIDD-0150610 is a better that can be used in combination with PZQ to mitigate drug resistance and improve efficacy. National Institute of General Medical Science (2k12GM111726), the National Institute of Allergy and Infectious Diseases (AI27219), Morrison Trust, the National Institutes of Health (P30 CA054174), the NIH-ORIP SIG (S10OD030374), the National Institute of General Medical Sciences (P30 GM124165), and the NIH-ORIP HEI (S10OD021527).

The antischistosomal drug oxamniquine, OXA, requires activation by a sulfotransferase within the parasitic worm to enable killing. Examination of the pharmacokinetic/pharmacodynamic (PK/PD) relationship for OXA identified an in vitro-in vivo paradox with the maximal clinical plasma concentrations five-to ten-times lower than the efficacious concentration for in vitro schistosomal killing. The parasite resides in the vasculature between the intestine and the liver, and modeling the PK data to determine portal concentrations fits with in vitro studies and explains the required human dose. In silico models were used to predict murine dosing to recapitulate human conditions for OXA portal concentration and time course. Follow-up PK studies verified in mice that a 50–100 mg/kg oral gavage dose of OXA formulated in acetate buffer recapitulates the 20–40 mg/kg dose common in patients. OXA was rapidly cleared through a combination of metabolism and excretion into bile. OXA absorbance and tissue distribution were similar in wild-type and P-gp efflux transporter knockout mice. The incorporation of in vitro efficacy data and portal concentration was demonstrated for an improved OXA-inspired analog that has been shown to kill S. mansoni, S. haematobium, and S. japonicum, whereas OXA is only effective against S. mansoni. Second-generation OXA analogs should optimize both in vitro killing and physiochemical properties to achieve high portal concentration via rapid oral absorption, facilitated by favorable solubility, permeability, and minimal intestinal metabolism.

Human schistosomiasis is a neglected tropical disease caused by Schistosoma mansoni, S. haematobium, and S. japonicum. Praziquantel (PZQ) is the method of choice for treatment. Due to constant selection pressure, there is an urgent need for new therapies for schistosomiasis. Previous treatment of S. mansoni included the use of oxamniquine (OXA), a drug that is activated by a schistosome sulfotransferase (SULT). Guided by data from X-ray crystallography and Schistosoma killing assays more than 350 OXA derivatives were designed, synthesized, and tested. We were able to identify CIDD-0150610 and CIDD-0150303 as potent derivatives in vitro that kill (100%) of all three Schistosoma species at a final concentration of 71.5 μM. We evaluated the efficacy of the best OXA derivates in an in vivo model after treatment with a single dose of 100 mg/kg by oral gavage. The highest rate of worm burden reduction was achieved by CIDD -150303 (81.8%) against S. mansoni, CIDD-0149830 (80.2%) against S. haematobium and CIDD-066790 (86.7%) against S. japonicum. We have also evaluated the ability of the derivatives to kill immature stages since PZQ does not kill immature schistosomes. CIDD-0150303 demonstrated (100%) killing for all life stages at a final concentration of 143 μM in vitro and effective reduction in worm burden in vivo against S. mansoni. To understand how OXA derivatives fit in the SULT binding pocket, X-ray crystal structures of CIDD-0150303 and CIDD-0150610 demonstrate that the SULT active site will accommodate further modifications to our most active compounds as we fine tune them to increase favorable pharmacokinetic properties. Treatment with a single dose of 100 mg/kg by oral gavage with co-dose of PZQ + CIDD-0150303 reduced the worm burden of PZQ resistant parasites in an animal model by 90.8%. Therefore, we conclude that CIDD-0150303, CIDD-0149830 and CIDD-066790 are novel drugs that overcome some of PZQ limitations, and CIDD-0150303 can be used with PZQ in combination therapy.

Oxamniquine (OXA) is a prodrug activated by a sulfotransferase (SULT) that was only active against Schistosoma mansoni. We have reengineered OXA to be effective against S. haematobium and S. japonicum. Three derivatives stand out, CIDD-0066790, CIDD-0072229, and CIDD-0149830 as they kill all three major human schistosome species. However, questions remain. Is the OXA mode of action conserved in derivatives? RNA-interference experiments demonstrate that knockdown of the SmSULT, ShSULT, and SjSULT results in resistance to CIDD-0066790. Confirming that the OXA-derivative mode of action is conserved. Next is the level of expression of the schistosome SULTs in each species, as well as changes in SULT expression throughout development in S. mansoni. Using multiple tools, our data show that SmSULT has higher expression compared to ShSULT and SjSULT. Third, is the localization of SULT in the adult, multicellular eucaryotic schistosome species. We utilized fluorescence in situ hybridization and uptake of radiolabeled OXA to determine that multiple cell types throughout the adult schistosome worm express SULT. Thus, we hypothesize the ability of many cells to express the sulfotransferase accounts for the ability of the OXA derivatives to kill adult worms. Our studies demonstrate that the OXA derivatives are able to kill all three human schistosome species and thus will be a useful complement to PZQ.

Human schistosomiasis is a debilitating, life-threatening disease affecting more than 229 million people in as many as 78 countries. There is only one drug of choice effective against all three major species of Schistosoma, praziquantel (PZQ). However, as with many monotherapies, evidence for resistance is emerging in the field and can be selected for in the laboratory. Previously used therapies include oxamniquine (OXA), but shortcomings such as drug resistance and affordability resulted in discontinuation. Employing a genetic, biochemical and molecular approach, a sulfotransferase (SULT-OR) was identified as responsible for OXA drug resistance. By crystallizing SmSULT- OR with OXA, the mode of action of OXA was determined. This information allowed a rational approach to novel drug design. Our team approach with schistosome biologists, medicinal chemists, structural biologists and geneticists has enabled us to develop and test novel drug derivatives of OXA to treat this disease. Using an iterative process for drug development, we have successfully identified derivatives that are effective against all three species of the parasite. One derivative CIDD-0149830 kills 100% of all three human schistosome species within 5 days. The goal is to generate a second therapeutic with a different mode of action that can be used in conjunction with praziquantel to overcome the ever-growing threat of resistance and improve efficacy. The ability and need to design, screen, and develop future, affordable therapeutics to treat human schistosomiasis is critical for successful control program outcomes.

During schistosomiasis, the paired Schistosoma mansoni female produces about 300 eggs each day. These eggs are responsible for the clinical picture and the transmission of the disease. During female development and egg production, fs800 is expressed only in female vitelline cells. Blast search of fs800 did not show similarities with any published sequences by NCBI. We hypothesize that the product of this gene plays a role in S. mansoni egg production. By using RNA interference to knockdown fs800 and quantitative PCR to measure the gene expression in the female schistosomes, we were able to demonstrate that fs800 product is crucial for viable egg production, it has no effect on worm health or male-female pairing. Our data suggest fs800 inhibition as a potential target to prevent transmission and pathology of schistosomiasis.

Human schistosomiasis is a neglected tropical disease caused by parasitic worms. It affects over 250 million people globally. Most human infections are caused by S. mansoni, S. haematobium, and S. japonicum. Currently there is only one method of treatment for human schistosomiasis, the drug praziquantel. Constant selection pressure has caused a serious concern for a rise in resistance to praziquantel leading to the necessity for additional pharmaceuticals, with a distinctly different mechanism of action, to be used in combination therapy with praziquantel.Previous treatment of Schistosoma mansoni included the use of oxamniquine (OXA), a prodrug that is enzymatically activated by a Sulfotransferase, an enzyme produced by S. mansoni (SmSULT). Although sulfotransferases are produced by S. haematobium and S. japonicum, OXA is not effective against these two species. By using information from the crystal structure of SmSULT bound to OXA, 250 OXA derivatives were designed and tested in vitro against the adult parasites. We were able to identify effective derivatives that kill Schistosoma mansoni (85%), S. haematobium (40%) and S. japonicum (83%). Recently, we identified CIDD‐149830 as an effective derivative that can kill all three schistosome species (100%) within 7 days in vitro. In vivo studies results show a significant reduction in the number of harvested worms from infected animals after treating them with CIDD‐149830.In this study, we re‐engineered and tested 42 derivatives based on the structure of CIDD‐149830 and we were able to identify CIDD‐0150610 as a very powerful derivative that kills 100 % of Schistosoma mansoni overnight in vitro.Support or Funding InformationR01 AI115691

Currently there is only one method of treatment for human schistosomiasis, the drug praziquantel. Strong selective pressure has caused a serious concern for a rise in resistance to praziquantel leading to the necessity for additional pharmaceuticals, with a distinctly different mechanism of action, to be used in combination therapy with praziquantel. Previous treatment of Schistosoma mansoni included the use of oxamniquine (OXA), a prodrug that is enzymatically activated in S. mansoni but is ineffective against S. haematobium and S. japonicum. The oxamniquine activating enzyme was identified as a S. mansoni sulfotransferase (SmSULT-OR). Structural data have allowed for directed drug development in reengineering oxamniquine to be effective against S. haematobium and S. japonicum. Guided by data from X-ray crystallographic studies and Schistosoma worm killing assays on oxamniquine, our structure-based drug design approach produced a robust SAR program that tested over 300 derivatives and identified several new lead compounds with effective worm killing in vitro. Previous studies resulted in the discovery of compound CIDD-0066790, which demonstrated broad-species activity in killing of schistosome species. As these compounds are racemic mixtures, we tested and demonstrate that the R enantiomer CIDD-007229 kills S. mansoni, S. haematobium and S. japonicum better than the parent drug (CIDD-0066790). The search for derivatives that kill better than CIDD-0066790 has resulted in a derivative (CIDD- 149830) that kills 100% of S. mansoni, S. haematobium and S. japonicum adult worms within 7 days. We hypothesize that the difference in activation and thus killing by the derivatives is due to the ability of the derivative to fit in the binding pocket of each sulfotransferase (SmSULT-OR, ShSULT-OR, SjSULT-OR) and to be efficiently sulfated. The purpose of this research is to develop a second drug to be used in conjunction with praziquantel to treat the major human species of Schistosoma. Collectively, our findings show that CIDD-00149830 and CIDD-0072229 are promising novel drugs for the treatment of human schistosomiasis and strongly support further development and in vivo testing.