Structure Elucidation and Characterization of the Venus Kinase Receptors of a Philippine Isolate of Schistosoma japonicum

Worldwide, millions of people suffer from the parasitic disease called schistosomiasis. This disease is caused by the digenetic trematode of the genus Schistosoma. The disease is endemic in the Philippines. Schistosomiasis is a parasitic disease caused by blood dwelling worms. This disease is spread by freshwater snails infected with the human infective stage of the parasite called schistosome cercariae. The disease is characterized by morbidities like inflammation of the liver and the spleen. Schistosomiasis is endemic in the Philippines. Due to its morbidities, economic loss amount to millions of Philippine Peso per year. Two and a half million people have active infections, posing 12 million individuals at risk of infection. In the Philippines, S. japonicum is the only causative agent of schistosomiasis. The disease is widespread in 12 Regions, corresponding to 28 Provinces. Schistosomiasis control program in the Philippines include mass drug administration. Endemic places with high disease prevalence rates are treated with a single drug, praziquantel. A single dose is given once annually or biannually. However, resistance to the drug poses legitimate fear. Under laboratory conditions, S. mansoni worms treated with subcurative drug concentration developed resistance against praziquantel. Moreover, in Senegal (1988), mass drug administration of praziquantel failed to meet the expected parasitological cure rate of 70-90%. The disease pathologies are caused by schistosome eggs deposited in different organs in the human host. Schistosome eggs are the causative agents of schistosomiasis. As such, interventions of schistosome egg production may lead to reduction of disease burden and transmission control. In recent years, the discovery of Venus Kinase Receptors (VKRs) in S. mansoni opened new directions in schistosomiasis research. It has been found that VKRs are directly involved in egg production. Thus, inhibition of VKR proteins can be exploited to develop anti-schistosomiasis therapeutics.
In order to develop new drugs active against schistosome worms, molecular understanding of VKR proteins will be needed. This proposal aims to undertake this complex task by employing cutting edge science and technology in the field of proteomics, drug design and membrane protein crystallography. We will express and purify the recombinant form of VKR proteins in either E. coli, S. cerivisiae or Bacullovirus expression systems. We will employ both experimental and computational approches to charcaterize VKR proteins in terms of their dimerization potential, ligand binding, and inhibitor interactions. Important to VKR inhibitor development is high resolution structure of VKR proteins. We will attempt to crystallize VKR proteins with and without their putative liagnds and inhibitors. The VKR structure will be used to screen small molecule VKR inhibitors, and will be tested for binding to VKR proteins and phosphorylase activity.
The impact of anti-schistosomiasis drug development takes years to deliver to end users. But, this proposal is a huge leap towards its realization. The immediate beneficiaries of study outcomes will be the academic groups, allowing them to access free data of the 3D structure of VKR proteins. With VKR coordinates available, simultaneous work on the development of anti-schistosomiasis drugs worldwide will be facilitated. Lead compounds identified in the study can be exploited by pharmaceutical companies to speed up delivery of impact to suffering individiduals due to schistosomiasis.

Aligned with UK's official development assistance programme, we propose and seek funding for a study aimed to develop novel anti-schistosome fecundity therapeutics through combined experimental and computational approaches. We will produce, characterise, and elucidate the structure of Venus Kinase Receptors (VKRs) from Philippine isolate of S. japonicum. Through computer-aided drug design, we will screen small molecule inhibitors of VKR proteins and test lead compounds on viable schistosome worms in vitro. We will solve the crystal structure of the VKRs. S. mansoni VKRs are activated by different ligands, L-arginine and calcium ions. We will be performing ligand binding assays using ITC and/or SPR spectroscopy to probe their binding affinities. We will perform Molecular Dynamics simulations of the whole VKR proteins with and without their respective ligands. Key interactions between the ligand binding site and the ligand will provide hints to the understanding of receptor activation and signalling in addition to the crystal structures. Through computer-aided drug design, we will screen small molecule inhibitors of VKR proteins and test lead compounds using kinase activity assay and mass spectrometry.
To carry out this complex task, our team will be composed of expertise in different specializations: molecular parasitology (Dr. Leonardo), molecular biology (Dr. Bascos), biochemistry (Dr. Yu-Roberto), structural biology (Dr. Beis), synthetic organic chemistry (Dr. Sabido), molecular biophysics (Dr. Nellas), and computational chemistry (Mr. Gomez). This study is collaborative in nature, a partnership between UK and the Philippines, allowing capacity building in the Philippines. Thus, we seek funding for procurement of instrument and training of personnel in membrane protein production and X-ray crystallography.

Schistosomiasis is a parasitic disease commonly spread in the Philippines by infected fresh water snails. Statistics shows the probability of contracting the disease is proportional to the frequency of exposure to infected waters. Moreover, the disease prevalence is higher in males than in females, owing to the propensity of males to work in agricultural and aquatic fields. The farmers are the most vulnerable group of people to contract the disease, followed by fishermen, unskilled laborers, and other job categories. Schistosomiasis has greatly affected the national agricultural output due to loss of working capacity of infected individuals. As such, this proposal aims to isolate and understand an important schistosome proteins called venus kinase receptors (VKR) in the hope of developing anti-schistosome drugs. Overall, in-depth understanding of the peculiarity of these proteins could potentially lead to the development of novel therapeutics for the control and management of schistosomiasis.
While many studies have been done to understand VKRs role in spreading schistosomiasis, no VKR has been isolated and characterized from species present in the Philippines. This proposal will holistically bridge that gap by employing both in vivo and in silico approaches. Due to the advent of powerful computer and smart algorithms, protein structure exploration through molecular dynamics studies and computer-aided drug design through electronic calculations or high-throughput ligand docking are now within reach. In order to accomplish our goal, a high-resolution structure elucidation of VKR proteins is needed in vitro. Since the structure coordinates will be made available to the public, rational drug design can be explored simultaneously worldwide. In addition, results here may be exploited by pharmaceutical companies, i.e. they can optimize lead compounds and develop cost-effective drugs with less side-effects.