Dataset Information

Identification of Inhibitors of the Schistosoma mansoni VKR2 Kinase Domain.

ABSTRACT: Schistosomiasis is a neglected tropical disease caused by parasitic flatworms. Current treatment relies on just one partially effective drug, praziquantel (PZQ). Schistosoma mansoni Venus Kinase Receptors 1 and 2 (SmVKR1 and SmVKR2) are important for parasite growth and egg production, and are potential targets for combating schistosomiasis. VKRs consist of an extracellular Venus Flytrap Module (VFTM) linked via a transmembrane helix to a kinase domain. Here, we initiated a drug discovery effort to inhibit the activity of the SmVKR2 kinase domain (SmVKR2_KD) by screening the GSK published kinase inhibitor set 2 (PKIS2). We identified several inhibitors, of which four were able to inhibit its enzymatic activity and induced phenotypic changes in ex vivo S. mansoni. Our crystal structure of the SmVKR2_KD displays an active-like state that sheds light on the activation process of VKRs. Our data provide a basis for the further exploration of SmVKR2 as a possible drug target.

SUBMITTER: Mathavan I

PROVIDER: S-EPMC9661718 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Identification of Inhibitors of the <i>Schistosoma mansoni</i> VKR2 Kinase Domain.

Mathavan Indran I Liu Lawrence J LJ Robinson Sean W SW El-Sakkary Nelly N Elatico Adam Jo J AJJ Gomez Darwin D Nellas Ricky R Owens Raymond J RJ Zuercher William W Navratilova Iva I Caffrey Conor R CR Beis Konstantinos K

ACS medicinal chemistry letters 20221005 11

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms. Current treatment relies on just one partially effective drug, praziquantel (PZQ). <i>Schistosoma mansoni</i> Venus Kinase Receptors 1 and 2 (SmVKR1 and SmVKR2) are important for parasite growth and egg production, and are potential targets for combating schistosomiasis. VKRs consist of an extracellular Venus Flytrap Module (VFTM) linked via a transmembrane helix to a kinase domain. Here, we initiated a drug discovery ...[more]

PMID: 36385939

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Project description:BackgroundMicroRNAs (miRNAs) constitute a class of single-stranded RNAs which play a crucial role in regulating development and controlling gene expression by targeting mRNAs and triggering either translation repression or messenger RNA (mRNA) degradation. miRNAs are widespread in eukaryotes and to date over 14,000 miRNAs have been identified by computational and experimental approaches. Several miRNAs are highly conserved across species. In Schistosoma, the full set of miRNAs and their expression patterns during development remain poorly understood. Here we report on the development and implementation of a homology-based detection strategy to search for miRNA genes in Schistosoma mansoni. In addition, we report results on the experimental detection of miRNAs by means of cDNA cloning and sequencing of size-fractionated RNA samples.ResultsHomology search using the high-throughput pipeline was performed with all known miRNAs in miRBase. A total of 6,211 mature miRNAs were used as reference sequences and 110 unique S. mansoni sequences were returned by BLASTn analysis. The existing mature miRNAs that produced these hits are reported, as well as the locations of the homologous sequences in the S. mansoni genome. All BLAST hits aligned with at least 95% of the miRNA sequence, resulting in alignment lengths of 19-24 nt. Following several filtering steps, 15 potential miRNA candidates were identified using this approach. By sequencing small RNA cDNA libraries from adult worm pairs, we identified 211 novel miRNA candidates in the S. mansoni genome. Northern blot analysis was used to detect the expression of the 30 most frequent sequenced miRNAs and to compare the expression level of these miRNAs between the lung stage schistosomula and adult worm stages. Expression of 11 novel miRNAs was confirmed by northern blot analysis and some presented a stage-regulated expression pattern. Three miRNAs previously identified from S. japonicum were also present in S. mansoni.ConclusionEvidence for the presence of miRNAs in S. mansoni is presented. The number of miRNAs detected by homology-based computational methods in S. mansoni is limited due to the lack of close relatives in the miRNA repository. In spite of this, the computational approach described here can likely be applied to the identification of pre-miRNA hairpins in other organisms. Construction and analysis of a small RNA library led to the experimental identification of 14 novel miRNAs from S. mansoni through a combination of molecular cloning, DNA sequencing and expression studies. Our results significantly expand the set of known miRNAs in multicellular parasites and provide a basis for understanding the structural and functional evolution of miRNAs in these metazoan parasites.

Project description:The Venus kinase receptor (VKR) is a single transmembrane molecule composed of an intracellular tyrosine kinase domain close to that of insulin receptor and an extracellular Venus Flytrap (VFT) structure similar to the ligand binding domain of many class C G protein coupled receptors. This receptor tyrosine kinase (RTK) was first discovered in the platyhelminth parasite Schistosoma mansoni, then in a large variety of invertebrates. A single vkr gene is found in most genomes, except in S. mansoni in which two genes Smvkr1 and Smvkr2 exist. VKRs form a unique family of RTKs present only in invertebrates and their biological functions are still to be discovered. In this work, we show that SmVKRs are expressed in the reproductive organs of S. mansoni, particularly in the ovaries of female worms. By transcriptional analyses evidence was obtained that both SmVKRs fulfill different roles during oocyte maturation. Suppression of Smvkr expression by RNA interference induced spectacular morphological changes in female worms with a strong disorganization of the ovary, which was dominated by the presence of primary oocytes, and a defect of egg formation. Following expression in Xenopus oocytes, SmVKR1 and SmVKR2 receptors were shown to be activated by distinct ligands which are L-Arginine and calcium ions, respectively. Signalling analysis in Xenopus oocytes revealed the capacity of SmVKRs to activate the PI3K/Akt/p70S6K and Erk MAPK pathways involved in cellular growth and proliferation. Additionally, SmVKR1 induced phosphorylation of JNK (c-Jun N-terminal kinase). Activation of JNK by SmVKR1 was supported by the results of yeast two-hybrid experiments identifying several components of the JNK pathway as specific interacting partners of SmVKR1. In conclusion, these results demonstrate the functions of SmVKR in gametogenesis, and particularly in oogenesis and egg formation. By eliciting signalling pathways potentially involved in oocyte proliferation, growth and migration, these receptors control parasite reproduction and can therefore be considered as potential targets for anti-schistosome therapies.

Project description:Schistosomiasis is a parasitic neglected disease with praziquantel (PZQ) utilized as the main drug for treatment, despite its low effectiveness against early stages of the worm. To aid in the search for new drugs to tackle schistosomiasis, computer-aided drug design has been proved a helpful tool to enhance the search and initial identification of schistosomicidal compounds, allowing fast and cost-efficient progress in drug discovery. The combination of high-throughput in silico data followed by in vitro phenotypic screening assays allows the assessment of a vast library of compounds with the potential to inhibit a single or even several biological targets in a more time- and cost-saving manner. Here, we describe the molecular docking for in silico screening of predicted homology models of five protein kinases (JNK, p38, ERK1, ERK2, and FES) of Schistosoma mansoni against approximately 85,000 molecules from the Managed Chemical Compounds Collection (MCCC) of the University of Nottingham (UK). We selected 169 molecules predicted to bind to SmERK1, SmERK2, SmFES, SmJNK, and/or Smp38 for in vitro screening assays using schistosomula and adult worms. In total, 89 (52.6%) molecules were considered active in at least one of the assays. This approach shows a much higher efficiency when compared to using only traditional high-throughput in vitro screening assays, where initial positive hits are retrieved from testing thousands of molecules. Additionally, when we focused on compound promiscuity over selectivity, we were able to efficiently detect active compounds that are predicted to target all kinases at the same time. This approach reinforces the concept of polypharmacology aiming for "one drug-multiple targets". Moreover, at least 17 active compounds presented satisfactory drug-like properties score when compared to PZQ, which allows for optimization before further in vivo screening assays. In conclusion, our data support the use of computer-aided drug design methodologies in conjunction with high-throughput screening approach.

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Identification of Inhibitors of the Schistosoma mansoni VKR2 Kinase Domain.

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Identification of Inhibitors of the <i>Schistosoma mansoni</i> VKR2 Kinase Domain.

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Identification of Inhibitors of the Schistosoma mansoni VKR2 Kinase Domain.

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Identification of Inhibitors of the &lt;i&gt;Schistosoma mansoni&lt;/i&gt; VKR2 Kinase Domain.

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Identification of Inhibitors of the <i>Schistosoma mansoni</i> VKR2 Kinase Domain.