Identification of potential inhibitors of casein kinase 2 alpha of Plasmodium falciparum with potent in vitro activity.

Drug resistance to commercially available antimalarials is a major obstacle in malaria control and elimination, creating the need to find new antiparasitic compounds with novel mechanisms of action. The success of kinase inhibitors for oncological treatments has paved the way for the exploitation of protein kinases as drug targets in various diseases, including malaria. Casein kinases are ubiquitous serine/threonine kinases involved in a wide range of cellular processes such as mitotic checkpoint signaling, DNA damage response, and circadian rhythm. In Plasmodium, it is suggested that these protein kinases are essential for both asexual and sexual blood-stage parasites, reinforcing their potential as targets for multi-stage antimalarials. To identify new putative PfCK2α inhibitors, we utilized an in silico chemogenomic strategy involving virtual screening with docking simulations and quantitative structure-activity relationship predictions. Our investigation resulted in the discovery of a new quinazoline molecule (542), which exhibited potent activity against asexual blood stages and a high selectivity index (>100). Subsequently, we conducted chemical-genetic interaction analysis on yeasts with mutations in casein kinases. Our chemical-genetic interaction results are consistent with the hypothesis that 542 inhibits yeast Cka1, which has a hinge region with high similarity to PfCK2α. This finding is in agreement with our in silico results suggesting that 542 inhibits PfCK2α via hinge region interaction.

Influence of trem-1 gene polymorphisms on cytokine levels during malaria by Plasmodium vivax in a frontier area of the Brazilian Amazon.

BACKGROUND: The immunopathology during malaria depends on the level of inflammatory response generated. In this scenario, the TREM-1 has been associated with the severity of infectious diseases and could play an important role in the inflammatory course of malaria. We aimed to describe the allelic and genotypic frequency of four polymorphisms in the trem-1 gene in Plasmodium vivax-infected patients and to verify the association of these polymorphisms with clinical and immunological factors in a frontier area of the Brazilian Amazon. METHODS: We included 76 individuals infected with P. vivax and 144 healthy controls living in the municipality of Oiapoque, Amapá, Brazil. The levels of TNF-α, IL-10, IL-2, IL-4, IL-5, and IFN-γ were measured by flow cytometry, while IL-6, sTREM-1, and antibodies against PvMSP-119 were evaluated by ELISA. The SNPs were genotyped by qPCR technique. Polymorphisms analysis, allelic and genotype, frequencies, and HWE calculation were determined by x2 test in R Software. The association between the parasitemia, gametocytes, antibodies, cytokines, and sTREM-1 with the genotypes of malaria and control groups was performed using the Kruskal-Wallis test, these analyzes were conducted in SPSS Software, at 5% significance level. RESULTS: All SNPs were successfully genotyped. Allelic and genotypic distribution was in Hardy-Weinberg Equilibrium. Furthermore, several associations were identified between malaria and control groups, with increased levels of IL-5, IL-6, IL-10, TNF-α, and IFN-γ in the infected individuals with rs6910730A, rs2234237T, rs2234246T, rs4711668C alleles compared to the homozygous wild-type and heterozygous genotypes of the controls (p-value < 0.05). No association was found for these SNPs and the levels of IL-2, and sTREM-1. CONCLUSIONS: The SNPs on the trem-1 gene are associated with the effector molecules of the innate immunity and may contribute to the identification and effective participation of trem-1 in the modulation of the immune response. This association may be essential for the establishment of immunization strategies against malaria.

Polymorphisms in Toll-Like receptors genes and their associations with immunological parameters in Plasmodium vivax malaria in the Brazil-French Guiana Border.

BACKGROUND: The innate immune response plays an important role during malaria. Toll-like receptors (TLR) are capable of recognizing pathogen molecules. We aimed to evaluate five polymorphisms in TLR-4, TLR-6, and TLR-9 genes and their association with cytokine levels and clinical parameters in malaria from the Brazil-French Guiana border. METHODS: A case-control study was conducted in Amapá, Brazil. P. vivax patients and individuals not infected were evaluated. Genotyping of five SNPs was carried out by qPCR. Circulating cytokines were measured by CBA. The MSP-119 IgG antibodies were performed by ELISA. RESULTS: An association between TLR4 A299G with parasitemia was observed. There was an increase for IFN-ɤ, TNF-ɑ, IL-6, and IL-10 in the TLR-4 A299G and T3911, TLR-6 S249P, and TLR-9 1486C/T, SNPs for the studied malarial groups. There were significant findings for the TLR-4 variants A299G and T3911, TLR-9 1237C/T, and 1486C/T. For the reactivity of MSP-119 antibodies levels, no significant results were found in malaria, and control groups. CONCLUSIONS: The profile of the immune response observed by polymorphisms in TLRs genes does not seem to be standard for all types of malaria infection around the world. This can depend on the human population and the species of Plasmodium.

Artificial Intelligence Applied to the Rapid Identification of New Antimalarial Candidates with Dual-Stage Activity.

Increasing reports of multidrug-resistant malaria parasites urge the discovery of new effective drugs with different chemical scaffolds. Protein kinases play a key role in many cellular processes such as signal transduction and cell division, making them interesting targets in many diseases. Protein kinase 7 (PK7) is an orphan kinase from the Plasmodium genus, essential for the sporogonic cycle of these parasites. Here, we applied a robust and integrative artificial intelligence-assisted virtual-screening (VS) approach using shape-based and machine learning models to identify new potential PK7 inhibitors with in vitro antiplasmodial activity. Eight virtual hits were experimentally evaluated, and compound LabMol-167 inhibited ookinete conversion of Plasmodium berghei and blood stages of Plasmodium falciparum at nanomolar concentrations with low cytotoxicity in mammalian cells. As PK7 does not have an essential role in the Plasmodium blood stage and our virtual screening strategy aimed for both PK7 and blood-stage inhibition, we conducted an in silico target fishing approach and propose that this compound might also inhibit P. falciparum PK5, acting as a possible dual-target inhibitor. Finally, docking studies of LabMol-167 with P. falciparum PK7 and PK5 proteins highlighted key interactions for further hit-to lead optimization.

Deep Learning-driven research for drug discovery: Tackling Malaria.

Malaria is an infectious disease that affects over 216 million people worldwide, killing over 445,000 patients annually. Due to the constant emergence of parasitic resistance to the current antimalarial drugs, the discovery of new drug candidates is a major global health priority. Aiming to make the drug discovery processes faster and less expensive, we developed binary and continuous Quantitative Structure-Activity Relationships (QSAR) models implementing deep learning for predicting antiplasmodial activity and cytotoxicity of untested compounds. Then, we applied the best models for a virtual screening of a large database of chemical compounds. The top computational predictions were evaluated experimentally against asexual blood stages of both sensitive and multi-drug-resistant Plasmodium falciparum strains. Among them, two compounds, LabMol-149 and LabMol-152, showed potent antiplasmodial activity at low nanomolar concentrations (EC50 <500 nM) and low cytotoxicity in mammalian cells. Therefore, the computational approach employing deep learning developed here allowed us to discover two new families of potential next generation antimalarial agents, which are in compliance with the guidelines and criteria for antimalarial target candidates.

Integrative Multi-Kinase Approach for the Identification of Potent Antiplasmodial Hits.

Malaria is a tropical infectious disease that affects over 219 million people worldwide. Due to the constant emergence of parasitic resistance to the current antimalarial drugs, the discovery of new antimalarial drugs is a global health priority. Multi-target drug discovery is a promising and innovative strategy for drug discovery and it is currently regarded as one of the best strategies to face drug resistance. Aiming to identify new multi-target antimalarial drug candidates, we developed an integrative computational approach to select multi-kinase inhibitors for Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4) and protein kinase 6 (PK6). For this purpose, we developed and validated shape-based and machine learning models to prioritize compounds for experimental evaluation. Then, we applied the best models for virtual screening of a large commercial database of drug-like molecules. Ten computational hits were experimentally evaluated against asexual blood stages of both sensitive and multi-drug resistant P. falciparum strains. Among them, LabMol-171, LabMol-172, and LabMol-181 showed potent antiplasmodial activity at nanomolar concentrations (EC50 ≤ 700 nM) and selectivity indices >15 folds. In addition, LabMol-171 and LabMol-181 showed good in vitro inhibition of P. berghei ookinete formation and therefore represent promising transmission-blocking scaffolds. Finally, docking studies with protein kinases CDPK1, CDPK4, and PK6 showed structural insights for further hit-to-lead optimization studies.

Chalcones as a basis for computer-aided drug design: innovative approaches to tackle malaria.

Aim: Computer-aided drug design approaches were applied to identify chalcones with antiplasmodial activity. Methodology: The virtual screening was performed as follows: structural standardization of in-house database of chalcones; identification of potential Plasmodium falciparum protein targets for the chalcones; homology modeling of the predicted P. falciparum targets; molecular docking studies; and in vitro experimental validation. Results: Using these models, we prioritized 16 chalcones with potential antiplasmodial activity, for further experimental evaluation. Among them, LabMol-86 and LabMol-87 showed potent in vitro antiplasmodial activity against P. falciparum, while LabMol-63 and LabMol-73 were potent inhibitors of Plasmodium berghei progression into mosquito stages. Conclusion: Our results encourage the exploration of chalcones in hit-to-lead optimization studies for tackling malaria.

A new heparan sulfate from the mollusk Nodipecten nodosus inhibits merozoite invasion and disrupts rosetting and cytoadherence of Plasmodium falciparum.

BACKGROUND: Despite treatment with effective antimalarial drugs, the mortality rate is still high in severe cases of the disease, highlighting the need to find adjunct therapies that can inhibit the adhesion of Plasmodium falciparum-infected erythrocytes (Pf-iEs). OBJECTIVES: In this context, we evaluated a new heparan sulfate (HS) from Nodipecten nodosus for antimalarial activity and inhibition of P. falciparum cytoadhesion and rosetting. METHODS: Parasite inhibition was measured by SYBR green using a cytometer. HS was assessed in rosetting and cytoadhesion assays under static and flow conditions using Chinese hamster ovary (CHO) and human lymphatic endothelial cell (HLEC) cells expressing intercellular adhesion molecule-1 (ICAM1) and chondroitin sulfate A (CSA), respectively. FINDINGS: This HS inhibited merozoite invasion similar to heparin. Moreover, mollusk HS decreased cytoadherence of P. falciparum to CSA and ICAM-1 on the surface of endothelial cells under static and flow conditions. In addition, this glycan efficiently disrupted rosettes. CONCLUSIONS: These findings support a potential use for mollusk HS as adjunct therapy for severe malaria.

Hydroxyazole scaffold-based Plasmodium falciparum dihydroorotate dehydrogenase inhibitors: Synthesis, biological evaluation and X-ray structural studies.

Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) has been clinically validated as a target for antimalarial drug discovery, as a triazolopyrimidine class inhibitor (DSM265) is currently undergoing clinical development. Here, we have identified new hydroxyazole scaffold-based PfDHODH inhibitors belonging to two different chemical series. The first series was designed by a scaffold hopping strategy that exploits the use of hydroxylated azoles. Within this series, the hydroxythiadiazole 3 was identified as the best selective PfDHODH inhibitor (IC50 12.0 µM). The second series was designed by modulating four different positions of the hydroxypyrazole scaffold. In particular, hydroxypyrazoles 7e and 7f were shown to be active in the low µM range (IC50 2.8 and 5.3 µM, respectively). All three compounds, 3, 7e and 7f showed clear selectivity over human DHODH (IC50 > 200 µM), low cytotoxicity, and retained micromolar activity in P. falciparum-infected erythrocytes. The crystallographic structures of PfDHODH in complex with compounds 3 and 7e proved their binding mode, supplying essential data for future optimization of these scaffolds.

A new heparan sulfate from the mollusk Nodipecten nodosus inhibits merozoite invasion and disrupts rosetting and cytoadherence of Plasmodium falciparum

BACKGROUND Despite treatment with effective antimalarial drugs, the mortality rate is still high in severe cases of the disease, highlighting the need to find adjunct therapies that can inhibit the adhesion of Plasmodium falciparum-infected erythrocytes (Pf-iEs). OBJECTIVES In this context, we evaluated a new heparan sulfate (HS) from Nodipecten nodosus for antimalarial activity and inhibition of P. falciparum cytoadhesion and rosetting. METHODS Parasite inhibition was measured by SYBR green using a cytometer. HS was assessed in rosetting and cytoadhesion assays under static and flow conditions using Chinese hamster ovary (CHO) and human lymphatic endothelial cell (HLEC) cells expressing intercellular adhesion molecule-1 (ICAM1) and chondroitin sulfate A (CSA), respectively. FINDINGS This HS inhibited merozoite invasion similar to heparin. Moreover, mollusk HS decreased cytoadherence of P. falciparum to CSA and ICAM-1 on the surface of endothelial cells under static and flow conditions. In addition, this glycan efficiently disrupted rosettes. CONCLUSIONS These findings support a potential use for mollusk HS as adjunct therapy for severe malaria.

Genetic sequence characterization and naturally acquired immune response to Plasmodium vivax Rhoptry Neck Protein 2 (PvRON2).

BACKGROUND: The genetic diversity of malaria antigens often results in allele variant-specific immunity, imposing a great challenge to vaccine development. Rhoptry Neck Protein 2 (PvRON2) is a blood-stage antigen that plays a key role during the erythrocyte invasion of Plasmodium vivax. This study investigates the genetic diversity of PvRON2 and the naturally acquired immune response to P. vivax isolates. RESULTS: Here, the genetic diversity of PvRON21828-2080 and the naturally acquired humoral immune response against PvRON21828-2080 in infected and non-infected individuals from a vivax malaria endemic area in Brazil was reported. The diversity analysis of PvRON21828-2080 revealed that the protein is conserved in isolates in Brazil and worldwide. A total of 18 (19%) patients had IgG antibodies to PvRON21828-2080. Additionally, the analysis of the antibody response in individuals who were not acutely infected with malaria, but had been infected with malaria in the past indicated that 32 patients (33%) exhibited an IgG immune response against PvRON2. CONCLUSIONS: PvRON2 was conserved among the studied isolates. The presence of naturally acquired antibodies to this protein in the absence of the disease suggests that PvRON2 induces a long-term antibody response. These results indicate that PvRON2 is a potential malaria vaccine candidate.

QSAR-Driven Design and Discovery of Novel Compounds With Antiplasmodial and Transmission Blocking Activities.

Malaria is a life-threatening infectious disease caused by parasites of the genus Plasmodium, affecting more than 200 million people worldwide every year and leading to about a half million deaths. Malaria parasites of humans have evolved resistance to all current antimalarial drugs, urging for the discovery of new effective compounds. Given that the inhibition of deoxyuridine triphosphatase of Plasmodium falciparum (PfdUTPase) induces wrong insertions in plasmodial DNA and consequently leading the parasite to death, this enzyme is considered an attractive antimalarial drug target. Using a combi-QSAR (quantitative structure-activity relationship) approach followed by virtual screening and in vitro experimental evaluation, we report herein the discovery of novel chemical scaffolds with in vitro potency against asexual blood stages of both P. falciparum multidrug-resistant and sensitive strains and against sporogonic development of P. berghei. We developed 2D- and 3D-QSAR models using a series of nucleosides reported in the literature as PfdUTPase inhibitors. The best models were combined in a consensus approach and used for virtual screening of the ChemBridge database, leading to the identification of five new virtual PfdUTPase inhibitors. Further in vitro testing on P. falciparum multidrug-resistant (W2) and sensitive (3D7) parasites showed that compounds LabMol-144 and LabMol-146 demonstrated fair activity against both strains and presented good selectivity versus mammalian cells. In addition, LabMol-144 showed good in vitro inhibition of P. berghei ookinete formation, demonstrating that hit-to-lead optimization based on this compound may also lead to new antimalarials with transmission blocking activity.

Cytokine gene polymorphisms are not associated with anti-PvDBP, anti-PvAMA-1 or anti-PvMSP-119 IgG antibody levels in a malaria-endemic area of the Brazilian Amazon.

BACKGROUND: The immune response against Plasmodium vivax immunogenic epitopes is regulated by pro- and anti-inflammatory cytokines that determine antibody levels and class switching. Cytokine gene polymorphisms may be responsible for changes in the humoral immune response against malaria. The aim of this study was to evaluate whether polymorphisms in the TNFA, IFNG and IL10 genes would alter the levels of anti-PvAMA1, PvDBP and -PvMSP-119 IgG antibodies in patients with vivax malaria. METHODS: Samples from 90 vivax malaria-infected and 51 uninfected subjects from an endemic area of the Brazilian Amazon were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to identify polymorphisms of the genes TNFA (-1031T > C, -308G > A, -238G > A), IFNG (+874T > A) and IL10 (-819C > T, -592C > A). The levels of total IgG against PvAMA1, PvDBP and PvMSP-119 were determined using an enzyme-linked immunosorbent assay (ELISA). Associations between the polymorphisms and the antibody response were assessed by means of logistic regression models. RESULTS: No significant differences were found in the levels of IgG antibodies against the PvAMA-1, PvDBP or PvMSP-119 proteins in relation to the studied polymorphisms. CONCLUSIONS: Although no associations were found among the evaluated genotypes and alleles and anti-merozoite IgG class P. vivax antibody levels, this study helps elucidate the immunogenic profile involved in the humoral immune response in malaria.

Polymorphisms in B Cell Co-Stimulatory Genes Are Associated with IgG Antibody Responses against Blood-Stage Proteins of Plasmodium vivax.

The development of an effective immune response can help decrease mortality from malaria and its clinical symptoms. However, this mechanism is complex and has significant inter-individual variation, most likely owing to the genetic contribution of the human host. Therefore, this study aimed to investigate the influence of polymorphisms in genes involved in the costimulation of B-lymphocytes in the naturally acquired humoral immune response against proteins of the asexual stage of Plasmodium vivax. A total of 319 individuals living in an area of malaria transmission in the Brazilian Amazon were genotyped for four SNPs in the genes CD40, CD40L, BLYS and CD86. In addition, IgG antibodies against P. vivax apical membrane antigen 1 (PvAMA-1), Duffy binding protein (PvDBP) and merozoite surface protein 1 (PvMSP-119) were detected by ELISA. The SNP BLYS -871C>T was associated with the frequency of IgG responders to PvAMA-1 and PvMSP-119. The SNP CD40 -1C>T was associated with the IgG response against PvDBP, whereas IgG antibody titers against PvMSP-119 were influenced by the polymorphism CD86 +1057G>A. These data may help to elucidate the immunological aspects of vivax malaria and consequently assist in the design of malaria vaccines.

No evidence for association of the CD40, CD40L and BLYS polymorphisms, B-cell co-stimulatory molecules, with Brazilian endemic Plasmodium vivax malaria.

BACKGROUND: Plasmodium vivax is the most prevalent malaria species in Brazil. The parasite-host coevolutionary process can be viewed as an 'arms race', in which adaptive genetic changes in one are eventually matched by alterations in the other. METHODS: Following the candidate gene approach we analyzed the CD40, CD40L and BLYS genes that participate in B-cell co-stimulation, for associations with P. vivax malaria. The study sample included 97 patients and 103 controls. We extracted DNA using the extraction and purification commercial kit and identified the following SNPs: -1C > T in the CD40 gene, -726T > C in the CD40L gene and the -871C > T in the BLyS gene using PCR-RFLP. We analyzed the genotype and allele frequencies by direct counting. We also compared the observed with the expected genotype frequencies using the Hardy-Weinberg equilibrium. RESULTS: The allele and genotype frequencies for these SNPs did not differ statistically between patient and control groups. Gene-gene interactions were not observed between the CD40 and BLYS and between the CD40L and BLYS genes. Overall, the genes were in Hardy-Weinberg equilibrium. Significant differences were not observed among the frequencies of antibody responses against P. vivax sporozoite and erythrocytic antigens and the CD40 and BLYS genotypes. CONCLUSIONS: The results of this study show that, although the investigated CD40, CD40L and BLYS alleles differ functionally, this variation does not alter the functionality of the molecules in a way that would interfere in susceptibility to the disease. The variants of these genes may influence the clinical course rather than simply increase or decrease susceptibility.

Evaluation of the naturally acquired antibody immune response to the Pv200L N-terminal fragment of Plasmodium vivax merozoite surface protein-1 in four areas of the Amazon Region of Brazil.

Frequency and levels of IgG antibodies to an N-terminal fragment of the Plasmodium vivax MSP-1 (Pv200L) protein, in individuals naturally exposed to malaria in four endemic areas of Brazil, were evaluated by enzyme-linked immunosorbent assay. Plasma samples of 261 P. vivax-infected individuals from communities of Macapá, Novo Repartimento, Porto Velho, and Plácido de Castro in the Amazonian region with different malaria transmission intensities. A high mean number of studied individuals (89.3%) presented with antibodies to the Pv200L that correlated with the number of previous malaria infections; there were significant differences in the frequency of the responders (71.9-98.7) and in the antibody levels (1:200-1:51,200) among the four study areas. Results of this study provide evidence that Pv200L is a naturally immunogenic fragment of the PvMSP-1 and is associated with the degree of exposure to parasites. The fine specificity of antibodies to Pv200L is currently being assessed.

Plasmodium vivax circumsporozoite genotypes: a limited variation or new subspecies with major biological consequences?

BACKGROUND: Plasmodium vivax circumsporozoite variants have been identified in several geographical areas. The real implication of the genetic variation in this region of the P. vivax genome has been questioned for a long time. Although previous studies have observed significant association between VK210 and the Duffy blood group, we present here that evidences of this variation are limited to the CSP central portion. METHODS: The phylogenetic analyses were accomplished starting from the amplification of conserved domains of 18 SSU RNAr and Cyt B. The antibodies responses against the CSP peptides, MSP-1, AMA-1 and DBP were detected by ELISA, in plasma samples of individuals infected with two P. vivax CS genotypes: VK210 and P. vivax-like. RESULTS: These analyses of the two markers demonstrate high similarity among the P. vivax CS genotypes and surprisingly showed diversity equal to zero between VK210 and P. vivax-like, positioning these CS genotypes in the same clade. A high frequency IgG antibody against the N- and C-terminal regions of the P. vivax CSP was found as compared to the immune response to the R- and V- repetitive regions (p = 0.0005, Fisher's Exact test). This difference was more pronounced when the P. vivax-like variant was present in the infection (p = 0.003, Fisher's Exact test). A high frequency of antibody response against MSP-1 and AMA-1 peptides was observed for all P. vivax CS genotypes in comparison to the same frequency for DBP. CONCLUSIONS: This results target that the differences among the P. vivax CS variants are restrict to the central repeated region of the protein, mostly nucleotide variation with important serological consequences.

Plasmodium vivax circumsporozoite variants and Duffy blood group genotypes in the Brazilian Amazon region.

The circumsporozoite protein (CSP) of the Plasmodium vivax infective sporozoite is considered to be a major target for the development of recombinant malaria vaccines. The Duffy blood group molecule acts as the red blood cell receptor for P. vivax. We review the frequency of P. vivax CSP variants and report their association with the Duffy blood group genotypes from Brazilian Amazon patients carrying P. vivax malaria. Peripheral blood samples were collected from 155 P. vivax-infected individuals from five Brazilian malaria-endemic areas. The P. vivax CSP variants and the Duffy blood group genotypes were assessed using PCR/RFLP. In single infections, the VK210 variant was the commonest followed by the P. vivax-like variant. The typing of P. vivax indicated that the frequency of variants among the study areas was significantly different from one to another. This is the first detection of the VK247 and P. vivax-like variant in single infections in endemic areas of Brazil. Association of the CSP P. vivax variants with the heterozygous Duffy blood group system genotype was significant for VK210 single infection. These observations provide additional data on the Plasmodium-host interactions concerning the Duffy blood group and P. vivax capability of causing human malaria.