Kailash C. Pandey

Vinyl Sulfones as Antiparasitic Agents and a Structural Basis for Drug Design

Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.

Structures of falcipain-2 and falcipain-3 bound to small molecule inhibitors: implications for substrate specificity.

Falcipain-2 and falcipain-3 are critical hemoglobinases of Plasmodium falciparum, the most virulent human malaria parasite. We have determined the 2.9 Å crystal structure of falcipain-2 in complex with the epoxysuccinate E64 and the 2.5 Å crystal structure of falcipain-3 in complex with the aldehyde leupeptin. These complexes represent the first crystal structures of plasmodial cysteine proteases with small molecule inhibitors and the first reported crystal structure of falcipain-3. Our structural analyses indicate that the relative shape and flexibility of the S2 pocket are affected by a number of discrete amino acid substitutions. The cumulative effect of subtle differences, including those at “gatekeeper” positions, may explain the observed kinetic differences between these two closely related enzymes.

Falstatin, a cysteine protease inhibitor of Plasmodium falciparum, facilitates erythrocyte invasion.

Erythrocytic malaria parasites utilize proteases for a number of cellular processes, including hydrolysis of hemoglobin, rupture of erythrocytes by mature schizonts, and subsequent invasion of erythrocytes by free merozoites. However, mechanisms used by malaria parasites to control protease activity have not been established. We report here the identification of an endogenous cysteine protease inhibitor of Plasmodium falciparum, falstatin, based on modest homology with the Trypanosoma cruzi cysteine protease inhibitor chagasin. Falstatin, expressed in Escherichia coli, was a potent reversible inhibitor of the P. falciparum cysteine proteases falcipain-2 and falcipain-3, as well as other parasite- and nonparasite-derived cysteine proteases, but it was a relatively weak inhibitor of the P. falciparum cysteine proteases falcipain-1 and dipeptidyl aminopeptidase 1. Falstatin is present in schizonts, merozoites, and rings, but not in trophozoites, the stage at which the cysteine protease activity of P. falciparum is maximal. Falstatin localizes to the periphery of rings and early schizonts, is diffusely expressed in late schizonts and merozoites, and is released upon the rupture of mature schizonts. Treatment of late schizionts with antibodies that blocked the inhibitory activity of falstatin against native and recombinant falcipain-2 and falcipain-3 dose-dependently decreased the subsequent invasion of erythrocytes by merozoites. These results suggest that P. falciparum requires expression of falstatin to limit proteolysis by certain host or parasite cysteine proteases during erythrocyte invasion. This mechanism of regulation of proteolysis suggests new strategies for the development of antimalarial agents that specifically disrupt erythrocyte invasion.

Identification and biochemical characterization of vivapains, cysteine proteases of the malaria parasite Plasmodium vivax

Cysteine proteases play important roles in the life cycles of malaria parasites. Cysteine protease inhibitors block haemoglobin hydrolysis and development in Plasmodium falciparum, suggesting that the cysteine proteases of this major human pathogen, termed falcipains, are appropriate therapeutic targets. To expand our understanding of plasmodial proteases to Plasmodium vivax, the other prevalent human malaria parasite, we identified and cloned genes encoding the P. vivax cysteine proteases, vivapain-2 and vivapain-3, and functionally expressed the proteases in Escherichia coli. The vivapain-2 and vivapain-3 genes predicted papain-family cysteine proteases, which shared a number of unusual features with falcipain-2 and falcipain-3, including large prodomains and short N-terminal extensions on the catalytic domain. Recombinant vivapain-2 and vivapain-3 shared properties with the falcipains, including acidic pH optima, requirements for reducing conditions for activity and hydrolysis of substrates with positively charged residues at P1 and Leu at P2. Both enzymes hydrolysed native haemoglobin at acidic pH and the erythrocyte cytoskeletal protein 4.1 at neutral pH, suggesting similar biological roles to the falcipains. Considering inhibitor profiles, the vivapains were inhibited by fluoromethylketone and vinyl sulphone inhibitors that also inhibited falcipains and have demonstrated potent antimalarial activity.

Salivary glands harbor more diverse microbial communities than gut in Anopheles culicifacies

BackgroundIn recent years, it has been well documented that gut flora not only influence mosquito physiology, but also significantly alter vector competency. Although, salivary gland and gut constitute key partners of the digestive system, it is still believed that salivary glands may harbor less flora than gut (Parasit Vectors 6: 146, 2013).MethodsUsing a metagenomic approach, we have identified for the first time the diverse microbial community associated with these two physiologically different tissues of the digestive system in the mosquito Anopheles culicifacies.ResultsA total of 17 different phyla could be assigned to the whole metagenomic dataset, predominated by the phylum Proteobacteria, Firmicutes, Bacteriodetes, Tenericutes and Actinomycetes. Common bacteria included the members of Enhydrobacter, Agromonas, Serratia, Ralsonia, Lactobacillus, Pseudomonas, Streptococcus, Rubrobacter, Anaerococcus, Methylobacterium, Turicibacter, Elizabethkingia etc. in both the tissues representing ‘core microbiota’ of the mosquito digestive system. Salivary associated unique bacterial community included the members of Chloriflexi, Chlorobi, Cyanobacteria, Nitrospira, TM7, Armatimonadetes, Planctomycetes, Fibrobacteres etc.ConclusionWe find that the salivary gland microbial community structure is more diverse than gut of the mosquito, probably due to differential feeding associated engagements such as food acquisition, ingestion and digestion processes.

Regulatory Elements within the Prodomain of Falcipain-2, a Cysteine Protease of the Malaria Parasite Plasmodium falciparum

Falcipain-2, a papain family cysteine protease of the malaria parasite Plasmodium falciparum, plays a key role in parasite hydrolysis of hemoglobin and is a potential chemotherapeutic target. As with many proteases, falcipain-2 is synthesized as a zymogen, and the prodomain inhibits activity of the mature enzyme. To investigate the mechanism of regulation of falcipain-2 by its prodomain, we expressed constructs encoding different portions of the prodomain and tested their ability to inhibit recombinant mature falcipain-2. We identified a C-terminal segment (Leu155–Asp243) of the prodomain, including two motifs (ERFNIN and GNFD) that are conserved in cathepsin L sub-family papain family proteases, as the mediator of prodomain inhibitory activity. Circular dichroism analysis showed that the prodomain including the C-terminal segment, but not constructs lacking this segment, was rich in secondary structure, suggesting that the segment plays a crucial role in protein folding. The falcipain-2 prodomain also efficiently inhibited other papain family proteases, including cathepsin K, cathepsin L, cathepsin B, and cruzain, but it did not inhibit cathepsin C or tested proteases of other classes. A structural model of pro-falcipain-2 was constructed by homology modeling based on crystallographic structures of mature falcipain-2, procathepsin K, procathepsin L, and procaricain, offering insights into the nature of the interaction between the prodomain and mature domain of falcipain-2 as well as into the broad specificity of inhibitory activity of the falcipain-2 prodomain.

Structure-Function of Falcipains: Malarial Cysteine Proteases

Evidence indicates that cysteine proteases play essential role in malaria parasites; therefore an obvious area of investigation is the inhibition of these enzymes to treat malaria. Studies with cysteine protease inhibitors and manipulating cysteine proteases genes have suggested a role for cysteine proteases in hemoglobin hydrolysis. The best characterized Plasmodium cysteine proteases are falcipains, which are papain family enzymes. Falcipain-2 and falcipain-3 are major hemoglobinases of P. falciparum. Structural and functional analysis of falcipains showed that they have unique domains including a refolding domain and a hemoglobin binding domain. Overall, the complexes of falcipain-2 and falcipain-3 with small and macromolecular inhibitors provide structural insight to facilitate the design or modification of effective drug treatment against malaria. Drug development targeting falcipains should be aided by a strong foundation of biochemical and structural studies.

The Ionic and Hydrophobic Interactions Are Required for the Auto Activation of Cysteine Proteases of Plasmodium falciparum

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 are major hemoglobinases and potential antimalarial drug targets. Our previous studies demonstrated that these enzymes are equipped with specific domains for specific functions. Structural and functional analysis of falcipains showed that they have unique domains including a refolding domain and a hemoglobin binding domain. As with many proteases, falcipain-2 and falcipain-3 are synthesized as inactive zymogens. However, it is not known how these enzymes get activated for hemoglobin hydrolysis. In this study, we are presenting the first evidence that salt bridges and hydrophobic interactions are required for the auto activation of cysteine proteases of P.falciparum. To investigate the mechanism of activation of these enzymes, we expressed the wild type protein as well as different mutants in E.coli. Refolding was assessed by circular dichroism. Both CD and trans activation data showed that the wild type enzymes and mutants are rich in secondary structures with similar folds. Our study revealed that prodomain-mature domain of falcipain-2 and falcipain-3 interacts via salt bridges and hydrophobic interactions. We mutated specific residues of falcipain-2 and falcipain-3, and evaluated their ability to undergo auto processing. Mutagenesis result showed that two salt bridges (Arg 185 - Glu 221, Glu 210 - Lys 403) in falcipain-2, and one salt bridge (Arg 202-Glu 238) in falcipain-3, play crucial roles in the activation of these enzymes. Further study revealed that hydrophobic interactions present both in falcipain-2 (Phe214, Trp449 Trp 453) and falcipain-3 (Phe 231 Trp 457 Trp 461) also play important roles in the activation of these enzymes. Our results revealed the interactions involved in auto processing of two major hemoglobinases of malaria parasite.

Salivary gland transcriptome analysis in response to sugar feeding in malaria vector Anopheles stephensi

In this study, we analyzed a small scale transcriptome of salivary glands in sugar fed female mosquitoes. Thirty five percent of the transcripts could not be assigned a function. Some of them may code for salivary gland specific products involved in sugar feeding. We identified and characterized two new putative cDNAs encoding a sugar transporter and a cAMP generating DAPIT (Diabetes-Associated proteins in insulin sensitive tissues). Down regulation of these two cDNAs in response to blood feeding suggest that both AsST and AsDAPIT salivary genes may specifically be involved in the facilitation of sugar metabolism and energy production. The inability to absorb or digest sugar may cause organ failure, improper functioning of nervous system, behavioral disorder and death. Further functional characterization of theses putative transcripts is under investigation to examine their role in the mosquito salivary glands.

Design, synthesis and biological evaluation of functionalized phthalimides: A new class of antimalarials and inhibitors of falcipain-2, a major hemoglobinase of malaria parasite

Phthalimides functionalized with cyclic amines were synthesized, characterized and screened for their in vitro antimalarial efficacy against Plasmodium falciparum (Pf3D7). Of all the listed phthalimides evaluated, 14 and 24 were identified as potent antimalarial agents as advocated by assessment of their ability to inhibit [(3)H] hypoxanthine incorporation in the nucleic acid of parasites. In addition, phthalimides 14 and 24 were incubated for 60 and 90h and an enhanced antimalarial effect was noticed with increase in time to great extent. A reduction in IC50 values was observed with increase in exposure time of the parasite to the compounds. A symmetric phthalimide, 24 possessing piperazine as linker unit was identified as the most potent antimalarial agent with IC50 values of 5.97±0.78, 2.0±1.09 and 1.1±0.75μM on incubation period of 42, 60 and 90h, respectively. The abnormal morphologies such as delay in developmental stages, growth arrest and condensed nuclei of parasite were observed with the aid of microscopic studies upon exposure with 14 and 24. The evaluation of 14 and 24 against chloroquine resistant strain, (Pf7GB) of P. falciparum afforded IC50 values, 13.29±1.20 and 7.21±0.98μM, respectively. The combination of 24 with artemisinin (ART) showed enhanced killing of parasite against Pf3D7. Further, all phthalimides were evaluated for their activity against falcipain-2 (FP2), a major hemoglobinase of malarial parasite. The enzymatic assay afforded 6 as most active member against FP2. To the best of our knowledge this is the initial study represents phthalimide protected amino acids functionalized with cyclic amines as potent antimalarial agents.