Cytoskeleton Organization in Formation and Motility of Apicomplexan Parasites.

Apicomplexan parasites are a group of eukaryotic protozoans with diverse biology that have affected human health like no other group of parasites. These obligate intracellular parasites rely on their cytoskeletal structures for giving them form, enabling them to replicate in unique ways and to migrate across tissue barriers. Recent progress in transgenesis and imaging tools allowed detailed insights into the components making up and regulating the actin and microtubule cytoskeleton as well as the alveolate-specific intermediate filament-like cytoskeletal network. These studies revealed interesting details that deviate from the cell biology of canonical model organisms. Here we review the latest developments in the field and point to a number of open questions covering the most experimentally tractable parasites: Plasmodium, the causative agent of malaria; Toxoplasma gondii, the causative agent of toxoplasmosis; and Cryptosporidium, a major cause of diarrhea.

Divergent Plasmodium actin residues are essential for filament localization, mosquito salivary gland invasion and malaria transmission.

Actin is one of the most conserved and ubiquitous proteins in eukaryotes. Its sequence has been highly conserved for its monomers to self-assemble into filaments that mediate essential cell functions such as trafficking, cell shape and motility. The malaria-causing parasite, Plasmodium, expresses a highly sequence divergent actin that is critical for its rapid motility at different stages within its mammalian and mosquito hosts. Each of Plasmodium actin's four subdomains have divergent regions compared to canonical vertebrate actins. We previously identified subdomains 2 and 3 as providing critical contributions for parasite actin function as these regions could not be replaced by subdomains of vertebrate actins. Here we probed the contributions of individual divergent amino acid residues in these subdomains on parasite motility and progression. Non-lethal changes in these subdomains did not affect parasite development in the mammalian host but strongly affected progression through the mosquito with striking differences in transmission to and through the insect. Live visualization of actin filaments showed that divergent amino acid residues in subdomains 2 and 4 enhanced localization associated with filaments, while those in subdomain 3 negatively affected actin filaments. This suggests that finely tuned actin dynamics are essential for efficient organ entry in the mosquito vector affecting malaria transmission. This work provides residue level insight on the fundamental requirements of actin in highly motile cells.

Inter-subunit interactions drive divergent dynamics in mammalian and Plasmodium actin filaments.

Cell motility is essential for protozoan and metazoan organisms and typically relies on the dynamic turnover of actin filaments. In metazoans, monomeric actin polymerises into usually long and stable filaments, while some protozoans form only short and highly dynamic actin filaments. These different dynamics are partly due to the different sets of actin regulatory proteins and partly due to the sequence of actin itself. Here we probe the interactions of actin subunits within divergent actin filaments using a comparative dynamic molecular model and explore their functions using Plasmodium, the protozoan causing malaria, and mouse melanoma derived B16-F1 cells as model systems. Parasite actin tagged to a fluorescent protein (FP) did not incorporate into mammalian actin filaments, and rabbit actin-FP did not incorporate into parasite actin filaments. However, exchanging the most divergent region of actin subdomain 3 allowed such reciprocal incorporation. The exchange of a single amino acid residue in subdomain 2 (N41H) of Plasmodium actin markedly improved incorporation into mammalian filaments. In the parasite, modification of most subunit-subunit interaction sites was lethal, whereas changes in actin subdomains 1 and 4 reduced efficient parasite motility and hence mosquito organ penetration. The strong penetration defects could be rescued by overexpression of the actin filament regulator coronin. Through these comparative approaches we identified an essential and common contributor, subdomain 3, which drives the differential dynamic behaviour of two highly divergent eukaryotic actins in motile cells.

Screening for potential prophylactics targeting sporozoite motility through the skin.

BACKGROUND: Anti-malarial compounds have not yet been identified that target the first obligatory step of infection in humans: the migration of Plasmodium sporozoites in the host dermis. This movement is essential to find and invade a blood vessel in order to be passively transported to the liver. Here, an imaging screening pipeline was established to screen for compounds capable of inhibiting extracellular sporozoites. METHODS: Sporozoites expressing the green fluorescent protein were isolated from infected Anopheles mosquitoes, incubated with compounds from two libraries (MMV Malaria Box and a FDA-approved library) and imaged. Effects on in vitro motility or morphology were scored. In vivo efficacy of a candidate drug was investigated by treating mice ears with a gel prior to infectious mosquito bites. Motility was analysed by in vivo imaging and the progress of infection was monitored by daily blood smears. RESULTS: Several compounds had a pronounced effect on in vitro sporozoite gliding or morphology. Notably, monensin sodium potently affected sporozoite movement while gramicidin S resulted in rounding up of sporozoites. However, pre-treatment of mice with a topical gel containing gramicidin did not reduce sporozoite motility and infection. CONCLUSIONS: This approach shows that it is possible to screen libraries for inhibitors of sporozoite motility and highlighted the paucity of compounds in currently available libraries that inhibit this initial step of a malaria infection. Screening of diverse libraries is suggested to identify more compounds that could serve as leads in developing 'skin-based' malaria prophylactics. Further, strategies need to be developed that will allow compounds to effectively penetrate the dermis and thereby prevent exit of sporozoites from the skin.

The Dynamic Nonprime Binding of Sampatrilat to the C-Domain of Angiotensin-Converting Enzyme.

Sampatrilat is a vasopeptidase inhibitor that inhibits both angiotensin I-converting enzyme (ACE) and neutral endopeptidase. ACE is a zinc dipeptidyl carboxypeptidase that contains two extracellular domains (nACE and cACE). In this study the molecular basis for the selectivity of sampatrilat for nACE and cACE was investigated. Enzyme inhibition assays were performed to evaluate the in vitro ACE domain selectivity of sampatrilat. The inhibition of the C-domain (Ki = 13.8 nM) by sampatrilat was 12.4-fold more potent than that for the N-domain (171.9 nM), indicating differences in affinities for the respective ACE domain binding sites. Interestingly, replacement of the P2 group of sampatrilat with an aspartate abrogated its C-selectivity and lowered the potency of the inhibitor to activities in the micromolar range. The molecular basis for this selective profile was evaluated using molecular modeling methods. We found that the C-domain selectivity of sampatrilat is due to occupation of the lysine side chain in the S1 and S2 subsites and interactions with Glu748 and Glu1008, respectively. This study provides new insights into ligand interactions with the nonprime binding site that can be exploited for the design of domain-selective ACE inhibitors.

Fragment-based design for the development of N-domain-selective angiotensin-1-converting enzyme inhibitors.

ACE (angiotensin-1-converting enzyme) is a zinc metallopeptidase that plays a prominent role in blood pressure regulation and electrolyte homeostasis. ACE consists of two homologous domains that despite similarities of sequence and topology display differences in substrate processing and inhibitor binding. The design of inhibitors that selectively inhibit the N-domain (N-selective) could be useful in treating conditions of tissue injury and fibrosis due to build-up of N-domain-specific substrate Ac-SDKP (N-acetyl-Ser-Asp-Lys-Pro). Using a receptor-based SHOP (scaffold hopping) approach with N-selective inhibitor RXP407, a shortlist of scaffolds that consisted of modified RXP407 backbones with novel chemotypes was generated. These scaffolds were selected on the basis of enhanced predicted interaction energies with N-domain residues that differed from their C-domain counterparts. One scaffold was synthesized and inhibitory binding tested using a fluorogenic ACE assay. A molecule incorporating a tetrazole moiety in the P2 position (compound 33RE) displayed potent inhibition (K(i)=11.21±0.74 nM) and was 927-fold more selective for the N-domain than the C-domain. A crystal structure of compound 33RE in complex with the N-domain revealed its mode of binding through aromatic stacking with His388 and a direct hydrogen bond with the hydroxy group of the N-domain specific Tyr369. This work further elucidates the molecular basis for N-domain-selective inhibition and assists in the design of novel N-selective ACE inhibitors that could be employed in treatment of fibrosis disorders.

Antifibrotic peptide N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP): opportunities for angiotensin-converting enzyme inhibitor design.

The renin-angiotensin system (RAS) is central to regulation of blood pressure and electrolyte homeostasis. Angiotensin-converting enzyme (ACE), a key protease in the RAS, has a range of substrates, including N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP). The peptide Ac-SDKP is cleared almost exclusively by ACE, and specifically by the N-domain active site of this enzyme. N-Acetyl-Ser-Asp-Lys-Pro is a negative regulator of haematopoietic stem cell differentiation and is a potent antifibrotic agent. In this review, the physiological actions of Ac-SDKP are presented, together with the potential clinical usefulness of raising Ac-SDKP levels. This emphasizes the possible opportunity of N-domain-selective ACE inhibitors or ACE-resistant Ac-SDKP analogues for the treatment of fibrosis.

New ketomethylene inhibitor analogues: synthesis and assessment of structural determinants for N-domain selective inhibition of angiotensin-converting enzyme.

Angiotensin-converting enzyme (ACE) is a zinc metallopeptidase containing two homologous domains. While the C-domain plays a major role in blood pressure regulation, the N-domain hydrolyzes the antifibrotic agent N-acetyl-Ser-Asp-Lys-Pro. Thus, N-domain selective (N-selective) inhibitors could be useful in the treatment of conditions relating to excessive tissue fibrosis. New keto-ACE analogues were designed that contained functionalities considered important for N-selective inhibitor RXP407 binding, namely, a P(2) Asp, N-acetyl group, and C-terminal amide. Such functionalities were incorporated to assess the structural determinants for N-selective binding in a novel inhibitor template. Inhibitors containing a C-terminal amide and modified P(2)' group were poor inhibitors of the N-domain, with several of these displaying improved inhibition of the C-domain. Molecules with both a C-terminal amide and P(2) Asp were also poor inhibitors and not N-selective. Compounds containing a free C-terminus, a P(2) Asp and protecting group displayed a change of more than 1000-fold N-selectivity compared with the parent molecule. Molecular docking models revealed interaction of these P(2) groups with S(2) residues Tyr369 and Arg381. This study emphasizes the importance of P(2) functionalities in allowing for improved N-selective binding and provides further rationale for the design of N-selective inhibitors, which could be useful in treating tissue fibrosis.

Characterization of angiotensin I-converting enzyme N-domain selectivity using positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides.

Somatic angiotensin I-converting enzyme (ACE)has two homologous active sites (N and C domains) that show differences in various biochemical properties.In a previous study, we described the use of positionals canning synthetic combinatorial (PS-SC) libraries of fluorescence resonance energy transfer (FRET) peptides to define the ACE C-domain versus N-domain substrate specificity and developed selective substrates for the C-domain(Bersanetti et al., 2004). In the present work, we used the results from the PS-SC libraries to define the N-domain preferences and designed selective substrates for this domain. The peptide Abz-GDDVAK(Dnp)-OH presented the most favorable residues for N-domain selectivity in the P 3 to P 1 ' positions. The fluorogenic analog Abz-DVAK(Dnp)-OH (Abz = ortho -aminobenzoic acid; Dnp = 2,4-dinitrophenyl)showed the highest selectivity for ACE N-domain( k cat /K m = 1.76 µ m -1 · s -1) . Systematic reduction of the peptide length resulted in a tripeptide that was preferentially hydrolyzed by the C-domain. The binding of Abz-DVAK(Dnp)-OH to the active site of ACE N-domain was examined using a combination of conformational analysis and molecular docking. Our results indicated that the binding energies for the N-domain-substrate complexes were lower than those for the C-domain-substrate, suggesting that the former complexes are more stable.

Investigating the domain specificity of phosphinic inhibitors RXPA380 and RXP407 in angiotensin-converting enzyme.

Human somatic angiotensin-converting enzyme (ACE) is a membrane-bound dipeptidyl carboxypeptidase that contains two extracellular domains (N and C). Although highly homologous, they exhibit different substrate and inhibition profiles. The phosphinic inhibitors RXPA380 and RXP407 are highly selective for the C- and N-domains, respectively. A number of residues, implicated by structural data, are likely to contribute to this selectivity. However, the extent to which these different interactions are responsible for domain selectivity is unclear. In this study, a series of C- and N-domain mutants containing conversions to corresponding domain residues were used to scrutinize the contribution of these residues to selective inhibitor binding. Enzyme kinetic analyses of the purified mutants indicated that the RXPA380 C-selectivity is particularly reliant on the interaction between the P2 substituent and Phe 391 (testis ACE numbering). Moreover, a C-domain mutant in which Phe 391 has been changed to a Tyr residue, in addition to containing an N-domain S2' pocket (S2'F/Y), displayed the greatest shift toward a more N-domain-like Ki. None of the single mutations within the N-domain caused a large shift in RXP407's affinity for these enzymes. However, the double mutant containing the Tyr 369 to Phe change as well as Arg 381 to Glu displayed a 100-fold decrease in binding affinity, confirming that the S2 pocket plays a major role in RXP407 selectivity. Taken together, these data advance our understanding regarding the molecular basis for the remarkable ACE domain selectivity exhibited by these inhibitors.