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1.
J Med Chem ; 67(16): 14493-14523, 2024 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-39134060

RESUMEN

To contribute to the global effort to develop new antimalarial therapies, we previously disclosed initial findings on the optimization of the dihydroquinazolinone-3-carboxamide class that targets PfATP4. Here we report on refining the aqueous solubility and metabolic stability to improve the pharmacokinetic profile and consequently in vivo efficacy. We show that the incorporation of heterocycle systems in the 8-position of the scaffold was found to provide the greatest attainable balance between parasite activity, aqueous solubility, and metabolic stability. Optimized analogs, including the frontrunner compound S-WJM992, were shown to inhibit PfATP4-associated Na+-ATPase activity, gave rise to a metabolic signature consistent with PfATP4 inhibition, and displayed altered activities against parasites with mutations in PfATP4. Finally, S-WJM992 showed appreciable efficacy in a malaria mouse model and blocked gamete development preventing transmission to mosquitoes. Importantly, further optimization of the dihydroquinazolinone class is required to deliver a candidate with improved pharmacokinetic and risk of resistance profiles.


Asunto(s)
Antimaláricos , Plasmodium falciparum , Quinazolinonas , Antimaláricos/farmacología , Antimaláricos/química , Antimaláricos/farmacocinética , Animales , Plasmodium falciparum/efectos de los fármacos , Quinazolinonas/farmacología , Quinazolinonas/química , Quinazolinonas/farmacocinética , Ratones , Administración Oral , Relación Estructura-Actividad , Humanos , Malaria/tratamiento farmacológico , Femenino , Solubilidad
2.
ChemMedChem ; : e202400549, 2024 Aug 30.
Artículo en Inglés | MEDLINE | ID: mdl-39210733

RESUMEN

The emergence of resistance against current antimalarial treatments has necessitated the need for the development of novel antimalarial chemotypes. Toward this goal, we recently optimised the antimalarial activity of the dihydroquinazolinone scaffold and showed it targeted PfATP4. Here, we deconstruct the lactam moiety of the tricyclic dihydroquinazolinone scaffold and investigate the structure-activity relationship of the truncated scaffold. It was shown that SAR between scaffolds was largely transferrable and generated analogues with potent asexual stage activity. Evaluation of the truncated analogues against PfATP4 mutant drug resistant parasite strains and in assays measuring PfATP4-associated ATPase activity demonstrated retention of PfATP4 as the molecular target. Analogues exhibited activity against both male and female gametes and multidrug resistant parasites. Limited efficacy of analogues in a P. berghei asexual stage mouse model was attributed to their moderate metabolic stability and low aqueous stability. Further development is required to address these attributes toward the potential use of the dihydroquinazolinone class in a curative and transmission blocking combination antimalarial therapy.

3.
Proc Natl Acad Sci U S A ; 121(28): e2403442121, 2024 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-38968107

RESUMEN

Plasmodium falciparum causes severe malaria and assembles a protein translocon (PTEX) complex at the parasitophorous vacuole membrane (PVM) of infected erythrocytes, through which several hundred proteins are exported to facilitate growth. The preceding liver stage of infection involves growth in a hepatocyte-derived PVM; however, the importance of protein export during P. falciparum liver infection remains unexplored. Here, we use the FlpL/FRT system to conditionally excise genes in P. falciparum sporozoites for functional liver-stage studies. Disruption of PTEX members ptex150 and exp2 did not affect sporozoite development in mosquitoes or infectivity for hepatocytes but attenuated liver-stage growth in humanized mice. While PTEX150 deficiency reduced fitness on day 6 postinfection by 40%, EXP2 deficiency caused 100% loss of liver parasites, demonstrating that PTEX components are required for growth in hepatocytes to differing degrees. To characterize PTEX loss-of-function mutations, we localized four liver-stage Plasmodium export element (PEXEL) proteins. P. falciparum liver specific protein 2 (LISP2), liver-stage antigen 3 (LSA3), circumsporozoite protein (CSP), and a Plasmodium berghei LISP2 reporter all localized to the periphery of P. falciparum liver stages but were not exported beyond the PVM. Expression of LISP2 and CSP but not LSA3 was reduced in ptex150-FRT and exp2-FRT liver stages, suggesting that expression of some PEXEL proteins is affected directly or indirectly by PTEX disruption. These results show that PTEX150 and EXP2 are important for P. falciparum development in hepatocytes and emphasize the emerging complexity of PEXEL protein trafficking.


Asunto(s)
Hepatocitos , Hígado , Malaria Falciparum , Plasmodium falciparum , Proteínas Protozoarias , Esporozoítos , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Animales , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Esporozoítos/metabolismo , Esporozoítos/crecimiento & desarrollo , Ratones , Hígado/parasitología , Hígado/metabolismo , Humanos , Hepatocitos/parasitología , Hepatocitos/metabolismo , Malaria Falciparum/parasitología
4.
Eur J Med Chem ; 276: 116677, 2024 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-39024967

RESUMEN

Emerging resistance to current antimalarials is reducing their effectiveness and therefore there is a need to develop new antimalarial therapies. Toward this goal, high throughput screens against the P. falciparum asexual parasite identified the pyrazolopyridine 4-carboxamide scaffold. Structure-activity relationship analysis of this chemotype defined that the N1-tert-butyl group and aliphatic foliage in the 3- and 6-positions were necessary for activity, while the inclusion of a 7'-aza-benzomorpholine on the 4-carboxamide motif resulted in potent anti-parasitic activity and increased aqueous solubility. A previous report that resistance to the pyrazolopyridine class is associated with the ABCI3 transporter was confirmed, with pyrazolopyridine 4-carboxamides showing an increase in potency against parasites when the ABCI3 transporter was knocked down. The low metabolic stability intrinsic to the pyrazolopyridine scaffold and the slow rate by which the compounds kill asexual parasites resulted in poor performance in a P. berghei asexual blood stage mouse model. Lowering the risk of resistance and mitigating the metabolic stability and cytochrome P450 inhibition will be challenges in the future development of the pyrazolopyrimidine antimalarial class.


Asunto(s)
Antimaláricos , Plasmodium falciparum , Pirazoles , Piridinas , Antimaláricos/farmacología , Antimaláricos/química , Antimaláricos/síntesis química , Plasmodium falciparum/efectos de los fármacos , Relación Estructura-Actividad , Pirazoles/química , Pirazoles/farmacología , Pirazoles/síntesis química , Animales , Piridinas/farmacología , Piridinas/química , Piridinas/síntesis química , Ratones , Pruebas de Sensibilidad Parasitaria , Estructura Molecular , Resistencia a Medicamentos/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos
5.
Nat Commun ; 15(1): 5219, 2024 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-38890312

RESUMEN

With resistance to most antimalarials increasing, it is imperative that new drugs are developed. We previously identified an aryl acetamide compound, MMV006833 (M-833), that inhibited the ring-stage development of newly invaded merozoites. Here, we select parasites resistant to M-833 and identify mutations in the START lipid transfer protein (PF3D7_0104200, PfSTART1). Introducing PfSTART1 mutations into wildtype parasites reproduces resistance to M-833 as well as to more potent analogues. PfSTART1 binding to the analogues is validated using organic solvent-based Proteome Integral Solubility Alteration (Solvent PISA) assays. Imaging of invading merozoites shows the inhibitors prevent the development of ring-stage parasites potentially by inhibiting the expansion of the encasing parasitophorous vacuole membrane. The PfSTART1-targeting compounds also block transmission to mosquitoes and with multiple stages of the parasite's lifecycle being affected, PfSTART1 represents a drug target with a new mechanism of action.


Asunto(s)
Acetamidas , Antimaláricos , Plasmodium falciparum , Proteínas Protozoarias , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Plasmodium falciparum/crecimiento & desarrollo , Acetamidas/farmacología , Acetamidas/química , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Antimaláricos/farmacología , Antimaláricos/química , Animales , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Mutación , Malaria Falciparum/parasitología , Malaria Falciparum/prevención & control , Malaria Falciparum/tratamiento farmacológico , Humanos , Resistencia a Medicamentos/genética , Resistencia a Medicamentos/efectos de los fármacos , Estadios del Ciclo de Vida/efectos de los fármacos
6.
Eur J Med Chem ; 270: 116354, 2024 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-38554474

RESUMEN

Malaria is a devastating disease that causes significant morbidity worldwide. The development of new antimalarial chemotypes is urgently needed because of the emergence of resistance to frontline therapies. Independent phenotypic screening campaigns against the Plasmodium asexual parasite, including our own, identified the aryl amino acetamide hit scaffold. In a prior study, we identified the STAR-related lipid transfer protein (PfSTART1) as the molecular target of this antimalarial chemotype. In this study, we combined structural elements from the different aryl acetamide hit subtypes and explored the structure-activity relationship. It was shown that the inclusion of an endocyclic nitrogen, to generate the tool compound WJM-715, improved aqueous solubility and modestly improved metabolic stability in rat hepatocytes. Metabolic stability in human liver microsomes remains a challenge for future development of the aryl acetamide class, which was underscored by modest systemic exposure and a short half-life in mice. The optimized aryl acetamide analogs were cross resistant to parasites with mutations in PfSTART1, but not to other drug-resistant mutations, and showed potent binding to recombinant PfSTART1 by biophysical analysis, further supporting PfSTART1 as the likely molecular target. The optimized aryl acetamide analogue, WJM-715 will be a useful tool for further investigating the druggability of PfSTART1 across the lifecycle of the malaria parasite.


Asunto(s)
Antimaláricos , Proteínas Portadoras , Malaria Falciparum , Malaria , Ratas , Ratones , Humanos , Animales , Antimaláricos/química , Plasmodium falciparum , Malaria Falciparum/tratamiento farmacológico , Malaria/tratamiento farmacológico , Acetamidas/farmacología , Lípidos
7.
Med Res Rev ; 43(6): 2303-2351, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-37232495

RESUMEN

Humans have lived in tenuous battle with malaria over millennia. Today, while much of the world is free of the disease, areas of South America, Asia, and Africa still wage this war with substantial impacts on their social and economic development. The threat of widespread resistance to all currently available antimalarial therapies continues to raise concern. Therefore, it is imperative that novel antimalarial chemotypes be developed to populate the pipeline going forward. Phenotypic screening has been responsible for the majority of the new chemotypes emerging in the past few decades. However, this can result in limited information on the molecular target of these compounds which may serve as an unknown variable complicating their progression into clinical development. Target identification and validation is a process that incorporates techniques from a range of different disciplines. Chemical biology and more specifically chemo-proteomics have been heavily utilized for this purpose. This review provides an in-depth summary of the application of chemo-proteomics in antimalarial development. Here we focus particularly on the methodology, practicalities, merits, and limitations of designing these experiments. Together this provides learnings on the future use of chemo-proteomics in antimalarial development.


Asunto(s)
Antimaláricos , Antagonistas del Ácido Fólico , Malaria , Humanos , Antimaláricos/química , Proteómica , Malaria/tratamiento farmacológico , Malaria/prevención & control , Resistencia a Medicamentos
8.
Nat Commun ; 14(1): 2219, 2023 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-37072430

RESUMEN

Plasmodium falciparum causes the most severe form of malaria in humans. The protozoan parasite develops within erythrocytes to mature schizonts, that contain more than 16 merozoites, which egress and invade fresh erythrocytes. The aspartic protease plasmepsin X (PMX), processes proteins and proteases essential for merozoite egress from the schizont and invasion of the host erythrocyte, including the leading vaccine candidate PfRh5. PfRh5 is anchored to the merozoite surface through a 5-membered complex (PCRCR), consisting of Plasmodium thrombospondin-related apical merozoite protein, cysteine-rich small secreted protein, Rh5-interacting protein and cysteine-rich protective antigen. Here, we show that PCRCR is processed by PMX in micronemes to remove the N-terminal prodomain of PhRh5 and this activates the function of the complex unmasking a form that can bind basigin on the erythrocyte membrane and mediate merozoite invasion. The ability to activate PCRCR at a specific time in merozoite invasion most likely masks potential deleterious effects of its function until they are required. These results provide an important understanding of the essential role of PMX and the fine regulation of PCRCR function in P. falciparum biology.


Asunto(s)
Malaria Falciparum , Plasmodium falciparum , Humanos , Animales , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Antígenos de Protozoos , Cisteína/metabolismo , Malaria Falciparum/parasitología , Eritrocitos/parasitología , Merozoítos/metabolismo
9.
ACS Infect Dis ; 9(3): 668-691, 2023 03 10.
Artículo en Inglés | MEDLINE | ID: mdl-36853190

RESUMEN

The development of new antimalarials is required because of the threat of resistance to current antimalarial therapies. To discover new antimalarial chemotypes, we screened the Janssen Jumpstarter library against the P. falciparum asexual parasite and identified the 7-N-substituted-3-oxadiazole quinolone hit class. We established the structure-activity relationship and optimized the antimalarial potency. The optimized analog WJM228 (17) showed robust metabolic stability in vitro, although the aqueous solubility was limited. Forward genetic resistance studies uncovered that WJM228 targets the Qo site of cytochrome b (cyt b), an important component of the mitochondrial electron transport chain (ETC) that is essential for pyrimidine biosynthesis and an established antimalarial target. Profiling against drug-resistant parasites confirmed that WJM228 confers resistance to the Qo site but not Qi site mutations, and in a biosensor assay, it was shown to impact the ETC via inhibition of cyt b. Consistent with other cyt b targeted antimalarials, WJM228 prevented pre-erythrocytic parasite and male gamete development and reduced asexual parasitemia in a P. berghei mouse model of malaria. Correcting the limited aqueous solubility and the high susceptibility to cyt b Qo site resistant parasites found in the clinic will be major obstacles in the future development of the 3-oxadiazole quinolone antimalarial class.


Asunto(s)
Antimaláricos , Antagonistas del Ácido Fólico , Malaria Falciparum , Quinolonas , Animales , Ratones , Antimaláricos/farmacología , Citocromos b , Antagonistas del Ácido Fólico/metabolismo , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Plasmodium falciparum , Quinolonas/farmacología
10.
J Med Chem ; 66(5): 3540-3565, 2023 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-36812492

RESUMEN

There is an urgent need to populate the antimalarial clinical portfolio with new candidates because of resistance against frontline antimalarials. To discover new antimalarial chemotypes, we performed a high-throughput screen of the Janssen Jumpstarter library against the Plasmodium falciparum asexual blood-stage parasite and identified the 2,3-dihydroquinazolinone-3-carboxamide scaffold. We defined the SAR and found that 8-substitution on the tricyclic ring system and 3-substitution of the exocyclic arene produced analogues with potent activity against asexual parasites equivalent to clinically used antimalarials. Resistance selection and profiling against drug-resistant parasite strains revealed that this antimalarial chemotype targets PfATP4. Dihydroquinazolinone analogues were shown to disrupt parasite Na+ homeostasis and affect parasite pH, exhibited a fast-to-moderate rate of asexual kill, and blocked gametogenesis, consistent with the phenotype of clinically used PfATP4 inhibitors. Finally, we observed that optimized frontrunner analogue WJM-921 demonstrates oral efficacy in a mouse model of malaria.


Asunto(s)
Antimaláricos , Malaria Falciparum , Malaria , Animales , Ratones , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Plasmodium falciparum , Homeostasis , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología
11.
Biochem J ; 479(24): 2529-2546, 2022 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-36520108

RESUMEN

Transmission blocking interventions can stop malaria parasite transmission from mosquito to human by inhibiting parasite infection in mosquitos. One of the most advanced candidates for a malaria transmission blocking vaccine is Pfs230. Pfs230 is the largest member of the 6-cysteine protein family with 14 consecutive 6-cysteine domains and is expressed on the surface of gametocytes and gametes. Here, we present the crystal structure of the first two 6-cysteine domains of Pfs230. We identified high affinity Pfs230-specific nanobodies that recognized gametocytes and bind to distinct sites on Pfs230, which were isolated from immunized alpacas. Using two non-overlapping Pfs230 nanobodies, we show that these nanobodies significantly blocked P. falciparum transmission and reduced the formation of exflagellation centers. Crystal structures of the transmission blocking nanobodies with the first 6-cysteine domain of Pfs230 confirm that they bind to different epitopes. In addition, these nanobodies bind to Pfs230 in the absence of the prodomain, in contrast with the binding of known Pfs230 transmission blocking antibodies. These results provide additional structural insight into Pfs230 domains and elucidate a mechanism of action of transmission blocking Pfs230 nanobodies.


Asunto(s)
Malaria , Anticuerpos de Dominio Único , Animales , Humanos , Plasmodium falciparum/química , Proteínas Protozoarias/química , Antígenos de Protozoos/química , Cisteína , Anticuerpos Antiprotozoarios
12.
Nat Microbiol ; 7(12): 2039-2053, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36396942

RESUMEN

The most severe form of malaria is caused by Plasmodium falciparum. These parasites invade human erythrocytes, and an essential step in this process involves the ligand PfRh5, which forms a complex with cysteine-rich protective antigen (CyRPA) and PfRh5-interacting protein (PfRipr) (RCR complex) and binds basigin on the host cell. We identified a heteromeric disulfide-linked complex consisting of P. falciparum Plasmodium thrombospondin-related apical merozoite protein (PfPTRAMP) and P. falciparum cysteine-rich small secreted protein (PfCSS) and have shown that it binds RCR to form a pentameric complex, PCRCR. Using P. falciparum lines with conditional knockouts, invasion inhibitory nanobodies to both PfPTRAMP and PfCSS, and lattice light-sheet microscopy, we show that they are essential for merozoite invasion. The PCRCR complex functions to anchor the contact between merozoite and erythrocyte membranes brought together by strong parasite deformations. We solved the structure of nanobody-PfCSS complexes to identify an inhibitory epitope. Our results define the function of the PCRCR complex and identify invasion neutralizing epitopes providing a roadmap for structure-guided development of these proteins for a blood stage malaria vaccine.


Asunto(s)
Antígenos de Grupos Sanguíneos , Vacunas contra la Malaria , Malaria Falciparum , Humanos , Plasmodium falciparum/genética , Cisteína , Eritrocitos , Epítopos
13.
ACS Med Chem Lett ; 13(11): 1745-1754, 2022 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-36385924

RESUMEN

Drug resistance to first-line antimalarials-including artemisinin-is increasing, resulting in a critical need for the discovery of new agents with novel mechanisms of action. In collaboration with the Walter and Eliza Hall Institute and with funding from the Wellcome Trust, a phenotypic screen of Merck's aspartyl protease inhibitor library identified a series of plasmepsin X (PMX) hits that were more potent than chloroquine. Inspired by a PMX homology model, efforts to optimize the potency resulted in the discovery of leads that, in addition to potently inhibiting PMX, also inhibit another essential aspartic protease, plasmepsin IX (PMIX). Further potency and pharmacokinetic profile optimization efforts culminated in the discovery of WM382, a very potent dual PMIX/X inhibitor with robust in vivo efficacy at multiple stages of the malaria parasite life cycle and an excellent resistance profile.

14.
J Med Chem ; 65(20): 14121-14143, 2022 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-36216349

RESUMEN

Plasmepsin X (PMX) is an essential aspartyl protease controlling malaria parasite egress and invasion of erythrocytes, development of functional liver merozoites (prophylactic activity), and blocking transmission to mosquitoes, making it a potential multistage drug target. We report the optimization of an aspartyl protease binding scaffold and the discovery of potent, orally active PMX inhibitors with in vivo antimalarial efficacy. Incorporation of safety evaluation early in the characterization of PMX inhibitors precluded compounds with a long human half-life (t1/2) to be developed. Optimization focused on improving the off-target safety profile led to the identification of UCB7362 that had an improved in vitro and in vivo safety profile but a shorter predicted human t1/2. UCB7362 is estimated to achieve 9 log 10 unit reduction in asexual blood-stage parasites with once-daily dosing of 50 mg for 7 days. This work demonstrates the potential to deliver PMX inhibitors with in vivo efficacy to treat malaria.


Asunto(s)
Antimaláricos , Antagonistas del Ácido Fólico , Malaria , Animales , Humanos , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Plasmodium falciparum/metabolismo , Ácido Aspártico Endopeptidasas , Malaria/tratamiento farmacológico
15.
ChemMedChem ; 17(18): e202200306, 2022 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-35906744

RESUMEN

Plasmepsin X (PMX) is an aspartyl protease that processes proteins essential for Plasmodium parasites to invade and egress from host erythrocytes during the symptomatic asexual stage of malaria. PMX substrates possess a conserved cleavage region denoted by the consensus motif, SFhE (h=hydrophobic amino acid). Peptidomimetics reflecting the P3 -P1 positions of the consensus motif were designed and showed potent and selective inhibition of PMX. It was established that PMX prefers Phe in the P1 position, di-substitution at the ß-carbon of the P2 moiety and a hydrophobic P3 group which was supported by modelling of the peptidomimetics in complex with PMX. The peptidomimetics were shown to arrest asexual P. falciparum parasites at the schizont stage by impairing PMX substrate processing. Overall, the peptidomimetics described will assist in further understanding PMX substrate specificity and have the potential to act as a template for future antimalarial design.


Asunto(s)
Antimaláricos , Antagonistas del Ácido Fólico , Malaria Falciparum , Peptidomiméticos , Aminoácidos , Antimaláricos/química , Antimaláricos/farmacología , Ácido Aspártico Endopeptidasas , Carbono , Humanos , Malaria Falciparum/tratamiento farmacológico , Peptidomiméticos/química , Peptidomiméticos/farmacología , Plasmodium falciparum/metabolismo , Inhibidores de Proteasas/química , Proteínas Protozoarias
16.
Mol Syst Biol ; 18(4): e10824, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35475529

RESUMEN

Clinical immunity to P. falciparum malaria is non-sterilizing, with adults often experiencing asymptomatic infection. Historically, asymptomatic malaria has been viewed as beneficial and required to help maintain clinical immunity. Emerging views suggest that these infections are detrimental and constitute a parasite reservoir that perpetuates transmission. To define the impact of asymptomatic malaria, we pursued a systems approach integrating antibody responses, mass cytometry, and transcriptional profiling of individuals experiencing symptomatic and asymptomatic P. falciparum infection. Defined populations of classical and atypical memory B cells and a TH2 cell bias were associated with reduced risk of clinical malaria. Despite these protective responses, asymptomatic malaria featured an immunosuppressive transcriptional signature with upregulation of pathways involved in the inhibition of T-cell function, and CTLA-4 as a predicted regulator in these processes. As proof of concept, we demonstrated a role for CTLA-4 in the development of asymptomatic parasitemia in infection models. The results suggest that asymptomatic malaria is not innocuous and might not support the induction of immune processes to fully control parasitemia or efficiently respond to malaria vaccines.


Asunto(s)
Malaria Falciparum , Parasitemia , Adulto , Infecciones Asintomáticas , Antígeno CTLA-4 , Humanos , Terapia de Inmunosupresión , Malaria Falciparum/genética , Malaria Falciparum/parasitología , Plasmodium falciparum
17.
Structure ; 30(7): 947-961.e6, 2022 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-35460613

RESUMEN

Plasmepsins IX (PMIX) and X (PMX) are essential aspartyl proteases for Plasmodium spp. egress, invasion, and development. WM4 and WM382 inhibit PMIX and PMX in Plasmodium falciparum and P. vivax. WM4 inhibits PMX, while WM382 is a dual inhibitor of PMIX and PMX. To understand their function, we identified protein substrates. Enzyme kinetic and structural analyses identified interactions responsible for drug specificity. PMIX and PMX have similar substrate specificity; however, there are distinct differences for peptide and protein substrates. Differences in WM4 and WM382 binding for PMIX and PMX map to variations in the S' region and engagement of the active site S3 pocket. Structures of PMX reveal interactions and mechanistic detail of drug binding important for development of clinical candidates against these targets.


Asunto(s)
Ácido Aspártico Endopeptidasas , Plasmodium falciparum , Ácido Aspártico Endopeptidasas/química , Cinética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Especificidad por Sustrato
18.
Mol Microbiol ; 117(5): 1245-1262, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35403274

RESUMEN

Infection with Plasmodium falciparum parasites results in approximately 627,000 deaths from malaria annually. Key to the parasite's success is their ability to invade and subsequently grow within human erythrocytes. Parasite proteins involved in parasite invasion and proliferation are therefore intrinsically of great interest, as targeting these proteins could provide novel means of therapeutic intervention. One such protein is P113 which has been reported to be both an invasion protein and an intracellular protein located within the parasitophorous vacuole (PV). The PV is delimited by a membrane (PVM) across which a plethora of parasite-specific proteins are exported via the Plasmodium Translocon of Exported proteins (PTEX) into the erythrocyte to enact various immune evasion functions. To better understand the role of P113 we isolated its binding partners from in vitro cultures of P. falciparum. We detected interactions with the protein export machinery (PTEX and exported protein-interacting complex) and a variety of proteins that either transit through the PV or reside on the parasite plasma membrane. Genetic knockdown or partial deletion of P113 did not significantly reduce parasite growth or protein export but did disrupt the morphology of the PVM, suggesting that P113 may play a role in maintaining normal PVM architecture.


Asunto(s)
Malaria Falciparum , Parásitos , Animales , Eritrocitos/parasitología , Humanos , Malaria Falciparum/parasitología , Parásitos/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Transporte de Proteínas/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Vacuolas/metabolismo
19.
Commun Biol ; 5(1): 333, 2022 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-35393572

RESUMEN

RhopH complexes consists of Clag3, RhopH2 and RhopH3 and are essential for growth of Plasmodium falciparum inside infected erythrocytes. Proteins are released from rhoptry organelles during merozoite invasion and trafficked to the surface of infected erythrocytes and enable uptake of nutrients. RhopH3, unlike other RhopH proteins, is required for parasite invasion, suggesting some cellular processes RhopH proteins function as single players rather than a complex. We show the RhopH complex has not formed during merozoite invasion. Clag3 is directly released into the host cell cytoplasm, whilst RhopH2 and RhopH3 are released into the nascent parasitophorous vacuole. Export of RhopH2 and RhopH3 from the parasitophorous vacuole into the infected erythrocyte cytoplasm enables assembly of Clag3/RhopH2/RhopH3 complexes and incorporation into the host cell membrane concomitant with activation of nutrient uptake. This suggests compartmentalisation prevents premature channel assembly before intact complex is assembled at the host cell membrane.


Asunto(s)
Membrana Eritrocítica , Malaria Falciparum , Membrana Eritrocítica/metabolismo , Eritrocitos/parasitología , Humanos , Malaria Falciparum/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo
20.
Front Cell Infect Microbiol ; 12: 1049065, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36605129

RESUMEN

Background: RH5 is the leading vaccine candidate for the Plasmodium falciparum blood stage and has shown impact on parasite growth in the blood in a human clinical trial. RH5 binds to Ripr and CyRPA at the apical end of the invasive merozoite form, and this complex, designated RCR, is essential for entry into human erythrocytes. RH5 has advanced to human clinical trials, and the impact on parasite growth in the blood was encouraging but modest. This study assessed the potential of a protein-in-adjuvant blood stage malaria vaccine based on a combination of RH5, Ripr and CyRPA to provide improved neutralizing activity against P. falciparum in vitro. Methods: Mice were immunized with the individual RCR antigens to down select the best performing adjuvant formulation and rats were immunized with the individual RCR antigens to select the correct antigen dose. A second cohort of rats were immunized with single, double and triple antigen combinations to assess immunogenicity and parasite neutralizing activity in growth inhibition assays. Results: The DPX® platform was identified as the best performing formulation in potentiating P. falciparum inhibitory antibody responses to these antigens. The three antigens derived from RH5, Ripr and CyRPA proteins formulated with DPX induced highly inhibitory parasite neutralising antibodies. Notably, RH5 either as a single antigen or in combination with Ripr and/or CyRPA, induced inhibitory antibodies that outperformed CyRPA, Ripr. Conclusion: An RCR combination vaccine may not induce substantially improved protective immunity as compared with RH5 as a single immunogen in a clinical setting and leaves the development pathway open for other antigens to be combined with RH5 as a next generation malaria vaccine.


Asunto(s)
Vacunas contra la Malaria , Malaria Falciparum , Humanos , Ratones , Ratas , Animales , Antígenos de Protozoos , Proteínas Protozoarias/metabolismo , Malaria Falciparum/parasitología , Plasmodium falciparum , Anticuerpos Antiprotozoarios , Vacunas Combinadas
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