Identification of covalent fragment inhibitors for Plasmodium falciparum UCHL3 with anti-malarial efficacy.

Malaria continues to be a major burden on global health, responsible for 619,000 deaths in 2021. The causative agent of malaria is the eukaryotic parasite Plasmodium. Resistance to artemisinin-based combination therapies (ACTs), the current first-line treatment for malaria, has emerged in Asia, South America, and more recently Africa, where >90% of all malaria-related deaths occur. This has necessitated the identification and investigation of novel parasite proteins and pathways as antimalarial targets, including components of the ubiquitin proteasome system. Here, we investigate Plasmodium falciparum deubiquitinase ubiquitin C-terminal hydrolase L3 (PfUCHL3) as one such target. We carried out a high-throughput screen with covalent fragments and identified seven scaffolds that selectively inhibit the plasmodial UCHL3, but not human UCHL3 or the closely related human UCHL1. After assessing toxicity in human cells, we identified four promising hits and demonstrated their efficacy against asexual P. falciparum blood stages and P. berghei sporozoite stages.

Characterization of Competitive Inhibitors of Plasmodium falciparum cGMP-Dependent Protein Kinase.

Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is an enticing antimalarial drug target. Novel chemotypes are needed because existing inhibitors have safety issues that may prevent further development. This work demonstrates isoxazole-based compounds are potent ATP competitive inhibitors of PfPKG and discloses a new analogue in this series. Isoxazoles 3 and 5 had Ki values that are comparable to a known standard, 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl] pyridine. They also exhibited excellent selectivity for PfPKG over the human orthologue and the gatekeeper mutant T618Q PfPKG, which mimics the less accessible binding site of the human orthologue. The human orthologue's larger binding site volume is predicted to explain the selectivity of the inhibitors for the P. falciparum enzyme.

Overlapping and distinct roles of CDPK family members in the pre-erythrocytic stages of the rodent malaria parasite, Plasmodium berghei.

Invasion of hepatocytes by Plasmodium sporozoites initiates the pre-erythrocytic step of a malaria infection. Subsequent development of the parasite within hepatocytes and exit from them is essential for starting the disease-causing erythrocytic cycle. Identification of signaling pathways that operate in pre-erythrocytic stages provides insight into a critical step of infection and potential targets for chemoprotection from malaria. We demonstrate that P. berghei homologs of Calcium Dependent Protein Kinase 1 (CDPK1), CDPK4 and CDPK5 play overlapping but distinct roles in sporozoite invasion and parasite egress from hepatocytes. All three kinases are expressed in sporozoites. All three are required for optimal motility of sporozoites and consequently their invasion of hepatocytes. Increased cGMP can compensate for the functional loss of CDPK1 and CDPK5 during sporozoite invasion but cannot overcome loss of CDPK4. CDPK1 and CDPK5 expression is downregulated after sporozoite invasion. CDPK5 reappears in a subset of late stage liver stages and is present in all merosomes. Chemical inhibition of CDPK4 and depletion of CDPK5 in liver stages implicate these kinases in the formation and/or release of merosomes from mature liver stages. Furthermore, depletion of CDPK5 in merosomes significantly delays initiation of the erythrocytic cycle without affecting infectivity of hepatic merozoites. These data suggest that CDPK5 may be required for the rupture of merosomes. Our work provides evidence that sporozoite invasion requires CDPK1 and CDPK5, and suggests that CDPK5 participates in the release of hepatic merozoites.

A Plasmodium homolog of ER tubule-forming proteins is required for parasite virulence.

Reticulon and REEP family of proteins stabilize the high curvature of endoplasmic reticulum (ER) tubules. Plasmodium berghei Yop1 (PbYop1) is a REEP5 homolog in Plasmodium. Here, we characterize its function using a gene-knockout (Pbyop1∆). Pbyop1∆ asexual stage parasites display abnormal ER architecture and an enlarged digestive vacuole. The erythrocytic cycle of Pbyop1∆ parasites is severely attenuated and the incidence of experimental cerebral malaria is significantly decreased in Pbyop1∆-infected mice. Pbyop1∆ sporozoites have reduced speed, are slower to invade host cells but give rise to equal numbers of infected HepG2 cells, as WT sporozoites. We propose that PbYOP1's disruption may lead to defects in trafficking and secretion of a subset of proteins required for parasite development and invasion of erythrocytes. Furthermore, the maintenance of ER morphology in different parasite stages is likely to depend on different proteins.

Antimalarial proteasome inhibitor reveals collateral sensitivity from intersubunit interactions and fitness cost of resistance.

We describe noncovalent, reversible asparagine ethylenediamine (AsnEDA) inhibitors of the Plasmodium falciparum proteasome (Pf20S) ß5 subunit that spare all active subunits of human constitutive and immuno-proteasomes. The compounds are active against erythrocytic, sexual, and liver-stage parasites, against parasites resistant to current antimalarials, and against P. falciparum strains from patients in Africa. The ß5 inhibitors synergize with a ß2 inhibitor in vitro and in mice and with artemisinin. P. falciparum selected for resistance to an AsnEDA ß5 inhibitor surprisingly harbored a point mutation in the noncatalytic ß6 subunit. The ß6 mutant was resistant to the species-selective Pf20S ß5 inhibitor but remained sensitive to the species-nonselective ß5 inhibitors bortezomib and carfilzomib. Moreover, resistance to the Pf20S ß5 inhibitor was accompanied by increased sensitivity to a Pf20S ß2 inhibitor. Finally, the ß5 inhibitor-resistant mutant had a fitness cost that was exacerbated by irradiation. Thus, used in combination, multistage-active inhibitors of the Pf20S ß5 and ß2 subunits afford synergistic antimalarial activity with a potential to delay the emergence of resistance to artemisinins and each other.

A novel quantitative PCR detects Babesia infection in patients not identified by currently available non-nucleic acid amplification tests.

BACKGROUND: Ticks transmit Babesia microti, the causative agents of babesiosis in North America and Europe. Babesiosis is now endemic in Northeastern USA and affects people of all ages. Babesia species infect erythrocytes and can be transmitted through blood transfusion. Whole blood and blood products, which are not tested for Babesia, can cause transfusion-transmitted babesiosis (TTB) resulting in severe consequences in the immuno-compromised patients. The purpose of this study was epidemiological evaluation of babesiosis in a tick-infested state. RESULTS: We examined blood samples from 192 patients who visited clinics during the active tick-borne diseases season, using a newly developed qPCR assay that uses the specific molecular beacon probe. Due to the absence of clear symptomology, clinical laboratories did not test 131 samples by IFA, FISH or microscopic examination of Giemsa-stained blood smears. Babesia infection was detected in all age groups by FISH and microscopy; notably patients >40 years of age represented 64% of tested samples and 13% were younger patients. We tested all samples using qPCR and found that 38% were positive for Babesia. Of 28 samples that were positive by FISH, 27 (96%) were also positive by qPCR indicating high congruency between nucleic acid based tests. Interestingly, of 78 asymptomatic samples not tested by FISH, 22 were positive by our qPCR. Direct detection of Babesia relies upon microscopic examination of patient blood smears, which is labor intensive, difficult to scale up, requires specific expertise and is hence, often not performed. In fact, a clinical laboratory examined only 23 of 86 blood samples obtained from two different counties by microscopy. By considering individuals positive for Babesia infection when results from currently available microscopy, FISH or serological tests were positive, we found that our qPCR is highly sensitive (96.2%) and showed a specificity of 70.5% for Babesia. CONCLUSION: Robust qPCR using specific probes can be highly useful for efficient and appropriate diagnosis of babesiosis in patients in conjunction with conventional diagnostics, or as a stand-alone test, especially for donated blood screening. The use of a nucleic acid amplification test based screening of blood and blood products could prevent TTB.

Structure-Activity Relationship of a Pyrrole Based Series of PfPKG Inhibitors as Anti-Malarials.

Controlling malaria requires new drugs against Plasmodium falciparum. The P. falciparum cGMP-dependent protein kinase (PfPKG) is a validated target whose inhibitors could block multiple steps of the parasite's life cycle. We defined the structure-activity relationship (SAR) of a pyrrole series for PfPKG inhibition. Key pharmacophores were modified to enable full exploration of chemical diversity and to gain knowledge about an ideal core scaffold. In vitro potency against recombinant PfPKG and human PKG were used to determine compound selectivity for the parasite enzyme. P. berghei sporozoites and P. falciparum asexual blood stages were used to assay multistage antiparasitic activity. Cellular specificity of compounds was evaluated using transgenic parasites expressing PfPKG carrying a substituted "gatekeeper" residue. The structure of PfPKG bound to an inhibitor was solved, and modeling using this structure together with computational tools was utilized to understand SAR and establish a rational strategy for subsequent lead optimization.

Chemical probes of a trisubstituted pyrrole to identify its protein target(s) in Plasmodium sporozoites.

Malaria is a disease that has a major impact in many developing nations, especially on the African continent. There is a need to develop new therapeutics and prophylactic treatments against it. A trisubstituted pyrrole was recently found to inhibit infection of mammalian hepatocytes by Plasmodium sporozoites, but the target of this agent is not known. In this study trisubstituted pyrrole derivatives with different substituents on a piperidinyl nitrogen were prepared. We determined if modifications of the piperidinyl nitrogen would accommodate a drug-biotin linking strategy for affinity purification of the trisubstituted pyrrole's target protein(s).

Target specific-trisubstituted pyrrole inhibits Babesia bovis erythrocytic growth.

Babesiosis, a significant veterinary disease and an emerging zoonotic human infection, is caused by certain species of the protozoan parasite, Babesia. Here we report that a trisubstituted pyrrole is a potent inhibitor of Babesia bovis, a bovine parasite. Furthermore, B. bovis expresses the known target of the compound, the cGMP dependent protein kinase. Target conservation and the in vitro efficacy support further investigation of this compound and validation of Babesia cGMP dependent protein kinase as its in vivo target.

Role of Plasmodium berghei cGMP-dependent protein kinase in late liver stage development.

The liver is the first organ infected by Plasmodium sporozoites during malaria infection. In the infected hepatocytes, sporozoites undergo a complex developmental program to eventually generate hepatic merozoites that are released into the bloodstream in membrane-bound vesicles termed merosomes. Parasites blocked at an early developmental stage inside hepatocytes elicit a protective host immune response, making them attractive targets in the effort to develop a pre-erythrocytic stage vaccine. Here, we generated parasites blocked at a late developmental stage inside hepatocytes by conditionally disrupting the Plasmodium berghei cGMP-dependent protein kinase in sporozoites. Mutant sporozoites are able to invade hepatocytes and undergo intracellular development. However, they remain blocked as late liver stages that do not release merosomes into the medium. These late arrested liver stages induce protection in immunized animals. This suggests that, similar to the well studied early liver stages, late stage liver stages too can confer protection from sporozoite challenge.

Discovery of Imidazole-Based Inhibitors of Plasmodium falciparum cGMP-Dependent Protein Kinase.

The discovery of new targets for the treatment of malaria, in particular those aimed at the pre-erythrocytic stage in the life cycle, advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, this paper presents initial structure-activity relationships in an imidazole scaffold at four positions, representative in vitro ADME, hERG characterization, and cell-based antiparasitic activity. This series of PfPKG inhibitors has good in vitro PfPKG potency, low hERG activity, and cell-based antiparasitic activity against multiple Plasmodium species that appears to be correlated with the in vitro potency.

Activity of a trisubstituted pyrrole in inhibiting sporozoite invasion and blocking malaria infection.

Malaria infection is initiated by Plasmodium sporozoites infecting the liver. Preventing sporozoite infection would block the obligatory first step of the infection and perhaps reduce disease severity. In addition, such an approach would decrease Plasmodium vivax hypnozoite formation and therefore disease relapses. Here we describe the activity of a trisubstituted pyrrole, 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl] pyridine, in inhibiting motility, invasion, and consequently infection by P. berghei sporozoites. In tissue culture, the compound was effective within the first 3 h of sporozoite addition to HepG2 cells. In vivo, intraperitoneal administration of the compound significantly inhibited liver-stage parasitemia in P. yoelii sporozoite-infected mice and prevented the appearance of blood-stage parasites. P. berghei sporozoites lacking the parasite cGMP-dependent protein kinase, the primary target of the compound in erythrocyte-stage parasites, remained infectious to HepG2 cells and sensitive to the drug. These results suggest that the drug has an additional target(s) in sporozoites. We propose that drugs that inhibit sporozoite infection offer a feasible approach to malaria prophylaxis.

Adenylyl cyclase alpha and cAMP signaling mediate Plasmodium sporozoite apical regulated exocytosis and hepatocyte infection.

Malaria starts with the infection of the liver of the host by Plasmodium sporozoites, the parasite form transmitted by infected mosquitoes. Sporozoites migrate through several hepatocytes by breaching their plasma membranes before finally infecting one with the formation of an internalization vacuole. Migration through host cells induces apical regulated exocytosis in sporozoites. Here we show that apical regulated exocytosis is induced by increases in cAMP in sporozoites of rodent (P. yoelii and P. berghei) and human (P. falciparum) Plasmodium species. We have generated P. berghei parasites deficient in adenylyl cyclase alpha (ACalpha), a gene containing regions with high homology to adenylyl cyclases. PbACalpha-deficient sporozoites do not exocytose in response to migration through host cells and present more than 50% impaired hepatocyte infectivity in vivo. These effects are specific to ACalpha, as re-introduction of ACalpha in deficient parasites resulted in complete recovery of exocytosis and infection. Our findings indicate that ACalpha and increases in cAMP levels are required for sporozoite apical regulated exocytosis, which is involved in sporozoite infection of hepatocytes.

Plasmodium falciparum cGMP-Dependent Protein Kinase - A Novel Chemotherapeutic Target.

The primary effector of cGMP signaling in Plasmodium is the cGMP-dependent protein kinase (PKG). Work in human-infective Plasmodium falciparum and rodent-infective Plasmodium berghei has provided biological validation of P. falciparum PKG (PfPKG) as a drug target for treating and/or protecting against malaria. PfPKG is essential in the asexual erythrocytic and sexual cycles as well as the pre-erythrocytic cycle. Medicinal chemistry efforts, both target-based and phenotype-based, have targeted PfPKG in the past few years. This review provides a brief overview of their results and challenges.

Investigating disease severity in an animal model of concurrent babesiosis and Lyme disease.

The incidence of babesiosis, Lyme disease and other tick-borne diseases has increased steadily in Europe and North America during the last five decades. Babesia microti is transmitted by species of Ixodes, the same ticks that transmit the Lyme disease-causing spirochete, Borrelia burgdorferi. B. microti can also be transmitted through transfusion of blood products and is the most common transfusion-transmitted infection in the U.S.A. Ixodes ticks are commonly infected with both B. microti and B. burgdorferi, and are competent vectors for transmitting them together into hosts. Few studies have examined the effects of coinfections on humans and they had somewhat contradictory results. One study linked coinfection with B. microti to a greater number of symptoms of overall disease in patients, while another report indicated that B. burgdorferi infection either did not affect babesiosis symptoms or decreased its severity. Mouse models of infection that manifest pathological effects similar to those observed in human babesiosis and Lyme disease offer a unique opportunity to thoroughly investigate the effects of coinfection on the host. Lyme disease has been studied using the susceptible C3H mouse infection model, which can also be used to examine B. microti infection to understand pathological mechanisms of human diseases, both during a single infection and during coinfections. We observed that high B. microti parasitaemia leads to low haemoglobin levels in infected mice, reflecting the anaemia observed in human babesiosis. Similar to humans, B. microti coinfection appears to enhance the severity of Lyme disease-like symptoms in mice. Coinfected mice have lower peak B. microti parasitaemia compared to mice infected with B. microti alone, which may reflect attenuation of babesiosis symptoms reported in some human coinfections. These findings suggest that B. burgdorferi coinfection attenuates parasite growth while B. microti presence exacerbates Lyme disease-like symptoms in mice.

Babesia microti-Borrelia Burgdorferi Coinfection.

The incidence and geographic distribution of human babesiosis is growing in the U.S. Its major causative agent is the protozoan parasite, Babesia microti. B. microti is transmitted to humans primarily through the bite of Ixodes scapularis ticks, which are vectors for a number of other pathogens. Other routes of B. microti transmission are blood transfusion and in rare cases of mother-to-foetus transmission, through the placenta. This review discusses the current literature on mammalian coinfection with B. microti and Borrelia burgdorferi, the causative agent Lyme disease.

Protozoan Parasite Babesia microti Subverts Adaptive Immunity and Enhances Lyme Disease Severity.

Lyme disease is the most prominent tick-borne disease in the United States. Co-infections with the tick-transmitted pathogens Babesia microti and Borrelia burgdorferi sensu stricto are becoming a serious health problem. B. burgdorferi is an extracellular spirochete that causes Lyme disease while B. microti is a protozoan that infects erythrocytes and causes babesiosis. Testing of donated blood for Babesia species is not currently mandatory due to unavailability of an FDA approved test. Transmission of this protozoan by blood transfusion often results in high morbidity and mortality in recipients. Infection of C3H/HeJ mice with B. burgdorferi and B. microti individually results in inflammatory Lyme disease and display of human babesiosis-like symptoms, respectively. Here we use this mouse model to provide a detailed investigation of the reciprocal influence of the two pathogens on each other during co-infection. We show that B. burgdorferi infection attenuates parasitemia in mice while B. microti subverts the splenic immune response, such that a marked decrease in splenic B and T cells, reduction in antibody levels and diminished functional humoral immunity, as determined by spirochete opsonophagocytosis, are observed in co-infected mice compared to only B. burgdorferi infected mice. Furthermore, immunosuppression by B. microti in co-infected mice showed an association with enhanced Lyme disease manifestations. This study demonstrates the effect of only simultaneous infection by B. burgdorferi and B. microti on each pathogen, immune response and on disease manifestations with respect to infection by the spirochete and the parasite. In our future studies, we will examine the overall effects of sequential infection by these pathogens on host immune responses and disease outcomes.

A Comprehensive Analysis of Plasmodium Circumsporozoite Protein Binding to Hepatocytes.

Circumsporozoite protein (CSP) is the dominant protein on the surface of Plasmodium sporozoites and plays a critical role in the invasion by sporozoites of hepatocytes. Contacts between CSP and heparin sulfate proteoglycans (HSPGs) lead to the attachment of sporozoites to hepatocytes and trigger signaling events in the parasite that promote invasion of hepatocytes. The precise sequence elements in CSP that bind HSPGs have not been identified. We performed a systematic in vitro analysis to dissect the association between Plasmodium falciparum CSP (PfCSP) and hepatocytes. We demonstrate that interactions between PfCSP and heparin or a cultured hepatoma cell line, HepG2, are mediated primarily by a lysine-rich site in the amino terminus of PfCSP. Importantly, the carboxyl terminus of PfCSP facilitates heparin-binding by the amino-terminus but does not interact directly with heparin. These findings provide insights into how CSP recognizes hepatocytes and useful information for further functional studies of CSP.

Defective sorting of the thrombospondin-related anonymous protein (TRAP) inhibits Plasmodium infectivity.

Thrombospondin-related anonymous protein (TRAP) is a type 1 transmembrane protein that plays an essential role in gliding motility and cell invasion by Plasmodium sporozoites. It is stored in micronemes-secretory organelles located primarily in the apical end of the parasites and is also found on the parasite surface. The mechanisms that target TRAP and other sporozoite proteins to micronemes and subsequently to the parasite surface are not known. Here we report that the micronemal and surface localization of TRAP requires a tyrosine-based motif located in its cytoplasmic tail. This motif is analogous to the YXXphi motif (Y: tyrosine, X: any amino acid; phi: hydrophobic amino acid) that targets eukaryotic proteins to certain sub-cellular compartments and to the plasma membrane. Abrogating the Y motif substantially reduces micronemal and cell surface localization of TRAP. The infectivity of mutant parasites is substantially inhibited. However, there is no significant difference in the amounts of TRAP secreted into the culture medium by wild type and mutant parasites, suggesting that TRAP destined for secretion bypasses micronemal localization.