Malaria: drugs developed for cancer that inactivate protein kinases are highly effective in killing the parasite

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The study, conducted by an international team and led by RMIT University’s Professor Christian Doerig, outlines a strategy that could save years of drug discovery research and millions of dollars in drug development by repurposing existing treatments designed for other diseases such as cancer.

The approach shows so much promise it has received government funding for its potential application in the fight against COVID-19.

The study, published in Nature Communications, demonstrated that the parasites that cause malaria are heavily dependent on enzymes in red blood cells where the parasites hide and proliferate.

It also revealed that drugs developed for cancer, and which inactivate these human enzymes, known as protein kinases, are highly effective in killing the parasite and represent an alternative to drugs that target the parasite itself.

Lead author, RMIT’s Dr. Jack Adderley, said the analysis revealed which of the host cell enzymes were activated during infection, revealing novel points of reliance of the parasite on its human host.

“This approach has the potential to considerably reduce the cost and accelerate the deployment of new and urgently needed antimalarials,” he said.

“These host enzymes are in many instances the same as those activated in cancer cells, so we can now jump on the back of existing cancer drug discovery and look to repurpose a drug that is already available or close to completion of the drug development process.”

As well as enabling the repurposing of drugs, the approach is likely to reduce the emergence of drug resistance, as the pathogen cannot escape by simply mutating the target of the drug, as is the case for most currently available antimalarials.

Doerig, Associate Dean for the Biomedical Sciences Cluster at RMIT and senior author of the paper, said the findings were exciting, as drug resistance is one of the biggest challenges in modern healthcare, not only in the case of malaria, but with most infectious agents, including a large number of highly pathogenic bacterial species.

“We are at risk of returning to the pre-antibiotic era if we don’t solve this resistance problem, which constitutes a clear and present danger for global public health. We need innovative ways to address this issue,” he said.

“By targeting the host and not the pathogen itself, we remove the possibility for the pathogen to rapidly become resistant by mutating the target of the drug, as the target is made by the human host, not the pathogen.”

Doerig’s team will now collaborate with the Peter Doherty Institute for Infection and Immunity (Doherty Institute) to investigate potential COVID-19 treatments using this approach, supported by funding from the Victorian Medical Research Acceleration Fund in partnership with the Bio Capital Impact Fund (BCIF).

Co-investigator on the grant, Royal Melbourne Hospital’s Dr. Julian Druce, from the Victorian Infectious Diseases Reference Laboratory (VIDRL) at the Doherty Institute, was part of the team that were first to grow and share the virus that causes COVID-19, and said the research was an important contribution to efforts to defeat the pandemic.

Royal Melbourne Hospital’s Professor Peter Revill, Senior Medical Scientist at the Doherty Institute and a leader on Hepatitis B research, said the approach developed by the RMIT team was truly exciting.

“This has proven successful for other human pathogens including malaria and Hepatitis C virus, and there are now very real prospects to use it to discover novel drug targets for Hepatitis B and COVID-19,” he said.

The paper, ‘Analysis of erythrocyte signalling pathways during Plasmodium falciparum infection identifies targets for host-directed antimalarial intervention’ and is published in Nature Communications.

PfCLK3: A new drug target for malaria.
Inhibition of the malaria parasite protein kinase CLK3 with our probe molecule TCMDC-135051 inhibits the development of liver-stage parasites, kills asexual blood-stage parasites at the trophozoite and schizont stages of the erythrocytic cycle, blocks the development of sexual gametocytes that infect mosquitoes, and blocks exflagellation that results in male gametes
link : https://science.sciencemag.org/content/365/6456/eaau1682?fbclid=IwAR3ZJJVBRqUW97J310gqP0vTwb8Ill7jBho1BAj_egSmvzWDzUXh-rDKYIs

To this day, the malaria parasite Plasmodium spp. remains a scourge particularly in less developed regions of the world. Of the five Plasmodium species that cause human malaria, Plasmodium falciparum (Pf), which is found predominantly in sub-Saharan Africa, was responsible for 50% of all malaria cases and 91% of the 446,000 deaths worldwide in 2016.

The next most widespread is P. vivax (Pv), the dominant species in Latin America and Asia causing a milder form of malaria.1 Other less problematic Plasmodium species are P. ovale (Po), P. malariae (Pm), and P. knowlesi (Pk), the latter much more common in nonhuman primates.

Eliminating malaria throughout the world, as has been achieved in many nations, is considered an achievable goal that will incorporate multipronged strategies including the development of new medicines.

Currently, the World Health Organization (WHO) recommends that artemisinin-based combination therapy (ACT) and vector control measures are key factors in relieving the burden of malaria.2 However, recent reports of emerging resistance toward the ACT regimen3 and other antimalarial drugs with known mechanisms of action4 emphasize the need to expand the diversity of chemical matter acting against novel targets toward more efficacious drugs with multistage parasite life-cycle activity.

In humans, the complex Plasmodium life-cycle encompasses a liver-stage infection wherein motile sporozoites differentiate and proliferate asexually to form merozoites and a blood-stage infection wherein the asexual merozoites replicate within red blood cells (through ring, trophozoite, and schizont substages), egress, and then reinfect red blood cells.5P. vivax and P. ovale sporozoites can also enter a hypnozoite stage that can remain quiescent in the liver for months if not longer before differentiating eventually into merozoites.6

A fraction of the merozoites in red blood cells differentiate and mature to female and male gametocytes that infect the mosquito after transmission from a bite.7 In the mosquito, the gametocytes further differentiate and eventually fuse to form a zygote that further evolves to form sporozoites that get transmitted to people in a mosquito bite.8

Notably, the expression of kinases and their importance to viability vary in the stages and substages of the life-cycle.9

Kinases are key controllers of signal transduction pathways that regulate essential cellular processes such as growth, development, and reproduction in eukaryotic cells.10,11 For this reason, human kinases are pursued as drug targets in a variety of diseases including cancers,12 inflammatory,13 and cardiovascular diseases.14

Since the approval of Gleevec 16 years ago,15 an additional 32 kinase inhibitors targeting the human kinome have been approved by the U.S. Food and Drug Administration (FDA) for clinical use.15

Given the success in developing drugs targeting human kinases, Plasmodium kinases are attractive targets for next generation antimalarials16 as both protein and lipid kinases are involved in key signaling pathways at various stages of the parasite life-cycle.17

The P. falciparum kinome encodes 86 to 99 protein kinase genes16 and a small set of lipid kinase genes. It is highly conserved between Plasmodium species and is much smaller than the human protein kinome of approximately 520 kinases.18Figure​Figure11A shows the phylogenetic tree with a subset of the well-characterized protein kinases of P. falciparum.

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Figure 1
(A) Phylogenetic tree for a set of 38 well-characterized protein kinases in P. falciparum. The bootstrap tree was generated from 500 replicates using the neighbor-joining method on the full protein sequence with evolutionary distances computed using the Poisson correction method in the Mega7 software package.19 Colored labels indicate kinases discussed in this Perspective. Lipid kinases PI3K and PI4K were not included in the analysis. (B) Crystal structure (PDB: 4RZ7) of P. vivax PKG with inhibitor illustrating key interaction in the ATP binding site. (C) Crystal structure (PDB: 4RZ7) of P. vivax PKG inhibitor (cyan) superimposed with dasatinib (purple) from the X-ray structure with activated ABL kinase (PDB: 2GQG). Both inhibitors access the “deep hydrophobic pocket” extending past the threonine gatekeeper residue.

A major challenge when targeting kinases is that inhibitors usually target the highly conserved adenosine triphosphate (ATP)-binding pocket of the enzyme (Figure​Figure11B), and therefore, target selectivity can be difficult to achieve.20

Fortunately, the long independent evolution of the malaria parasite allowed the emergence of distinct features in the malarial kinome.

These include kinases that clearly cluster within groups found in the human genome but that can be distinguished from their mammalian homologues (Figure​Figure11C). This would include Plasmodium kinases from given groups that contain characteristics of other families, such as PfPK6 or PfPK7, and composites between mitogen-activated protein kinase (MAPK) and cyclin dependent kinases (CDKs)21 and cyclic adenosine monophosphate (cAMP)-dependent kinase (PKA) and mitogen-activated protein kinase (MEK), respectively.22

This would also include kinases, such as CDPKs, that belong to a specific group but do not have a clear orthologue in mammalians and kinases that do not cluster within any of the established families, for example, Phe (F)–Ile (I)–Lys (K)–Lys (K) (FIKK). These important divergences can be exploited to synthesize compounds that selectively inhibit Plasmodium kinases over mammalian enzymes.23

The path to delivering a new antimalarial based on inhibiting a Plasmodium kinase is a multistep process. First, kinase essentiality must be validated by determining the effect of disrupting function or diminishing expression in an organism on proliferation in culture or in the host.

This has been achieved for the Plasmodium kinome through kinome-wide reverse genetics studies leading to the identification of 36 protein kinases that are essential (or likely essential) for completion of the erythrocytic cycle in P. falciparum in vitro(20) and of 12 protein kinases that are required for transmission of the rodent malaria parasite P. berghei (Pb) to the mosquito in vivo.(24) Phenotypic validation is a second level of validation defined as a chemical compound inhibiting a kinase target and also demonstrating an effect on the organism (most often cell kill).

Target engagement studies need to be carried out to show that the phenotypic effect is due to binding to the intended kinase and not a different mode-of-action. The third level, in vivo validation, oftentimes denoted as in vivo proof-of-concept (POC), refers to the capability of a compound to create the intended pharmacodynamic (PD) effect in an animal model. For an antimalarial drug, this most often is the reduction of parasitaemia in a mouse model of infection.

To show efficacy, the compound needs favorable in vivo pharmacokinetic (PK) properties for sufficient exposure in the blood to produce the intended PD response. Finally, as the key goal of drug discovery programs, clinical validation represents the fourth level with a drug molecule working effectively in malaria patients.

To achieve clinical validation, the human PK must be favorable, whether it would be exposure in the blood for asexual stages of the malaria parasite or in the liver for liver stage disease.

Importantly, sufficient safety for drug must also be demonstrated preclinically to justify human administration in the clinic and progression through Phases 1, 2, and 3. Table 1 represents a summary of the protein and lipid kinases discussed in this Perspective and their respective levels of validation achieved.

Table 1

Level of Validation Achieved for Plasmodium Targets Covered in This Perspectivea

P. falciparum protein kinasegenetic validationbphenotypic validationcin vivo efficacyclinical validation
CDPK1d 
CDPK4e 
PKG 
PKAe   
MRK  
GSK-3  
NEK-1 
FIKK8   
PI3Kf  
PI4K
a√ Indicates level of validation was achieved.
bGenetic validation of kinases refers to where potential essentiality has been confirmed by knockout, chemical-genetic, or overexpression methods.
cThe fields are checked if an inhibitor of the kinase target also displayed whole cell activity recognizing that the activity may or may not be due solely to inhibition of the target.
dThere are conflicting data in the literature as to whether the target has been genetically validated.
eTransmission-blocking was shown in an animal model.
fDHA, implicated as a PI3K inhibitor, has other modes-of-action that are thought to be primarily responsible for antiplasmodium activity.

In this perspective, the emphasis has been placed on compound series and kinase targets that have a phenotypic level of validation and therefore in vitro antiplasmodial activity due to inhibition of an identified kinase target.

Most interesting are those series that show POC in animal models of infection. Beyond animal models, there is a single example of a kinase target, P. falciparum phosphatidylinositol 4-kinase (PI4K), with inhibitor 37 (MMV048) that has progressed to human clinical trials.25

reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166223/


More information: Analysis of erythrocyte signalling pathways during Plasmodium falciparum infection identifies targets for host-directed antimalarial intervention, Nature Communications (2020). DOI: 10.1038/s41467-020-17829-7

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