Identification of a novel putative inhibitor of the Plasmodium falciparum purine nucleoside phosphorylase: exploring the purine salvage pathway to design new antimalarial drugs

Abstract Purine nucleoside phosphorylase (PNP), the enzyme schematically next to adenosine deaminase in the purine salvage pathway, has been demonstrated cytochemically in peripheral blood lymphocytes of healthy subjects and chronic lymphocytic leukemia (CLL) patients. The enzyme activity is confined to the cytosol. In healthy subjects the majority of lymphocytes are strongly reactive for PNP, whereas the rest are devoid of cytochemically demonstrable activity. The percentage of PNP- positive cells largely corresponds to the number of E rosette-forming cells and is inversely proportional to the number of Ig-bearing cells. In six of seven CLL patients studied only a minor percentage of the lymphocytes showed strong PNP activity, whereas the large majority (88%- -98%) possessed trace activity. Such patients have a high number of Ig- bearing cells and a low number of E rosette-forming cells. A different pattern of markers was found in the lymphocytes of the seventh CLL patient: 66% were strongly reactive for PNP, an important number formed E rosettes, and a minor percentage were Ig bearing. These data indicate that PNP can be useful as a “nonmembrane” marker in the differentiation of the B and T cell origin in CLL and deserves to be studied in other lymphoproliferative disorders.

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Purine nucleoside phosphorylase in chronic lymphocytic leukemia (CLL)

Blood ◽

10.1182/blood.v52.5.886.bloodjournal525886 ◽

1978 ◽

Vol 52 (5) ◽

pp. 886-895

Author(s):

M Borgers ◽

H Verhaegen ◽

M De Brabander ◽

J De Cree ◽

W De Cock ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Healthy Subjects ◽

Purine Nucleoside Phosphorylase ◽

Peripheral Blood Lymphocytes ◽

Lymphocytic Leukemia ◽

Purine Nucleoside ◽

Salvage Pathway ◽

Purine Salvage ◽

Nucleoside Phosphorylase ◽

A Minor

Purine nucleoside phosphorylase (PNP), the enzyme schematically next to adenosine deaminase in the purine salvage pathway, has been demonstrated cytochemically in peripheral blood lymphocytes of healthy subjects and chronic lymphocytic leukemia (CLL) patients. The enzyme activity is confined to the cytosol. In healthy subjects the majority of lymphocytes are strongly reactive for PNP, whereas the rest are devoid of cytochemically demonstrable activity. The percentage of PNP- positive cells largely corresponds to the number of E rosette-forming cells and is inversely proportional to the number of Ig-bearing cells. In six of seven CLL patients studied only a minor percentage of the lymphocytes showed strong PNP activity, whereas the large majority (88%- -98%) possessed trace activity. Such patients have a high number of Ig- bearing cells and a low number of E rosette-forming cells. A different pattern of markers was found in the lymphocytes of the seventh CLL patient: 66% were strongly reactive for PNP, an important number formed E rosettes, and a minor percentage were Ig bearing. These data indicate that PNP can be useful as a “nonmembrane” marker in the differentiation of the B and T cell origin in CLL and deserves to be studied in other lymphoproliferative disorders.

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Exploring new inhibitors of Plasmodium falciparum purine nucleoside phosphorylase

European Journal of Medicinal Chemistry ◽

10.1016/j.ejmech.2010.08.026 ◽

2010 ◽

Vol 45 (11) ◽

pp. 5140-5149 ◽

Cited By ~ 20

Author(s):

Huaqing Cui ◽

Gian Filippo Ruda ◽

Juana Carrero-Lérida ◽

Luis M. Ruiz-Pérez ◽

Ian H. Gilbert ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Purine Nucleoside Phosphorylase ◽

Purine Nucleoside ◽

Nucleoside Phosphorylase

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Microwave-assisted synthesis of C-8 aryl and heteroaryl inosines and determination of their inhibitory activities against Plasmodium falciparum purine nucleoside phosphorylase

European Journal of Medicinal Chemistry ◽

10.1016/j.ejmech.2014.05.073 ◽

2014 ◽

Vol 82 ◽

pp. 459-465 ◽

Cited By ~ 10

Author(s):

Alba Gigante ◽

Eva-María Priego ◽

Paula Sánchez-Carrasco ◽

Luis Miguel Ruiz-Pérez ◽

Johan Vande Voorde ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Purine Nucleoside Phosphorylase ◽

Purine Nucleoside ◽

Microwave Assisted Synthesis ◽

Nucleoside Phosphorylase ◽

Microwave Assisted ◽

Inhibitory Activities

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Adenosine binding to low-molecular-weight purine nucleoside phosphorylase: the structural basis for recognition based on its complex with the enzyme fromSchistosoma mansoni

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444909045715 ◽

2009 ◽

Vol 66 (1) ◽

pp. 73-79 ◽

Cited By ~ 16

Author(s):

Humberto M. Pereira ◽

Martha M. Rezende ◽

Marcelo Santos Castilho ◽

Glaucius Oliva ◽

Richard C. Garratt

Keyword(s):

Molecular Weight ◽

Purine Nucleoside Phosphorylase ◽

De Novo ◽

Purine Nucleoside ◽

Structural Basis ◽

Low Molecular Weight ◽

Salvage Pathway ◽

Structural Explanation ◽

Nucleoside Phosphorylase ◽

Transition State Analogues

Schistosomes are unable to synthesize purinesde novoand depend exclusively on the salvage pathway for their purine requirements. It has been suggested that blockage of this pathway could lead to parasite death. The enzyme purine nucleoside phosphorylase (PNP) is one of its key components and molecules designed to inhibit the low-molecular-weight (LMW) PNPs, which include both the human and schistosome enzymes, are typically analogues of the natural substrates inosine and guanosine. Here, it is shown that adenosine both binds toSchistosoma mansoniPNP and behaves as a weak micromolar inhibitor of inosine phosphorolysis. Furthermore, the first crystal structures of complexes of an LMW PNP with adenosine and adenine are reported, together with those with inosine and hypoxanthine. These are used to propose a structural explanation for the selective binding of adenosine to some LMW PNPs but not to others. The results indicate that transition-state analogues based on adenosine or other 6-amino nucleosides should not be discounted as potential starting points for alternative inhibitors.

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Identifying purine nucleoside phosphorylase as the target of quinine using cellular thermal shift assay

Science Translational Medicine ◽

10.1126/scitranslmed.aau3174 ◽

2019 ◽

Vol 11 (473) ◽

pp. eaau3174 ◽

Cited By ~ 41

Author(s):

Jerzy M. Dziekan ◽

Han Yu ◽

Dan Chen ◽

Lingyun Dai ◽

Grennady Wirjanata ◽

...

Keyword(s):

Purine Nucleoside Phosphorylase ◽

Cysteine Proteinase ◽

Antimalarial Drugs ◽

Purine Nucleoside ◽

Substantial Contribution ◽

Cysteine Proteinase Inhibitor ◽

Nucleoside Phosphorylase ◽

Thermal Shift Assay ◽

Cellular Thermal Shift Assay ◽

Thermal Shift

Mechanisms of action (MoAs) have been elusive for most antimalarial drugs in clinical use. Decreasing responsiveness to antimalarial treatments stresses the need for a better resolved understanding of their MoAs and associated resistance mechanisms. In the present work, we implemented the cellular thermal shift assay coupled with mass spectrometry (MS-CETSA) for drug target identification inPlasmodium falciparum, the main causative agent of human malaria. We validated the efficacy of this approach for pyrimethamine, a folic acid antagonist, and E64d, a broad-spectrum cysteine proteinase inhibitor. Subsequently, we applied MS-CETSA to quinine and mefloquine, two important antimalarial drugs with poorly characterized MoAs. Combining studies in theP. falciparumparasite lysate and intact infected red blood cells, we foundP. falciparumpurine nucleoside phosphorylase (PfPNP) as a common binding target for these two quinoline drugs. Biophysical and structural studies with a recombinant protein further established that both compounds bind within the enzyme’s active site. Quinine binds to PfPNP at low nanomolar affinity, suggesting a substantial contribution to its therapeutic effect. Overall, we demonstrated that implementation of MS-CETSA forP. falciparumconstitutes a promising strategy to elucidate the MoAs of existing and candidate antimalarial drugs.

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A resistant mutant of Plasmodium falciparum purine nucleoside phosphorylase uses wild-type neighbors to maintain parasite survival

Journal of Biological Chemistry ◽

10.1016/j.jbc.2021.100342 ◽

2021 ◽

pp. 100342

Author(s):

Yacoba V.T. Minnow ◽

Rajesh K. Harijan ◽

Vern L. Schramm

Keyword(s):

Plasmodium Falciparum ◽

Purine Nucleoside Phosphorylase ◽

Resistant Mutant ◽

Purine Nucleoside ◽

Wild Type ◽

Nucleoside Phosphorylase ◽

Parasite Survival

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Genetic resistance to purine nucleoside phosphorylase inhibition in Plasmodium falciparum

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1525670115 ◽

2018 ◽

Vol 115 (9) ◽

pp. 2114-2119 ◽

Cited By ~ 8

Author(s):

Rodrigo G. Ducati ◽

Hilda A. Namanja-Magliano ◽

Rajesh K. Harijan ◽

J. Eduardo Fajardo ◽

Andras Fiser ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Transition State ◽

Gene Amplification ◽

Purine Nucleoside Phosphorylase ◽

Point Mutations ◽

Catalytic Site ◽

Purine Nucleoside ◽

Drug Pressure ◽

Nucleoside Phosphorylase ◽

Transition State Analog

Plasmodium falciparum causes the most lethal form of human malaria and is a global health concern. The parasite responds to antimalarial therapies by developing drug resistance. The continuous development of new antimalarials with novel mechanisms of action is a priority for drug combination therapies. The use of transition-state analog inhibitors to block essential steps in purine salvage has been proposed as a new antimalarial approach. Mutations that reduce transition-state analog binding are also expected to reduce the essential catalytic function of the target. We have previously reported that inhibition of host and P. falciparum purine nucleoside phosphorylase (PfPNP) by DADMe-Immucillin-G (DADMe-ImmG) causes purine starvation and parasite death in vitro and in primate infection models. P. falciparum cultured under incremental DADMe-ImmG drug pressure initially exhibited increased PfPNP gene copy number and protein expression. At increased drug pressure, additional PfPNP gene copies appeared with point mutations at catalytic site residues involved in drug binding. Mutant PfPNPs from resistant clones demonstrated reduced affinity for DADMe-ImmG, but also reduced catalytic efficiency. The catalytic defects were partially overcome by gene amplification in the region expressing PfPNP. Crystal structures of native and mutated PfPNPs demonstrate altered catalytic site contacts to DADMe-ImmG. Both point mutations and gene amplification are required to overcome purine starvation induced by DADMe-ImmG. Resistance developed slowly, over 136 generations (2136 clonal selection). Transition-state analog inhibitors against PfPNP are slow to induce resistance and may have promise in malaria therapy.

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