Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development

TY - JOUR

T1 - Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development

AU - Cavazzuti, Antonio

AU - Paglietti, Giuseppe

AU - Hunter, William N.

AU - Gamarro, Francisco

AU - Piras, Sandra

AU - Loriga, Mario

AU - Alleca, Sergio

AU - Corona, Paola

AU - McLuskey, Karen

AU - Tulloch, Lindsay

AU - Gibellini, Federica

AU - Ferrari, Stefania

AU - Costin, Maria Paola

PY - 2008/2/5

Y1 - 2008/2/5

N2 - Pteridine reductase (PTR1) is essential for salvage of pterins by parasitic trypanosomatids and is a target for the development of improved therapies. To identify inhibitors of Leishmania major and Trypanosoma cruzi PTR1, we combined a rapid-screening strategy using a folate-based library with structure-based design. Assays were carried out against folate-dependent enzymes including PTR1, dihydrofolate reductase (DHFR), and thymidylate synthase. Affinity profiling determined selectivity and specificity of a series of quinoxaline and 2,4-diaminopteridine derivatives, and nine compounds showed greater activity against parasite enzymes compared with human enzymes. Compound 6a displayed a K of 100 nM toward LmPTR1, and the crystal structure of the LmPTR1:NADPH:6a ternary complex revealed a substrate-like binding mode distinct from that previously observed for similar compounds. A second round of design, synthesis, and assay produced a compound (6b) with a significantly improved K (37 nM) against LmPTR1, and the structure of this complex was also determined. Biological evaluation of selected inhibitors was performed against the extracellular forms of T. cruzi and L. major, both wild-type and overexpressing PTR1 lines, as a model for PTR1-driven antifolate drug resistance and the intracellular form of T, cruzi. An additive profile was observed when PTR1 inhibitors were used in combination with known DHFR inhibitors, and a reduction in toxicity of treatment was observed with respect to administration of a DHFR inhibitor alone. The successful combination of antifolates targeting two enzymes indicates high potential for such an approach in the development of previously undescribed antiparasitic drugs.

AB - Pteridine reductase (PTR1) is essential for salvage of pterins by parasitic trypanosomatids and is a target for the development of improved therapies. To identify inhibitors of Leishmania major and Trypanosoma cruzi PTR1, we combined a rapid-screening strategy using a folate-based library with structure-based design. Assays were carried out against folate-dependent enzymes including PTR1, dihydrofolate reductase (DHFR), and thymidylate synthase. Affinity profiling determined selectivity and specificity of a series of quinoxaline and 2,4-diaminopteridine derivatives, and nine compounds showed greater activity against parasite enzymes compared with human enzymes. Compound 6a displayed a K of 100 nM toward LmPTR1, and the crystal structure of the LmPTR1:NADPH:6a ternary complex revealed a substrate-like binding mode distinct from that previously observed for similar compounds. A second round of design, synthesis, and assay produced a compound (6b) with a significantly improved K (37 nM) against LmPTR1, and the structure of this complex was also determined. Biological evaluation of selected inhibitors was performed against the extracellular forms of T. cruzi and L. major, both wild-type and overexpressing PTR1 lines, as a model for PTR1-driven antifolate drug resistance and the intracellular form of T, cruzi. An additive profile was observed when PTR1 inhibitors were used in combination with known DHFR inhibitors, and a reduction in toxicity of treatment was observed with respect to administration of a DHFR inhibitor alone. The successful combination of antifolates targeting two enzymes indicates high potential for such an approach in the development of previously undescribed antiparasitic drugs.

KW - antitrypanosomatid agents

KW - antifolates

KW - drug discovery

KW - PARASITE LEISHMANIA-MAJOR

KW - DIHYDROFOLATE-REDUCTASE

KW - THYMIDYLATE SYNTHASE

KW - TRYPANOSOMA-CRUZI

KW - ANTIFOLATE METHOTREXATE

KW - CRYSTAL-STRUCTURE

KW - ESCHERICHIA-COLI

KW - RESISTANCE

KW - PTR1

KW - PURIFICATION

U2 - 10.1073/pnas.0704384105

DO - 10.1073/pnas.0704384105

M3 - Article

C2 - 18245389

SN - 0027-8424

VL - 105

SP - 1448

EP - 1453

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 5

ER -