Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development

Abstract

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 Ki 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 Ki (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.antitrypanosomatid agents ͉ antifolates ͉ drug discovery P rotozoan parasites of the order Kinetoplastida are the causal agents of serious human diseases, including African sleeping sickness, Chagas' disease, and leishmaniasis. There is an urgent need for new, more effective drugs targeting these neglected diseases, because those in current use are toxic, expensive, and often difficult to administer. The problem is compounded by an increase in drug resistance and lack of progress in drug development. Only a single new effective treatment has been developed in the last 25 years, Miltefosine (hexadecylphosphocholine), recently approved in India (1).Enzymes involved in the provision and use of reduced folate cofactors such as dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are valued drug targets for the treatment of bacterial infections (2), cancer (3), and certain parasitic diseases, notably malaria (4). DHFR catalyzes the two-step reduction of folate to tetrahydrofolate, which is then transformed to N 5 ,N 10 -methylene tetrahydrofolate and is used by TS as a methyl donor and reducing agent in the conversion of 2Ј-deoxyuridine-5Ј-monophosphate to 2Ј-deoxythymidine-5Ј-monophosphate. Inhibition of DHFR or TS reduces the cellular pool of 2Ј-deoxythymidine-5Ј-monophosphate, impairing DNA replication and resulting ...

Structure-Based Drug Discovery for Tropical Diseases

“…To confirm this assumption, a database of 440 synthetic folate-like compounds was analyzed aiming at identifying new inhibitors of both enzymes [83]. A series of physicochemical-and pharmacokinetic-based filters was applied to the database retrieving 131 molecules that fit the selection criteria.…”

Diterpenos de Núcleo Kaurano como Inhibidores de la PTR1 de Leishmania: un Estudio In-Silico

“…El experimento de acoplamiento sobre la PTR1 se llevó a cabo entre el ligando-minimizado en la enzima 2QHX a través de un cubo en el centro geométrico del ligando nativo presente en la estructura PDB evaluada, con las dimensiones 24×24×24 Å, que cubre el sitio de unión del ligando con un espaciado de rejilla de 0,375 Å. Las poses de acoplamiento se clasificaron de acuerdo a sus puntajes de docking (como la energía libre de acoplamiento o afinidad) y tanto la lista clasificada de ligandos acoplados y sus correspondientes poses de docking se exportaron como un archivo CSV para su posterior análisis. Los valores de RMSD no superaron los 1,2 Å. Como control del estudio in silico se utilizó el inhibidor (Cavazzuti, et al, 2008).…”

The Potential of Secondary Metabolites from Plants as Drugs or Leads against Protozoan Neglected Diseases—Part III: In-Silico Molecular Docking Investigations

“…An AutoDock study by Saha and Sharma revealed the withanolide 18-acetoxy-5,6-deoxy-5-withenolide D and the steroidal alkaloid sarachine (Figure 9) to be strongly docking phytochemical ligands for T. cruzi trypanothione reductase [397]. Parasitic trypanosomatids salvage pterins from their host organisms using pteridine reductase (PTR1), and this enzyme is a potential target for antiparasitic drug development [242]. Sahi and coworkers have carried out in vitro antileishmanial screening of constituents from Piper longum fruit and found the alkaloid piperlongumide (Figure 10) to be the most active compound [398].…”

“…Plasmepsin II (PMII) 1SME [224]; 1LEE, 1LF2 [225]; 1LF3, 1LF4 [226]; 2BJU [227]; 2IGX, 2IGY [228]; 3F9Q [229]; 1M43 [230]; 1ME6 [231]; 1W6H, 1W6I [232]; 1XDH, 1XE5, 1XE6 [233] Plasmepsin IV (PMIV) 1LS5 [225] Proline racemase (PRACA) 1W61, 1W62 [234] Protein Kinase 5 (PK5) 1OB3, 1V0O, 1V0P [235] Protein tyrosine phosphatase 1 (PTP1) 3M4U [236] 4AZ1 [237] Pteridine reductase 1 (PTR1) 2XOX [238] 1E7W, 1E92 [239]; 1W0C [240]; 2BF7, 2BFA,2BFM, 2BFO, 2BFP [241]; 2QHX, 3H4V [242] 2C7V [243]; 2WD7, 2WD8, 3GN1, 3GN2 [244]; 2VZ0 [245]; 3BMC, 3BMN, 3BMO, 3BMQ, 3JQ6, 3JQ7, 3JQ8, 3JQ9, 3JQA, 3JQB, 3JQC, 3JQD, 3JQE, 3JQF, 3JQG [246]; 2X9N, 2X9G, 2X9V, 3MCV [247]; 2YHI [248] Pteridine reductase 2 (PTR2) 1MXF, 1MXH [249] Purine nucleoside phosphorylase (PNP) 1NW4, 1Q1G [250]; 2BSX, 1SQ6 [251]; 3ENZ [252] Pyridoxal kinase (PdxK) 3ZS7 [253] Pyruvate kinase (PYK)…”