Lindsay B. Tulloch

Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development

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.

One Scaffold, Three Binding Modes: Novel and Selective Pteridine Reductase 1 Inhibitors Derived from Fragment Hits Discovered by Virtual Screening

The enzyme pteridine reductase 1 (PTR1) is a potential target for new compounds to treat human African trypanosomiasis. A virtual screening campaign for fragments inhibiting PTR1 was carried out. Two novel chemical series were identified containing aminobenzothiazole and aminobenzimidazole scaffolds, respectively. One of the hits (2-amino-6-chloro-benzimidazole) was subjected to crystal structure analysis and a high resolution crystal structure in complex with PTR1 was obtained, confirming the predicted binding mode. However, the crystal structures of two analogues (2-amino-benzimidazole and 1-(3,4-dichloro-benzyl)-2-amino-benzimidazole) in complex with PTR1 revealed two alternative binding modes. In these complexes, previously unobserved protein movements and water-mediated protein−ligand contacts occurred, which prohibited a correct prediction of the binding modes. On the basis of the alternative binding mode of 1-(3,4-dichloro-benzyl)-2-amino-benzimidazole, derivatives were designed and selective PTR1 inhibitors with low nanomolar potency and favorable physicochemical properties were obtained.

Structure of Staphylococcus aureus 1,4‐dihydroxy‐2‐naphthoyl‐CoA synthase (MenB) in complex with acetoacetyl‐CoA

Vitamin K(2), or menaquinone, is an essential cofactor for many organisms and the enzymes involved in its biosynthesis are potential antimicrobial drug targets. One of these enzymes, 1,4-dihydroxy-2-naphthoyl-CoA synthase (MenB) from the pathogen Staphylococcus aureus, has been obtained in recombinant form and its quaternary structure has been analyzed in solution. Cubic crystals of the enzyme allowed a low-resolution structure (2.9 A) to be determined. The asymmetric unit consists of two subunits and a crystallographic threefold axis of symmetry generates a hexamer consistent with size-exclusion chromatography. Analytical ultracentrifugation indicates the presence of six states in solution, monomeric through to hexameric, with the dimer noted as being particularly stable. MenB displays the crotonase-family fold with distinct N- and C-terminal domains and a flexible segment of structure around the active site. The smaller C-terminal domain plays an important role in oligomerization and also in substrate binding. The presence of acetoacetyl-CoA in one of the two active sites present in the asymmetric unit indicates how part of the substrate binds and facilitates comparisons with the structure of Mycobacterium tuberculosis MenB.

Regulation of the cardiac sodium pump.

In cardiac muscle, the sarcolemmal sodium/potassium ATPase is the principal quantitative means of active transport at the myocyte cell surface, and its activity is essential for maintaining the trans-sarcolemmal sodium gradient that drives ion exchange and transport processes that are critical for cardiac function. The 72-residue phosphoprotein phospholemman regulates the sodium pump in the heart: unphosphorylated phospholemman inhibits the pump, and phospholemman phosphorylation increases pump activity. Phospholemman is subject to a remarkable plethora of post-translational modifications for such a small protein: the combination of three phosphorylation sites, two palmitoylation sites, and one glutathionylation site means that phospholemman integrates multiple signaling events to control the cardiac sodium pump. Since misregulation of cytosolic sodium contributes to contractile and metabolic dysfunction during cardiac failure, a complete understanding of the mechanisms that control the cardiac sodium pump is vital. This review explores our current understanding of these mechanisms.

Design, synthesis and trypanocidal activity of lead compounds based on inhibitors of parasite glycolysis

The glycolytic pathway has been considered a potential drug target against the parasitic protozoan species of Trypanosoma and Leishmania. We report the design and the synthesis of inhibitors targeted against Trypanosoma brucei phosphofructokinase (PFK) and Leishmania mexicana pyruvate kinase (PyK). Stepwise library synthesis and inhibitor design from a rational starting point identified furanose sugar amino amides as a novel class of inhibitors for both enzymes with IC(50) values of 23microM and 26microM against PFK and PyK, respectively. Trypanocidal activity also showed potency in the low micromolar range and confirms these inhibitors as promising candidates for the development towards the design of anti-trypanosomal drugs.

The inhibitory effect of phospholemman on the sodium pump requires its palmitoylation.

Background: Phospholemman regulates the plasmalemmal sodium pump in excitable tissues such as the heart. Results: Phospholemman is palmitoylated at two intracellular cysteines, and this reduces ion transport by the sodium pump. Conclusion: Phospholemman must be palmitoylated to inhibit the sodium pump. Significance: This is a potentially new way to regulate the sodium pump, an enzyme expressed in most eukaryotic cells. Phospholemman (PLM), the principal sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. We investigated post-translational modifications of PLM additional to phosphorylation in adult rat ventricular myocytes (ARVM). LC-MS/MS of tryptically digested PLM immunoprecipitated from ARVM identified cysteine 40 as palmitoylated in some peptides, but no information was obtained regarding the palmitoylation status of cysteine 42. PLM palmitoylation was confirmed by immunoprecipitating PLM from ARVM loaded with [3H]palmitic acid and immunoblotting following streptavidin affinity purification from ARVM lysates subjected to fatty acyl biotin exchange. Mutagenesis identified both Cys-40 and Cys-42 of PLM as palmitoylated. Phosphorylation of PLM at serine 68 by PKA in ARVM or transiently transfected HEK cells increased its palmitoylation, but PKA activation did not increase the palmitoylation of S68A PLM-YFP in HEK cells. Wild type and unpalmitoylatable PLM-YFP were all correctly targeted to the cell surface membrane, but the half-life of unpalmitoylatable PLM was reduced compared with wild type. In cells stably expressing inducible PLM, PLM expression inhibited the sodium pump, but PLM did not inhibit the sodium pump when palmitoylation was inhibited. Hence, palmitoylation of PLM controls its turnover, and palmitoylated PLM inhibits the sodium pump. Surprisingly, phosphorylation of PLM enhances its palmitoylation, probably through the enhanced mobility of the phosphorylated intracellular domain increasing the accessibility of cysteines for the palmitoylating enzyme, with interesting theoretical implications. All FXYD proteins have conserved intracellular cysteines, so FXYD protein palmitoylation may be a universal means to regulate the sodium pump.

Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate

The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH‐dependent short‐chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad‐spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP+ and the inhibitor determined at 2.2 Å resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the β6‐α6 loop and α6 helix at one side of the substrate‐binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C‐terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.

Diversity oriented syntheses of fused pyrimidines designed as potential antifolates

Diversity oriented syntheses of some furo[2,3-d]pyrimidines and pyrrolo[2,3-d]pyrimidines related to folate, guanine, and diaminopyrimidine-containing drugs have been developed for the preparation of potential anti-infective and anticancer compounds. Amide couplings and Suzuki couplings on the basic heterocyclic templates were used, in the latter case yields being especially high using aromatic trifluoroborates as the coupling partner. A new ring synthesis of 6-aryl-substituted deazaguanines bearing 2-alkylthio groups has been developed using Michael addition of substituted nitrostyrenes. Diversity at C-2 has been introduced by oxidation and substitution with a range of amino nucleophiles. The chemical reactivity of these pyrrolopyrimidines with respect to both electrophilic substitution in ring synthesis and nucleophilic substitution for diversity is discussed. Several compounds were found to inhibit pteridine reductases from the protozoan parasites Trypanosoma brucei and Leishmania major at the micromolar level and to inhibit the growth of Trypanosma brucei brucei in cell culture at higher concentrations. From these results, significant structural features required for inhibition of this important drug target enzyme have been identified.

Structure-based design of pteridine reductase inhibitors targeting african sleeping sickness and the leishmaniases.

Pteridine reductase (PTR1) is a target for drug development against Trypanosoma and Leishmania species, parasites that cause serious tropical diseases and for which therapies are inadequate. We adopted a structure-based approach to the design of novel PTR1 inhibitors based on three molecular scaffolds. A series of compounds, most newly synthesized, were identified as inhibitors with PTR1-species specific properties explained by structural differences between the T. brucei and L. major enzymes. The most potent inhibitors target T. brucei PTR1, and two compounds displayed antiparasite activity against the bloodstream form of the parasite. PTR1 contributes to antifolate drug resistance by providing a molecular bypass of dihydrofolate reductase (DHFR) inhibition. Therefore, combining PTR1 and DHFR inhibitors might improve therapeutic efficacy. We tested two new compounds with known DHFR inhibitors. A synergistic effect was observed for one particular combination highlighting the potential of such an approach for treatment of African sleeping sickness.

A Separate Pool of Cardiac Phospholemman That Does Not Regulate or Associate with the Sodium Pump: MULTIMERS OF PHOSPHOLEMMAN IN VENTRICULAR MUSCLE

Background: Phospholemman regulates the plasmalemmal sodium pump in excitable tissues. Results: In cardiac muscle, a subpopulation of phospholemman with a unique phosphorylation signature associates with other phospholemman molecules but not with the pump. Conclusion: Phospholemman oligomers exist in cardiac muscle. Significance: Much like phospholamban regulation of SERCA, phospholemman exists as both a sodium pump inhibiting monomer and an unassociated oligomer. Phospholemman (PLM), the principal quantitative sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. Much like phospholamban, which regulates the related ATPase SERCA, PLM is reported to oligomerize. We investigated subpopulations of PLM in adult rat ventricular myocytes based on phosphorylation status. Co-immunoprecipitation identified two pools of PLM: one not associated with the sodium pump phosphorylated at Ser63 and one associated with the pump, both phosphorylated at Ser68 and unphosphorylated. Phosphorylation of PLM at Ser63 following activation of PKC did not abrogate association of PLM with the pump, so its failure to associate with the pump was not due to phosphorylation at this site. All pools of PLM co-localized to cell surface caveolin-enriched microdomains with sodium pump α subunits, despite the lack of caveolin-binding motif in PLM. Mass spectrometry analysis of phosphospecific immunoprecipitation reactions revealed no unique protein interactions for Ser63-phosphorylated PLM, and cross-linking reagents also failed to identify any partner proteins for this pool. In lysates from hearts of heterozygous transgenic animals expressing wild type and unphosphorylatable PLM, Ser63-phosphorylated PLM co-immunoprecipitated unphosphorylatable PLM, confirming the existence of PLM multimers. Dephosphorylation of the PLM multimer does not change sodium pump activity. Hence like phospholamban, PLM exists as a pump-inhibiting monomer and an unassociated oligomer. The distribution of different PLM phosphorylation states to different pools may be explained by their differential proximity to protein phosphatases rather than a direct effect of phosphorylation on PLM association with the pump.