Crystal structure of the ternary complex of Leishmania major pteridine reductase 1 with the cofactor NADP+/NADPH and the substrate folic acid

Pteridine reductase 1 (PTR1) is a key enzyme of the folate pathway in protozoan parasites of the genera Leishmania and Trypanosoma and is a valuable drug target for tropical diseases. This enzyme is able to catalyze the NADPH-dependent reduction of both conjugated (folate) and unconjugated (biopterin) pterins to their tetrahydro forms, starting from oxidized- or dihydrostate substrates. The currently available X-ray structures of Leishmania major PTR1 (LmPTR1) show the enzyme in its unbound, unconjugated substrate-bound (with biopterin derivatives) and inhibitor-bound forms. However, no structure has yet been determined of LmPTR1 bound to a conjugated substrate. Here, the high-resolution crystal structure of LmPTR1 in complex with folic acid is presented and the intermolecular forces that drive the binding of the substrate in the catalytic pocket are described. By expanding the collection of LmPTR1 structures in complex with process intermediates, additional insights into the active-site rearrangements that occur during the catalytic process are provided. In contrast to previous structures with biopterin derivatives, a small but significant difference in the orientation of Asp181 and Tyr194 of the catalytic triad is found. This feature is shared by PTR1 from T. brucei (TbPTR1) in complex with the same substrate molecule and may be informative in deciphering the importance of such residues at the beginning of the catalytic process

Chemistry at the protein-mineral interface in L-ferritin assists the assembly of a functional (μ3-oxo)Tris[(μ2-peroxo)] triiron(III) cluster

X-ray structures of homopolymeric L-ferritin obtained by freezing protein crystals at increasing exposure times to a ferrous solution showed the progressive formation of a triiron cluster on the inner cage surface of each subunit. After 60 min exposure, a fully assembled (μ3-oxo)Tris[(μ2-peroxo)(μ2-glutamato-κO:κO′)](glutamato-κO)(diaquo)triiron(III) anionic cluster appears in human L-ferritin. Glu60, Glu61, and Glu64 provide the anchoring of the cluster to the protein cage. Glu57 shuttles incoming iron ions toward the cluster. We observed a similar metallocluster in horse spleen L-ferritin, indicating that it represents a common feature of mammalian L-ferritins. The structures suggest a mechanism for iron mineral formation at the protein interface. The functional significance of the observed patch of carboxylate side chains and resulting metallocluster for biomineralization emerges from the lower iron oxidation rate measured in the E60AE61AE64A variant of human L-ferritin, leading to the proposal that the observed metallocluster corresponds to the suggested, but yet unobserved, nucleation site of L-ferritin

Chroman-4-One Derivatives Targeting Pteridine Reductase 1 and Showing Anti-Parasitic Activity

Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1 (PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one analogues (1-3) of previously published chromen-4-one derivatives were synthesized and biologically evaluated against parasitic enzymes (Trypanosoma brucei PTR1-TbPTR1 and Leishmania major-LmPTR1) and parasites (Trypanosoma brucei and Leishmania infantum). A crystal structure of TbPTR1 in complex with compound 1 and the first crystal structures of LmPTR1-flavanone complexes (compounds 1 and 3) were solved. The inhibitory activity of the chroman-4-one and chromen-4-one derivatives was explained by comparison of observed and predicted binding modes of the compounds. Compound 1 showed activity both against the targeted enzymes and the parasites with a selectivity index greater than 7 and a low toxicity. Our results provide a basis for further scaffold optimization and structure-based drug design aimed at the identification of potent anti-trypanosomatidic compounds targeting multiple PTR1 variants

Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs

Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 \u3bcM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 \u3bcM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability

Exploiting the 2-Amino-1,3,4-thiadiazole Scaffold To Inhibit <i>Trypanosoma brucei </i>Pteridine Reductase in Support of Early-Stage Drug Discovery

Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 μM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained

Accelerating Drug Discovery Efforts for Trypanosomatidic Infections Using an Integrated Transnational Academic Drug Discovery Platform

According to the World Health Organization, more than 1 billion people are at risk of or are affected by neglected tropical diseases. Examples of such diseases include trypanosomiasis, which causes sleeping sickness; leishmaniasis; and Chagas disease, all of which are prevalent in Africa, South America, and India. Our aim within the New Medicines for Trypanosomatidic Infections project was to use (1) synthetic and natural product libraries, (2) screening, and (3) a preclinical absorption, distribution, metabolism, and excretion\u2013toxicity (ADME-Tox) profiling platform to identify compounds that can enter the trypanosomatidic drug discovery value chain. The synthetic compound libraries originated from multiple scaffolds with known antiparasitic activity and natural products from the Hypha Discovery MycoDiverse natural products library. Our focus was first to employ target-based screening to identify inhibitors of the protozoan Trypanosoma brucei pteridine reductase 1 (TbPTR1) and second to use a Trypanosoma brucei phenotypic assay that made use of the T. brucei brucei parasite to identify compounds that inhibited cell growth and caused death. Some of the compounds underwent structure-activity relationship expansion and, when appropriate, were evaluated in a preclinical ADME-Tox assay panel. This preclinical platform has led to the identification of lead-like compounds as well as validated hits in the trypanosomatidic drug discovery value chain

Elucidating the 3D Structure of a Surface Membrane Antigen from Trypanosoma cruzi as a Serodiagnostic Biomarker of Chagas Disease

Chagas disease (CD) is a vector-borne parasitosis, caused by the protozoan parasite Trypanosoma cruzi, that affects millions of people worldwide. Although endemic in South America, CD is emerging throughout the world due to climate change and increased immigratory flux of infected people to non-endemic regions. Containing of the diffusion of CD is challenged by the asymptomatic nature of the disease in early infection stages and by the lack of a rapid and effective diagnostic test. With the aim of designing new serodiagnostic molecules to be implemented in a microarray-based diagnostic set-up for early screening of CD, herein, we report the recombinant production of the extracellular domain of a surface membrane antigen from T. cruzi (TcSMP) and confirm its ability to detect plasma antibodies from infected patients. Moreover, we describe its high-resolution (1.62 &Aring;) crystal structure, to which in silico epitope predictions were applied in order to locate the most immunoreactive regions of TcSMP in order to guide the design of epitopes that may be used as an alternative to the full-length antigen for CD diagnosis. Two putative, linear epitopes, belonging to the same immunogenic region, were synthesized as free peptides, and their immunological properties were tested in vitro. Although both peptides were shown to adopt a structural conformation that allowed their recognition by polyclonal antibodies raised against the recombinant protein, they were not serodiagnostic for T. cruzi infections. Nevertheless, they represent good starting points for further iterative structure-based (re)design cycles

Release of Soybean Isoflavones by Using a β‐Glucosidase from Alicyclobacillus herbarius

β‐Glucosidases are used in the food industry to hydrolyse glycosidic bonds in complex sugars, with enzymes sourced from extremophiles better able to tolerate the process conditions. In this work, a novel β‐glycosidase from the acidophilic organism Alicyclobacillus herbarius was cloned and heterologously expressed in Escherichia coli BL21(DE3). AheGH1 was stable over a broad range of pH values (5–11) and temperatures (4–55 °C). The enzyme exhibited excellent tolerance to fructose and good tolerance to glucose, retaining 65 % activity in the presence of 10 % (w/v) glucose. It also tolerated organic solvents, some of which appeared to have a stimulating effect, in particular ethanol with a 1.7‐fold increase in activity at 10 % (v/v). The enzyme was then applied for the cleavage of isoflavone from isoflavone glucosides in an ethanolic extract of soy flour, to produce soy isoflavones, which constitute a valuable food supplement, full conversion was achieved within 15 min at 30 °C

The structure of the human glutaminyl cyclase–SEN177 complex indicates routes for developing new potent inhibitors as possible agents for the treatment of neurological disorders

Abstract: Recent evidence links the role of human glutaminyl cyclase (hQC) to the amyloidogenic process involved in Alzheimer’s disease (AD). hQC is a zinc enzyme present in neuronal tissue and its activity is responsible for the cyclization of N-terminal Gln or Glu β-amyloid peptides, leading to N-pyroglutamic acid peptides (pE-Aβ) that is probably a crucial event in the initiation and progress of the disease. Indeed, pE-containing peptides exhibit an elevated neurotoxicity and a tendency to aggregate. These observations render hQC inhibition an attractive strategy for developing new molecules active against AD. We present here the crystal structure of hQC in complex with SEN177, a newly designed molecule. The SEN177-binding mode to hQC differs from that of the known hQC inhibitors. SEN177 Ki on hQC is 20 nM, comparable or better than that of the most potent known hQC inhibitors PBD150 and PQ912. In addition, SEN177 already demonstrated relevant pharmacological properties in in vivo models of Huntington’s disease. All these properties make SEN177 an important scaffold for developing molecules acting on AD and related diseases. Graphical abstract: [Figure not available: see fulltext.]