Teppo O. Leino

Pharmacophore Model To Discover OX1 and OX2 Orexin Receptor Ligands

Small molecule agonists and antagonists of the orexinergic system have key implications for research and therapeutic purposes. We report a pharmacophore model trained on ∼200 antagonists and prospectively validated by screening a collection of ∼137,000 compounds. The resulting hit list, 395 compounds, was tested for OX1 and OX2 receptor activity using calcium mobilization assay in recombinant cell lines. Validation was conducted using both calcium mobilization and [(125)I]-orexin-A competition binding. Compounds 4-7 have weak agonist activity and Kis in the 1-30 μM range; compounds 8-14 are antagonists with Kis in the 0.1-10 μM range for OX2 and 1-50 μM for the OX1 receptor. Docking simulations were used to devise a working hypothesis where two subpockets are important for activation, one between TM5 and TM6 lined by Phe5.42, Tyr5.47, and Tyr6.48 and another above the orthosteric pocket lined by Asp2.65 and Tyr7.32.

Synthesis of 1,3,6-Trisubstituted Azulenes

We have developed a short, general synthetic route to 1,3,6-trisubstituted azulenes. The key intermediate, 6-methylazulene, was synthesized from readily available and inexpensive starting materials in 63% yield over two steps. The methyl group of 6-methylazulene was then used as a synthetic handle to introduce different substituents at the 6-position via two different methods. Subsequently, the 1- and 3-positions were substituted with additional functional handles, such as formyl, chloromethylketone, and iodide. The efficiency of the synthetic route was demonstrated by preparing a collection of three different products with the best demonstrated yield 33% over seven steps.

Synthesis and biological evaluation of 2-arylbenzimidazoles targeting Leishmania donovani

A set of 56 2-arylbenzimidazoles was designed, synthesized and tested against Leishmania donovani amastigotes. The left- and right-hand side rings of the molecule, as well as the amide linker were modified. Structurally different derivatives were screened on L. donovani axenic amastigotes at concentrations of 5, 15 and 50 μM, and the ten most active derivatives were selected for further testing. 2-Arylbenzimidazole derivative 24 was active against L. donovani-infected THP-1 cells showing 46% parasite inhibition at 5 μM.

A Developability-Focused Optimization Approach Allows Identification of in Vivo Fast-Acting Antimalarials: N-[3-[(Benzimidazol-2-yl)amino]propyl]amides

Malaria continues to be a major global health problem, being particularly devastating in the African population under the age of five. Artemisinin-based combination therapies (ACTs) are the first-line treatment recommended by the WHO to treat Plasmodium falciparum malaria, but clinical resistance against them has already been reported. As a consequence, novel chemotypes are urgently needed. Herein we report a novel, in vivo active, fast-acting antimalarial chemotype based on a benzimidazole core. This discovery is the result of a medicinal chemistry plan focused on improving the developability profile of an antichlamydial chemical class previously reported by our group.

Synthesis of carboxyimidamide-substituted benzo[c][1,2,5]oxadiazoles and their analogs, and evaluation of biological activity against Leishmania donovani

A facile synthesis route to carboxyimidamide-substituted benzoxadiazoles and related derivatives was developed. A total of 25 derivatives were synthesized. They were evaluated for antileishmanial activity by inhibition of Leishmania donovani axenic amastigote growth using a fluorescent viability microplate assay. The most promising derivative (14) demonstrated an antileishmanial EC50 of 4.0 μM, and it also showed activity in infected macrophages (EC50 5.92 μM) without signs of cytotoxicity.

Stapled truncated orexin peptides as orexin receptor agonists

HIGHLIGHTSPeptide stapling of the neuropeptide orexin‐A15–33 lowers the potency significantly.The peptide C‐terminus is crucial for activity, and modifications were not allowed.Central and N‐terminal modifications yielded bioactive peptides. ABSTRACT The peptides orexin‐A and −B, the endogenous agonists of the orexin receptors, have similar 19‐amino‐acid C‐termini which retain full maximum response as truncated peptides with only marginally reduced potency, while further N‐terminal truncations successively reduce the activity. The peptides have been suggested to bind in an &agr;‐helical conformation, and truncation beyond a certain critical length is likely to disrupt the overall helical structure. In this study, we set out to stabilize the &agr;‐helical conformation of orexin‐A15–33 via peptide stapling at four different sites. At a suggested hinge region, we varied the length of the cross‐linker as well as replaced the staple with two &agr;‐aminoisobutyric acid residues. Modifications close to the peptide C‐terminus, which is crucial for activity, were not allowed. However, central and N‐terminal modifications yielded bioactive peptides, albeit with decreased potencies. This provides evidence that the orexin receptors can accommodate and be activated by &agr;‐helical peptides. The decrease in potency is likely linked to a stabilization of suboptimal peptide conformation or blocking of peptide backbone–receptor interactions at the hinge region by the helical stabilization or the modified amino acids.

Synthesis and Biological Evaluation of 2‐Aminobenzothiazole and Benzimidazole Analogs Based on the Clathrodin Structure

A series of 2‐aminobenzothiazole and benzimidazole analogs based on the clathrodin scaffold was synthesized and investigated for their antimicrobial and antiproliferative activities as well as for their effects in hepatitis C virus (HCV) replicon model. Compound 7, derived from 2‐aminobenzothiazole, exhibited moderate antimicrobial activity only against the Gram‐positive bacterium, Enterococcus faecalis. In the antiviral assay, compounds 4d and 7 were found to suppress the HCV replicon by >70%, but also to exhibit cytotoxicity against the host cells (35 and 44%, respectively). Compounds 4a and 7 demonstrated good activity in the antiproliferative assays on the human melanoma cell line A‐375. To assess the selectivity of the effects between cancerous and noncancerous cells, a mouse fibroblast cell line was used. The IC50 values for compound 7 against the melanoma cell line A‐375 and the fibroblast cell line BALB/c 3T3 were 16 and 71 µM, respectively, yielding fourfold selectivity toward the cancer cell line. These results suggest that compound 7 should be studied further in order to fully explore its potential for drug development.

Asymmetry in catalysis by Thermotoga maritima membrane-bound pyrophosphatase demonstrated by a non-phosphorous allosteric inhibitor

Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyse pyrophosphate into orthophosphates, coupled to the active transport of protons or sodium ions across membranes. They are important in the life cycle of bacteria, archaea, plants, and protist parasites, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first non-phosphorous allosteric inhibitor (Ki of 1.8 ± 0.3 μM) of the thermophilic bacterium Thermotoga maritima membrane-bound pyrophosphatase and its bound structure at 3.7 Å resolution together with the substrate analogue imidodiphosphate. The unit cell contains two protein homodimers, each binding a single inhibitor dimer near the exit channel, creating a hydrophobic clamp that inhibits the movement of β-strand 1–2 during pumping, and thus preventing the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provide the first clear demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.

Pharmacological characterization of the orexin/hypocretin receptor agonist Nag 26

ABSTRACT One promising series of small‐molecule orexin receptor agonists has been described, but the molecular pharmacological properties, i.e. ability and potency to activate the different orexin receptor‐regulated signal pathways have not been reported for any of these ligands. We have thus here assessed these properties for the most potent ligand of the series, 4′‐methoxy‐N,N‐dimethyl‐3′‐[N‐(3‐{[2‐(3‐methylbenzamido)ethyl]amino}phenyl sulfamoyl]‐(1,1′‐biphenyl)‐3‐carboxamide (Nag 26). Chinese hamster ovary‐K1 cells expressing human orexin receptor subtypes OX1 and OX2 were used. Ca2+ elevation and cell viability and death were assessed by fluorescent methods, the extracellular signal‐regulated kinase pathway by a luminescent Elk‐1 reporter assay, and phospholipase C and adenylyl cyclase activities by radioactive methods. The data suggest that for the Gq‐dependent responses, Ca2+, phospholipase C and Elk‐1, Nag 26 is a full agonist for both receptors, though of much lower potency. However, saturation was not always reached for OX1, partially due to Nag 26s low solubility and partially because the response decreased at high concentrations. The latter occurs in the same range as some reduction of cell viability, which is independent of orexin receptors. Based on the EC50, Nag 26 was OX2‐selective by 20–200 fold in different assays, with some indication of biased agonism (as compared to orexin‐A). Nag 26 is a potent orexin receptor agonist with a largely similar pharmacological profile as orexin‐A. However, its weaker potency (low–mid micromolar) and low water solubility as well as the non‐specific effect in the mid‐micromolar range may limit its usefulness under physiological conditions.

Azulene-based compounds for targeting orexin receptors

A library of 70 000 synthetically accessible azulene-based compounds was virtually screened at the OX2 receptor. Based on the results, a series of azulene derivatives was synthesized and the binding to and activation of both orexin receptor subtypes were assessed. Two most promising binders were determined to have inhibition constants in the 3-9 μM range and two other compounds showed weak OX2 receptor agonism. Furthermore, three compounds exhibited a concentration-dependent potentiation of the response to orexin-A at the OX1 but not the OX2 receptors. Altogether this data opens new approaches for further development of antagonists, agonists, and potentiators of orexin response based on the azulene scaffold.