Crystal structure of the M5 muscarinic acetylcholine receptor.

The human M5 muscarinic acetylcholine receptor (mAChR) has recently emerged as an exciting therapeutic target for treating a range of disorders, including drug addiction. However, a lack of structural information for this receptor subtype has limited further drug development and validation. Here we report a high-resolution crystal structure of the human M5 mAChR bound to the clinically used inverse agonist, tiotropium. This structure allowed for a comparison across all 5 mAChR family members that revealed important differences in both orthosteric and allosteric sites that could inform the rational design of selective ligands. These structural studies, together with chimeric swaps between the extracellular regions of the M2 and M5 mAChRs, provided structural insight into kinetic selectivity, where ligands show differential residency times between related family members. Collectively, our study provides important insights into the nature of orthosteric and allosteric ligand interaction across the mAChR family that could be exploited for the design of selective drugs.

Drug discovery and optimization based on the co-crystal structure of natural product with target.

Due to their structural diversities and prevalent biological activities, natural products (NPs) are momentous resources for drug discovery. Although NPs have a wide range of biological activities, many exhibit structural complexity that leads to synthetic difficulties, which combines with inefficient biological activity, toxicity, and unfavorable pharmacokinetic characteristics and ultimately imparts poor safety and efficacy outcomes. Progress in crystallization and computational techniques allow crystallography to have a seasonable influences on drug discovery. By co-crystallizing with proteins, therapeutic targets of NPs in specific diseases can be identified. By analyzing the co-crystal information, the structure-activity relationships (SARs) of NPs targeting specific proteins can be grasped. Under the guidance of co-crystal information, directional structural modification and simplification are powerful strategies for overcoming limitations of NPs, improving the success rate of NP-based drug discovery, and obtaining NP-based drugs with high selectivity, low toxicity and favorable pharmacokinetic characteristics. Here, we review the co-crystal information of a selection of NPs, focusing on the SARs of NPs reflected by co-crystal information and the modification and simplification strategies of NPs, and discuss how to apply co-crystal information in the optimization of NP-based lead compound.

Novel aroyl guanidine anti-trypanosomal compounds that exert opposing effects on parasite energy metabolism.

Human African trypanosomiasis (HAT), or sleeping sickness, is a neglected tropical disease with current treatments marred by severe side effects or delivery issues. To identify novel classes of compounds for the treatment of HAT, high throughput screening (HTS) had previously been conducted on bloodstream forms of T. b. brucei, a model organism closely related to the human pathogens T. b. gambiense and T. b. rhodesiense. This HTS had identified a number of structural classes with potent bioactivity against T. b. brucei (IC50 ≤ 10 µM) with selectivity over mammalian cell-lines (selectivity index of ≥10). One of the confirmed hits was an aroyl guanidine derivative. Deemed to be chemically tractable with attractive physicochemical properties, here we explore this class further to develop the SAR landscape. We also report the influence of the elucidated SAR on parasite metabolism, to gain insight into possible modes of action of this class. Of note, two sub-classes of analogues were identified that generated opposing metabolic responses involving disrupted energy metabolism. This knowledge may guide the future design of more potent inhibitors, while retaining the desirable physicochemical properties and an excellent selectivity profile of the current compound class.

Hit-to-lead optimization of 2-aminoquinazolines as anti-microbial agents against Leishmania donovani.

Visceral leishmaniasis is a potentially fatal disease caused by infection by the intracellular protist pathogens Leishmania donovani or Leishmania infantum. Present therapies are ineffective because of high costs, variable efficacy against different species, the requirement for hospitalization, toxicity and drug resistance. Detailed analysis of previously published hit molecules suggested a crucial role of 'guanidine' linkage for their efficacy against L. donovani. Here we report the design of 2-aminoquinazoline heterocycle as a basic pharmacophore-bearing guanidine linkage. The introduction of various groups and functionality at different positions of the quinazoline scaffold results in enhanced antiparasitic potency with modest host cell cytotoxicity using a physiologically relevant THP-1 transformed macrophage infection model. In terms of the ADME profile, the C7 position of quinazoline was identified as a guiding tool for designing better molecules. The good ADME profile of the compounds suggests that they merit further consideration as lead compounds for treating visceral leishmaniasis.

Enhanced stabilization of the Tobacco mosaic virus using protic ionic liquids.

We report on the use of protic ionic liquids, pILs, as solvents for the solubilisation and stabilization of viruses. We show that the shelf life of the pIL stabilized tobacco mosaic virus is significantly enhanced when compared to traditional phosphate buffer. This has new opportunities for the preparation, characterization and storage of viruses and virus based technologies.

Hit-to-lead optimization of novel phenyl imidazole carboxamides that are active against Leishmania donovani.

Visceral leishmaniasis is a potentially fatal disease caused by the parasitic protists, Leishmania donovani and L. infantum. Current treatments remain unsuitable due to cost, the need for hospitalization, variable efficacy against different species, toxicity and emerging resistance. Herein, we report the SAR exploration of the novel hit 4-Fluoro-N-(5-(4-methoxyphenyl)-1-methyl-1H-imidazole-2-yl)benzamide [1] previously identified from a high throughput screen against Trypanosoma brucei, Trypanosoma cruzi and Leishmania donovani. An extensive and informative set of analogues were synthesized incorporating key modifications around the scaffold resulting in improved potency, whilst the majority of compounds maintained low cytotoxicity against human THP-1 macrophages that are target cells for these pathogens. New lead compounds identified within this study also maintained desirable physicochemical properties, improved metabolic stability in vitro and displayed no significant mitotoxicity against HepG2 cell lines. This compound class warrants continued investigation towards development as a novel treatment for Visceral Leishmaniasis.

Structure-Activity Studies of Truncated Latrunculin Analogues with Antimalarial Activity.

Malarial parasites employ actin dynamics for motility, and any disruption to these dynamics renders the parasites unable to effectively establish infection. Therefore, actin presents a potential target for malarial drug discovery, and naturally occurring actin inhibitors such as latrunculins are a promising starting point. However, the limited availability of the natural product and the laborious route for synthesis of latrunculins have hindered their potential development as drug candidates. In this regard, we recently described novel truncated latrunculins, with superior actin binding potency and selectivity towards P. falciparum actin than the canonical latrunculin B. In this paper, we further explore the truncated latrunculin core to summarize the SAR for inhibition of malaria motility. This study helps further understand the binding pattern of these analogues in order to develop them as drug candidates for malaria.

Discovery of Potent N-Ethylurea Pyrazole Derivatives as Dual Inhibitors of Trypanosoma brucei and Trypanosoma cruzi.

Trypanosoma brucei (T. brucei) and Trypanosoma cruzi (T. cruzi) are causative agents of parasitic diseases known as human African trypanosomiasis and Chagas disease, respectively. Together, these diseases affect 68 million people around the world. Current treatments are unsatisfactory, frequently associated with intolerable side-effects, and generally inadequate in treating all stages of disease. In this paper, we report the discovery of N-ethylurea pyrazoles that potently and selectively inhibit the viability of T. brucei and T. cruzi. Sharp and logical SAR led to the identification of 54 as the best compound, with an in vitro IC50 of 9 nM and 16 nM against T. b. brucei and T. cruzi, respectively. Compound 54 demonstrates favorable physicochemical properties and was efficacious in a murine model of Chagas disease, leading to undetectable parasitemia within 6 days when CYP metabolism was inhibited.

Synthesis and structure-activity relationship study of pyrrolidine-oxadiazoles as anthelmintics against Haemonchus contortus.

Parasitic roundworms (nematodes) are significant pathogens of humans and animals and cause substantive socioeconomic losses due to the diseases that they cause. The control of nematodes in livestock animals relies heavily on the use of anthelmintic drugs. However, their extensive use has led to a widespread problem of drug resistance in these worms. Thus, the discovery and development of novel chemical entities for the treatment of parasitic worms of humans and animals is needed. Herein, we describe our medicinal chemistry optimization efforts of a phenotypic hit against Haemonchus contortus based on a pyrrolidine-oxadiazole scaffold. This led to the identification of compounds with potent inhibitory activities (IC50 = 0.78-22.4 µM) on the motility and development of parasitic stages of H. contortus, and which were found to be highly selective in a mammalian cell counter-screen. These compounds could be used as suitable chemical tools for drug target identification or as lead compounds for further optimization.

Discovery of Acylsulfonohydrazide-Derived Inhibitors of the Lysine Acetyltransferase, KAT6A, as Potent Senescence-Inducing Anti-Cancer Agents.

A high-throughput screen designed to discover new inhibitors of histone acetyltransferase KAT6A uncovered CTX-0124143 (1), a unique aryl acylsulfonohydrazide with an IC50 of 1.0 µM. Using this acylsulfonohydrazide as a template, we herein disclose the results of our extensive structure-activity relationship investigations, which resulted in the discovery of advanced compounds such as 55 and 80. These two compounds represent significant improvements on our recently reported prototypical lead WM-8014 (3) as they are not only equivalently potent as inhibitors of KAT6A but are less lipophilic and significantly more stable to microsomal degradation. Furthermore, during this process, we discovered a distinct structural subclass that contains key 2-fluorobenzenesulfonyl and phenylpyridine motifs, culminating in the discovery of WM-1119 (4). This compound is a highly potent KAT6A inhibitor (IC50 = 6.3 nM; KD = 0.002 µM), competes with Ac-CoA by binding to the Ac-CoA binding site, and has an oral bioavailability of 56% in rats.

Discovery of Benzoylsulfonohydrazides as Potent Inhibitors of the Histone Acetyltransferase KAT6A.

A high-throughput screen for inhibitors of the histone acetyltransferase, KAT6A, led to identification of an aryl sulfonohydrazide derivative (CTX-0124143) that inhibited KAT6A with an IC50 of 1.0 µM. Elaboration of the structure-activity relationship and medicinal chemistry optimization led to the discovery of WM-8014 (97), a highly potent inhibitor of KAT6A (IC50 = 0.008 µM). WM-8014 competes with acetyl-CoA (Ac-CoA), and X-ray crystallographic analysis demonstrated binding to the Ac-CoA binding site. Through inhibition of KAT6A activity, WM-8014 induces cellular senescence and represents a unique pharmacological tool.

Structural Determinants for Small-Molecule Activation of Skeletal Muscle AMPK α2ß2γ1 by the Glucose Importagog SC4.

The AMP-activated protein kinase (AMPK) αßγ heterotrimer regulates cellular energy homeostasis with tissue-specific isoform distribution. Small-molecule activation of skeletal muscle α2ß2 AMPK complexes may prove a valuable treatment strategy for type 2 diabetes and insulin resistance. Herein, we report the small-molecule SC4 is a potent, direct AMPK activator that preferentially activates α2 complexes and stimulates skeletal muscle glucose uptake. In parallel with the term secretagog, we propose "importagog" to define a substance that induces or augments cellular uptake of another substance. Three-dimensional structures of the glucose importagog SC4 bound to activated α2ß2γ1 and α2ß1γ1 complexes reveal binding determinants, in particular a key interaction between the SC4 imidazopyridine 4'-nitrogen and ß2-Asp111, which provide a design paradigm for ß2-AMPK therapeutics. The α2ß2γ1/SC4 structure reveals an interaction between a ß2 N-terminal α helix and the α2 autoinhibitory domain. Our results provide a structure-function guide to accelerate development of potent, but importantly tissue-specific, ß2-AMPK therapeutics.

Pyridyl benzamides as a novel class of potent inhibitors for the kinetoplastid Trypanosoma brucei.

A whole-organism screen of approximately 87000 compounds against Trypanosoma brucei brucei identified a number of promising compounds for medicinal chemistry optimization. One of these classes of compounds we termed the pyridyl benzamides. While the initial hit had an IC50 of 12 µM, it was small enough to be attractive for further optimization, and we utilized three parallel approaches to develop the structure-activity relationships. We determined that the physicochemical properties for this class are generally favorable with particular positions identified that appear to block metabolism when substituted and others that modulate solubility. Our most active compound is 79, which has an IC50 of 0.045 µM against the human pathogenic strain Trypanosoma brucei rhodesiense and is more than 4000 times less active against the mammalian L6 cell line.