Design of the Global Health chemical diversity library v2 for screening against infectious diseases.

There is a need for novel chemical matter for phenotypic and target-based screens to find starting points for drug discovery programmes in neglected infectious diseases and non-hormonal contraceptives that disproportionately affect Low- and Middle-Income Countries (LMICs). In some disease areas multiple screens of corporate and other libraries have been carried out, giving rise to some valuable starting points and leading to preclinical candidates. Whilst in other disease areas, little screening has been carried out. Much screening against pathogens has been conducted phenotypically as there are few robustly validated protein targets. However, many of the active compound series identified share the same molecular targets. To address the need for new chemical material, in this article we describe the design of a new library, designed for screening in drug discovery programmes for neglected infectious diseases. The compounds have been selected from the Enamine REAL (REadily AccessibLe) library, a virtual library which contains approximately 4.5 billion molecules. The molecules theoretically can be synthesized quickly using commercially available intermediates and building blocks. The vast majority of these have not been prepared before, so this is a source of novel compounds. In this paper we describe the design of a diverse library of 30,000 compounds from this collection (graphical abstract). The new library will be made available to laboratories working in neglected infectious diseases, subject to a review process. The project has been supported by the Bill & Melinda Gates Foundation and the Wellcome Trust (Wellcome).

Energetics of a protein disorder-order transition in small molecule recognition.

Many proteins recognise other proteins via mechanisms that involve the folding of intrinsically disordered regions upon complex formation. Here we investigate how the selectivity of a drug-like small molecule arises from its modulation of a protein disorder-to-order transition. Binding of the compound AM-7209 has been reported to confer order upon an intrinsically disordered 'lid' region of the oncoprotein MDM2. Calorimetric measurements revealed that truncation of the lid region of MDM2 increases the apparent dissociation constant of AM-7209 250-fold. By contrast, lid truncation has little effect on the binding of the ligand Nutlin-3a. Insights into these differential binding energetics were obtained via a complete thermodynamic analysis that featured adaptive absolute alchemical free energy of binding calculations with enhanced-sampling molecular dynamics simulations. The simulations reveal that in apo MDM2 the ordered lid state is energetically disfavoured. AM-7209, but not Nutlin-3a, shows a significant energetic preference for ordered lid conformations, thus shifting the balance towards ordering of the lid in the AM-7209/MDM2 complex. The methodology reported herein should facilitate broader targeting of intrinsically disordered regions in medicinal chemistry.

Genotoxicity assessment of four novel quinazoline-derived trypanocidal agents in the Drosophila wing somatic mutation and recombination test.

Chagas disease, caused by the protozoan Trypanosoma cruzi, has increased in the world due to migration, travelling and climate change; at present, the principal problem is that common trypanocidal agents have resulted in toxic or inconvenient side effects. We tested for genotoxicity in the standard (ST) and high bioactivation (HB) crosses of Drosophila wing somatic mutation and recombination test, four novel trypanocidal agents derived from 2, 4, 6-triaminquinazoline (TAQ): 2,4-diamino-6 nitro-1,3 diazonaftalene (S-1QN2-1), 2,4-diacetamino-6-amino 1,3 diazonaftalene (D-1), N6-(4,methoxybenzyl)quinazoline-2,4,6-triamine (GHPM) and N6-[4-(trifluoromethoxy)benzyl]quinazoline-2,4,6-triamine (GHPMF) at 1.9, 3.9, 7.9 and 15 µM, respectively. Also, high-pressure liquid chromatography (HPLC) analysis was run to determine the remanence of either drug in flare, and Oregon R(R)-flare flies emerged from treated larvae. S-1QN2-1 showed genotoxicity only in the ST cross, increasing the small, large and total spot frequencies at all concentrations and twin spots only at 1.9 µM; D-1 and GHPM showed significant increments of large spots only at 15 µM in the ST cross; GHPMF was not genotoxic at any concentration or either cross. In the mwh clones accumulated distribution frequencies analysis, associated with disrupted cell division, S-1QN2-1 caused alterations in the ST cross at all concentrations but only at 15 µM in the HB cross; D-1 caused alterations at 3.9, 7.9 and 15 µM in the ST cross and at 1.9 and 15 µM in the HB cross; GHPM caused alterations at 7.9 and 15 µM in the ST cross and also at 1.9, 3.9 and 7.9 µM in the HB cross; GHPMF caused those alterations at all concentrations in the ST cross and at 1.9, 3.9 and 7.9 µM in the HB cross. The HPLC results indicated no traces of either agent in the flare and Oregon R(R)-flare flies. We conclude that S-1QN2-1 is clearly genotoxic, D-1 and GHPM have an unclear genotoxicity and GHPMF was not genotoxic; all quinazoline derivatives disrupted cell division. GHPMF is a good candidate to be tested in other genotoxicity and cytotoxic bioassays. The differences in the genotoxic activity of these trypanocidal agents are correlated with differences in their chemical structure.

Design, synthesis and biological evaluation of quinazoline derivatives as anti-trypanosomatid and anti-plasmodial agents.

In this paper, the design, synthesis and biological evaluation of a set of quinazoline-2,4,6-triamine derivatives (1-9) as trypanocidal, antileishmanial and antiplasmodial agents are explained. The compounds were rationalized basing on docking studies of the dihydrofolate reductase (DHFR from Trypanosoma cruzi, Leishmania major and Plasmodium vivax) and pteridin reductase (PTR from T. cruzi and L. major) structures. All compounds were in vitro screened against both bloodstream trypomastigotes of T. cruzi (NINOA and INC-5 strains) and promatigotes of Leishmania mexicana (MHOM/BZ/61/M379 strain), and also for cytotoxicity using Vero cell line. Against T. cruzi, three compounds (5, 6 and 8) were the most effective showing a better activity profile than nifurtimox and benznidazole (reference drugs). Against L. mexicana, four compounds (5, 6, 8, and 9) exhibited the highest activity, even than glucantime (reference drug). In the cytotoxicity assay, protozoa were more susceptible than Vero cells. In vivo Plasmodium berghei assay (ANKA strain), the compounds 1, 5, 6 and 8 showed a more comparable activity than chloroquine and pyrimethamine (reference drugs) when they were administrated by the oral route. The antiprotozoal activity of these substances, endowed with redox properties, represented a good starting point for a medicinal chemistry program aiming for chemotherapy of Chagas' disease, leishmaniosis and malaria.

Antileishmanial activity of quinazoline derivatives: synthesis, docking screens, molecular dynamic simulations and electrochemical studies.

A series of quinazoline-2,4,6-triamine were synthesized and evaluated in vitro against Leishmania mexicana. Among them, N(6)-(ferrocenmethyl)quinazolin-2,4,6-triamine (H2) showed activity on promastigotes and intracellular amastigotes, as well as low cytotoxicity in mammalian cells. Docking and electrochemical studies showed the importance of both the ferrocene and the heterocyclic nucleus to the observed activity. H2 is readily oxidized electrochemically, indicating that the mechanism of action probably involves redox reactions.

Effects of an antimalarial quinazoline derivative on human erythrocytes and on cell membrane molecular models.

Plasmodium, the parasite which causes malaria in humans multiplies in the liver and then infects circulating erythrocytes. Thus, the role of the erythrocyte cell membrane in antimalarial drug activity and resistance has key importance. The effects of the antiplasmodial N(6)-(4-methoxybenzyl)quinazoline-2,4,6-triamine (M4), and its inclusion complex (M4/HPßCD) with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) on human erythrocytes and on cell membrane molecular models are herein reported. This work evidences that M4/HPßCD interacts with red cells as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes induced shape changes at a 10µM concentration; b) in isolated unsealed human erythrocyte membranes (IUM) a concentration as low as 1µM induced sharp DPH fluorescence anisotropy decrease whereas increasing concentrations produced a monotonically decrease of DPH fluorescence lifetime at 37°C; c) X-ray diffraction studies showed that 200µM induced a complete structural perturbation of dimyristoylphosphatidylcholine (DMPC) bilayers whereas no significant effects were detected in dimyristoylphosphatidylethanolamine (DMPE) bilayers, classes of lipids present in the outer and inner monolayers of the human erythrocyte membrane, respectively; d) fluorescence spectroscopy data showed that increasing concentrations of the complex interacted with the deep hydrophobic core of DMPC large unilamellar vesicles (LUV) at 18°C. All these experiments are consistent with the insertion of M4/HPßCD in the outer monolayer of the human erythrocyte membrane; thus, it can be considered a promising and novel antimalarial agent.