Discovery of Amoebicidal Compounds by Combining Computational and Experimental Approaches.

Pathogenic and opportunistic free-living amoebae such as Acanthamoeba spp. can cause keratitis (Acanthamoeba keratitis [AK]), which may ultimately lead to permanent visual impairment or blindness. Acanthamoeba can also cause rare but usually fatal granulomatous amoebic encephalitis (GAE). Current therapeutic options for AK require a lengthy treatment with nonspecific drugs that are often associated with adverse effects. Recent developments in the field led us to target cAMP pathways, specifically phosphodiesterase. Guided by computational tools, we targeted the Acanthamoeba phosphodiesterase RegA. Computational studies led to the construction and validation of a homology model followed by a virtual screening protocol guided by induced-fit docking and chemical scaffold analysis using our medicinal and biological chemistry (MBC) chemical library. Subsequently, 18 virtual screening hits were prioritized for further testing in vitro against Acanthamoeba castellanii, identifying amoebicidal hits containing piperidine and urea imidazole cores. Promising activities were confirmed in the resistant cyst form of the amoeba and in additional clinical Acanthamoeba strains, increasing their therapeutic potential. Mechanism-of-action studies revealed that these compounds produce apoptosis through reactive oxygen species (ROS)-mediated mitochondrial damage. These chemical families show promise for further optimization to produce effective antiacanthamoebal drugs.

Towards discovery of new leishmanicidal scaffolds able to inhibit Leishmania GSK-3.

Previous reports have validated the glycogen synthase kinase-3 (GSK-3) as a druggable target against the human protozoan parasite Leishmania. This prompted us to search for new leishmanicidal scaffolds as inhibitors of this enzyme from our in-house library of human GSK-3ß inhibitors, as well as from the Leishbox collection of leishmanicidal compounds developed by GlaxoSmithKline. As a result, new leishmanicidal inhibitors acting on Leishmania GSK-3 at micromolar concentrations were found. These inhibitors belong to six different chemical classes (thiadiazolidindione, halomethylketone, maleimide, benzoimidazole, N-phenylpyrimidine-2-amine and oxadiazole). In addition, the binding mode of the most active compounds into Leishmania GSK-3 was approached using computational tools. On the whole, we have uncovered new chemical scaffolds with an appealing prospective in the development and use of Leishmania GSK-3 inhibitors against this infectious protozoan.

Deciphering the enzymatic target of a new family of antischistosomal agents bearing a quinazoline scaffold using complementary computational tools.

A previous phenotypic screening campaign led to the identification of a quinazoline derivative with promising in vitro activity against Schistosoma mansoni. Follow-up studies of the antischistosomal potential of this candidate are presented here. The in vivo studies in a S. mansoni mouse model show a significant reduction of total worms and a complete disappearance of immature eggs when administered concomitantly with praziquantel in comparison with the administration of praziquantel alone. This fact is of utmost importance because eggs are responsible for the pathology and transmission of the disease. Subsequently, the chemical optimisation of the structure in order to improve the metabolic stability of the parent compound was carried out leading to derivatives with improved drug-like properties. Additionally, the putative target of this new class of antischistosomal compounds was envisaged by using computational tools and the binding mode to the target enzyme, aldose reductase, was proposed.

Imidazole Derivatives as Promising Agents for the Treatment of Chagas Disease.

More than 100 years after being first described, Chagas disease remains endemic in 21 Latin American countries and has spread to other continents. Indeed, this disease, which is caused by the protozoan parasite Trypanosoma cruzi, is no longer just a problem for the American continents but has become a global health threat. Current therapies, i.e., nifurtimox and benznidazole (Bz), are far from being adequate, due to their undesirable effects and their lack of efficacy in the chronic phases of the disease. In this work, we present an in-depth phenotypic evaluation in T. cruzi of a new class of imidazole compounds, which were discovered in a previous phenotypic screen against different trypanosomatids and were designed as potential inhibitors of cAMP phosphodiesterases (PDEs). The confirmation of several activities similar or superior to that of Bz prompted a synthesis program of hit optimization and extended structure-activity relationship aimed at improving drug-like properties such as aqueous solubility, which resulted in additional hits with 50% inhibitory concentration (IC50) values similar to that of Bz. The cellular effects of one representative hit, compound 9, on bloodstream trypomastigotes were further investigated. Transmission electron microscopy revealed cellular changes, after just 2 h of incubation with the IC50 concentration, that were consistent with induced autophagy and osmotic stress, mechanisms previously linked to cAMP signaling. Compound 9 induced highly significant increases in both cellular and medium cAMP levels, confirming that inhibition of T. cruzi PDE(s) is part of its mechanism of action. The potent and selective activity of this imidazole-based PDE inhibitor class against T. cruzi constitutes a successful repurposing of research into inhibitors of mammalian PDEs.

Computer-aided molecular design of pyrazolotriazines targeting glycogen synthase kinase 3.

Numerous studies have highlighted the implications of the glycogen synthase kinase 3 (GSK-3) in several processes associated with Alzheimer's disease (AD). Therefore, GSK-3 has become a crucial therapeutic target for the treatment of this neurodegenerative disorder. Hereby, we report the design and multistep synthesis of ethyl 4-oxo-pyrazolo[4,3-d][1-3]triazine-7-carboxylates and their biological evaluation as GSK-3 inhibitors. Molecular modelling studies allow us to develop this new scaffold optimising the chemical structure. Potential binding mode determination in the enzyme and the analysis of the key features in the catalytic site are also described. Furthermore, the ability of pyrazolotriazinones to cross the blood-brain barrier (BBB) was evaluated by passive diffusion and those who showed great GSK-3 inhibition and permeation to the central nervous system (CNS) showed neuroprotective properties against tau hyperphosphorylation in a cell-based model. These new brain permeable pyrazolotriazinones may be used for key in vivo studies and may be considered as new leads for further optimisation for the treatment of AD.

Cyclic Nucleotide-Specific Phosphodiesterases as Potential Drug Targets for Anti-Leishmania Therapy.

The available treatments for leishmaniasis are less than optimal due to inadequate efficacy, toxic side effects, and the emergence of resistant strains, clearly endorsing the urgent need for discovery and development of novel drug candidates. Ideally, these should act via an alternative mechanism of action to avoid cross-resistance with the current drugs. As cyclic nucleotide-specific phosphodiesterases (PDEs) of Leishmania major have been postulated as putative drug targets, a series of potential inhibitors of Leishmania PDEs were explored. Several displayed potent and selective in vitro activity against L. infantum intracellular amastigotes. One imidazole derivative, compound 35, was shown to reduce the parasite loads in vivo and to increase the cellular cyclic AMP (cAMP) level at in a dose-dependent manner at just 2× and 5× the 50% inhibitory concentration (IC50), indicating a correlation between antileishmanial activity and increased cellular cAMP levels. Docking studies and molecular dynamics simulations pointed to imidazole 35 exerting its activity through PDE inhibition. This study establishes for the first time that inhibition of cAMP PDEs can potentially be exploited for new antileishmanial chemotherapy.

Identification of new allosteric sites and modulators of AChE through computational and experimental tools.

Allosteric sites on proteins are targeted for designing more selective inhibitors of enzyme activity and to discover new functions. Acetylcholinesterase (AChE), which is most widely known for the hydrolysis of the neurotransmitter acetylcholine, has a peripheral allosteric subsite responsible for amyloidosis in Alzheimer's disease through interaction with amyloid ß-peptide. However, AChE plays other non-hydrolytic functions. Here, we identify and characterise using computational tools two new allosteric sites in AChE, which have allowed us to identify allosteric inhibitors by virtual screening guided by structure-based and fragment hotspot strategies. The identified compounds were also screened for in vitro inhibition of AChE and three were observed to be active. Further experimental (kinetic) and computational (molecular dynamics) studies have been performed to verify the allosteric activity. These new compounds may be valuable pharmacological tools in the study of non-cholinergic functions of AChE.

Medicinal and Biological Chemistry (MBC) Library: An Efficient Source of New Hits.

Identification of new hits is one of the biggest challenges in drug discovery. Creating a library of well-characterized drug-like compounds is a key step in this process. Our group has developed an in-house chemical library called the Medicinal and Biological Chemistry (MBC) library. This collection has been successfully used to start several medicinal chemistry programs and developed in an accumulation of more than 30 years of experience in drug design and discovery of new drugs for unmet diseases. It contains over 1000 compounds, mainly heterocyclic scaffolds. In this work, analysis of drug-like properties and comparative study with well-known libraries by using different computer software are presented here.

Benzodioxane-Benzamides as FtsZ Inhibitors: Effects of Linker's Functionalization on Gram-Positive Antimicrobial Activity.

FtsZ is an essential bacterial protein abundantly studied as a novel and promising target for antimicrobials. FtsZ is highly conserved among bacteria and mycobacteria, and it is crucial for the correct outcome of the cell division process, as it is responsible for the division of the parent bacterial cell into two daughter cells. In recent years, the benzodioxane-benzamide class has emerged as very promising and capable of targeting both Gram-positive and Gram-negative FtsZs. In this study, we explored the effect of including a substituent on the ethylenic linker between the two main moieties on the antimicrobial activity and pharmacokinetic properties. This substitution, in turn, led to the generation of a second stereogenic center, with both erythro and threo isomers isolated, characterized, and evaluated. With this work, we discovered how the hydroxy group slightly affects the antimicrobial activity, while being an important anchor for the exploitation and development of prodrugs, probes, and further derivatives.

Naphthoquinone as a New Chemical Scaffold for Leishmanicidal Inhibitors of Leishmania GSK-3.

More than 1 billion people live in areas endemic for leishmaniasis, which is a relevant threat for public health worldwide. Due to the inadequate treatments, there is an urgent need to develop novel alternative drugs and to validate new targets to fight this disease. One appealing approach is the selective inhibition of protein kinases (PKs), enzymes involved in a wide range of processes along the life cycle of Leishmania. Several PKs, including glycogen synthase kinase 3 (GSK-3), have been validated as essential for this parasite by genetic or pharmacological methods. Recently, novel chemical scaffolds have been uncovered as Leishmania GSK-3 inhibitors with antiparasitic activity. In order to find new inhibitors of this enzyme, a virtual screening of our in-house chemical library was carried out on the structure of the Leishmania GSK-3. The virtual hits identified were experimentally assayed both for leishmanicidal activity and for in vitro inhibition of the enzyme. The best hits have a quinone scaffold. Their optimization through a medicinal chemistry approach led to a set of new compounds, provided a frame to establish biochemical and antiparasitic structure-activity relationships, and delivered molecules with an improved selectivity index. Altogether, this study paves the way for a systemic search of this class of inhibitors for further development as potential leishmanicidal drugs.

Staphylococcus aureus RnpA Inhibitors: Computational-Guided Design, Synthesis and Initial Biological Evaluation.

Antibiotic resistance is spreading worldwide and it has become one of the most important issues in modern medicine. In this context, the bacterial RNA degradation and processing machinery are essential processes for bacterial viability that may be exploited for antimicrobial therapy. In Staphylococcus aureus, RnpA has been hypothesized to be one of the main players in these mechanisms. S. aureus RnpA is able to modulate mRNA degradation and complex with a ribozyme (rnpB), facilitating ptRNA maturation. Corresponding small molecule screening campaigns have recently identified a few classes of RnpA inhibitors, and their structure activity relationship (SAR) has only been partially explored. Accordingly, in the present work, using computational modeling of S. aureus RnpA we identified putative crucial interactions of known RnpA inhibitors, and we used this information to design, synthesize, and biologically assess new potential RnpA inhibitors. The present results may be beneficial for the overall knowledge about RnpA inhibitors belonging to both RNPA2000-like thiosemicarbazides and JC-like piperidine carboxamides molecular classes. We evaluated the importance of the different key moieties, such as the dichlorophenyl and the piperidine of JC2, and the semithiocarbazide, the furan, and the i-propylphenyl ring of RNPA2000. Our efforts could provide a foundation for further computational-guided investigations.

Computational Design and Development of Benzodioxane-Benzamides as Potent Inhibitors of FtsZ by Exploring the Hydrophobic Subpocket.

Multidrug resistant Staphylococcus aureus is a severe threat, responsible for most of the nosocomial infections globally. This resistant strain is associated with a 64% increase in death compared to the antibiotic-susceptible strain. The prokaryotic protein FtsZ and the cell division cycle have been validated as potential targets to exploit in the general battle against antibiotic resistance. Despite the discovery and development of several anti-FtsZ compounds, no FtsZ inhibitors are currently used in therapy. This work further develops benzodioxane-benzamide FtsZ inhibitors. We seek to find more potent compounds using computational studies, with encouraging predicted drug-like profiles. We report the synthesis and the characterization of novel promising derivatives that exhibit very low MICs towards both methicillin-susceptible and -resistant S. aureus, as well as another Gram positive species, Bacillus subtilis, while possessing good predicted physical-chemical properties in terms of solubility, permeability, and chemical and physical stability. In addition, we demonstrate by fluorescence microscopy that Z ring formation and FtsZ localization are strongly perturbed by our derivatives, thus validating the target.

A fragment-based approach to assess the ligandability of ArgB, ArgC, ArgD and ArgF in the L-arginine biosynthetic pathway of Mycobacterium tuberculosis.

The L-arginine biosynthesis pathway consists of eight enzymes that catalyse the conversion of L-glutamate to L-arginine. Arginine auxotrophs (argB/argF deletion mutants) of Mycobacterium tuberculosis are rapidly sterilised in mice, while inhibition of ArgJ with Pranlukast was found to clear chronic M. tuberculosis infection in a mouse model. Enzymes in the arginine biosynthetic pathway have therefore emerged as promising targets for anti-tuberculosis drug discovery. In this work, the ligandability of four enzymes of the pathway ArgB, ArgC, ArgD and ArgF is assessed using a fragment-based approach. We identify several hits against these enzymes validated with biochemical and biophysical assays, as well as X-ray crystallographic data, which in the case of ArgB were further confirmed to have on-target activity against M. tuberculosis. These results demonstrate the potential for more enzymes in this pathway to be targeted with dedicated drug discovery programmes.

Benzamide Derivatives Targeting the Cell Division Protein FtsZ: Modifications of the Linker and the Benzodioxane Scaffold and Their Effects on Antimicrobial Activity.

Filamentous temperature-sensitive Z (FtsZ) is a prokaryotic protein with an essential role in the bacterial cell division process. It is widely conserved and expressed in both Gram-positive and Gram-negative strains. In the last decade, several research groups have pointed out molecules able to target FtsZ in Staphylococcus aureus, Bacillus subtilis and other Gram-positive strains, with sub-micromolar Minimum Inhibitory Concentrations (MICs). Conversely, no promising derivatives active on Gram-negatives have been found up to now. Here, we report our results on a class of benzamide compounds, which showed comparable inhibitory activities on both S. aureus and Escherichia coli FtsZ, even though they proved to be substrates of E. coli efflux pump AcrAB, thus affecting the antimicrobial activity. These surprising results confirmed how a single molecule can target both species while maintaining potent antimicrobial activity. A further computational study helped us decipher the structural features necessary for broad spectrum activity and assess the drug-like profile and the on-target activity of this family of compounds.

Benzodioxane-Benzamides as Antibacterial Agents: Computational and SAR Studies to Evaluate the Influence of the 7-Substitution in FtsZ Interaction.

FtsZ is a crucial prokaryotic protein involved in bacterial cell replication. It recently arose as a promising target in the search for antimicrobial agents able to fight antimicrobial resistance. In this work, going on with our structure-activity relationship (SAR) study, we developed variously 7-substituted 1,4-benzodioxane compounds, linked to the 2,6-difluorobenzamide by a methylenoxy bridge. Compounds exhibit promising antibacterial activities not only against multidrug-resistant Staphylococcus aureus, but also on mutated Escherichia coli strains, thus enlarging their spectrum of action toward Gram-negative bacteria as well. Computational studies elucidated, through a validated FtsZ binding protocol, the structural features of new promising derivatives as FtsZ inhibitors.

QSAR Modelling to Identify LRRK2 Inhibitors for Parkinson's Disease.

Parkinson's disease is one of the most common neurodegenerative illnesses in older persons and the leucine-rich repeat kinase 2 (LRRK2) is an auspicious target for its pharmacological treatment. In this work, quantitative structure-activity relationship (QSAR) models for identification of putative inhibitors of LRRK2 protein are developed by using an in-house chemical library and several machine learning techniques. The methodology applied in this paper has two steps: first, alternative subsets of molecular descriptors useful for characterizing LRRK2 inhibitors are chosen by a multi-objective feature selection method; secondly, QSAR models are learned by using these subsets and three different strategies for supervised learning. The qualities of all these QSAR models are compared by classical metrics and the best models are discussed in statistical and physicochemical terms.

QSAR Classification Models for Predicting the Activity of Inhibitors of Beta-Secretase (BACE1) Associated with Alzheimer's Disease.

Alzheimer's disease is one of the most common neurodegenerative disorders in elder population. The ß-site amyloid cleavage enzyme 1 (BACE1) is the major constituent of amyloid plaques and plays a central role in this brain pathogenesis, thus it constitutes an auspicious pharmacological target for its treatment. In this paper, a QSAR model for identification of potential inhibitors of BACE1 protein is designed by using classification methods. For building this model, a database with 215 molecules collected from different sources has been assembled. This dataset contains diverse compounds with different scaffolds and physical-chemical properties, covering a wide chemical space in the drug-like range. The most distinctive aspect of the applied QSAR strategy is the combination of hybridization with backward elimination of models, which contributes to improve the quality of the final QSAR model. Another relevant step is the visual analysis of the molecular descriptors that allows guaranteeing the absence of information redundancy in the model. The QSAR model performances have been assessed by traditional metrics, and the final proposed model has low cardinality, and reaches a high percentage of chemical compounds correctly classified.

Discovery of novel Schistosoma mansoni PDE4A inhibitors as potential agents against schistosomiasis.

Aim: Due to the urgent need for effective drugs to treat schistosomiasis that act through a known molecular mechanism of action, we focused on a target-based approach with the aim to discover inhibitors of a cyclic nucleotide phosphodiesterase from Schistosoma mansoni (SmPDE4A). Materials & methods: To discover new inhibitors of SmPDE4A homology models of the enzyme structure were constructed based on known human and protozoan homologs. The best two models were selected for subsequent virtual screening of our in-house chemical library. Results & conclusion: A total of 25 library compounds were selected for experimental confirmation as SmPDE4A inhibitors and after dose-response experiments, three top hits were identified. The results presented validate the virtual screening approach to identify new inhibitors for clinically relevant phosphodiesterases.

Screening of a PDE-focused library identifies imidazoles with in vitro and in vivo antischistosomal activity.

We report the evaluation of 265 compounds from a PDE-focused library for their antischistosomal activity, assessed in vitro using Schistosoma mansoni. Of the tested compounds, 171 (64%) displayed selective in vitro activity, with 16 causing worm hypermotility/spastic contractions and 41 inducing various degrees of worm killing at 100 µM, with the surviving worms displaying sluggish movement, worm unpairing and complete absence of eggs. The compounds that did not affect worm viability (n = 72) induced a complete cessation of ovipositing. 82% of the compounds had an impact on male worms whereas female worms were barely affected. In vivo evaluation in S. mansoni-infected mice with the in vitro 'hit' NPD-0274 at 20 mg/kg/day orally for 5 days resulted in worm burden reductions of 29% and intestinal tissue egg load reduction of 35% at 10 days post-treatment. Combination of praziquantel (PZQ) at 10 mg/kg/day for 5 days with NPD-0274 or NPD-0298 resulted in significantly higher worm killing than PZQ alone, as well as a reduction in intestinal tissue egg load, disappearance of immature eggs and an increase in the number of dead eggs.

Chameleon-like behavior of indolylpiperidines in complex with cholinesterases targets: Potent butyrylcholinesterase inhibitors.

Alzheimer's disease (AD) is the most common form of dementia worldwide with an increasing prevalence for the next years. The multifactorial nature of AD precludes the design of new drugs directed to a single target being probably one of the reasons for recent failures. Therefore, dual binding site acetylcholinesterase (AChE) inhibitors have been revealed as cognitive enhancers and ß-amyloid modulators offering an alternative in AD therapy field. Based on the dual ligands NP61 and donepezil, the present study reports the synthesis of a series of indolylpiperidines hybrids to optimize the NP61 structure preserving the indole nucleus, but replacing the tacrine moiety of NP61 by benzyl piperidine core found in donepezil. Surprisingly, this new family of indolylpiperidines derivatives showed very potent and selective hBuChE inhibition. Further studies of NMR and molecular dynamics have showed the capacity of these hybrid molecules to change their bioactive conformation depending on the binding site, being capable to inhibit with different shapes BuChE and residually AChE.