Salvatore Bongarzone

The concept of privileged structures in rational drug design: focus on acridine and quinoline scaffolds in neurodegenerative and protozoan diseases

Introduction: For nearly 20 years, privileged structures have offered an optimal source of core scaffolds and capping fragments for the design of combinatorial libraries directed at a broad spectrum of targets. From describing structures promiscuous within a given target family, the concept has evolved to include frameworks that can modulate proteins lacking a strict target class relation. Areas covered: Based on a literature search from 2000 to 2010, we discuss how two privileged motifs, quinolines and acridines, are particularly recurrent in compounds active against two quite different pathologies, neurodegenerative and protozoan diseases. Expert opinion: As privileged structures, quinolines and acridines could improve the productivity of drug discovery projects in the field of neurodegenerative and protozoan diseases. They could be particularly relevant for protozoan diseases because of the importance of cost-effective strategies and less stringent intellectual property concerns. Furthermore, because of their inherent affinity for various targets, privileged structures could offer a viable starting point in the search for novel multi-target ligands. Finally, from a broader perspective, they can serve as effective probes for investigating unknown but interrelated mechanisms of action.

Molecular motions in drug design: the coming age of the metadynamics method

Metadynamics is emerging as a useful free energy method in physics, chemistry and biology. Recently, it has been applied also to investigate ligand binding to biomolecules of pharmacological interest. Here, after introducing the basic idea of the method, we review applications to challenging targets for pharmaceutical intervention. We show that this methodology, especially when combined with a variety of other computational approaches such as molecular docking and/or molecular dynamics simulation, may be useful to predict structure and energetics of ligand/target complexes even when the targets lack a deep binding cavity, such as DNA and proteins undergoing fibrillation in neurodegenerative diseases. Furthermore, the method allows investigating the routes of molecular recognition and the associated binding energy profiles, providing a molecular interpretation to experimental data.

Parallel Synthesis, Evaluation, and Preliminary Structure—Activity Relationship of 2,5-Diamino-1,4-benzoquinones as a Novel Class of Bivalent Anti-Prion Compound

A library of 11 entries, featuring a 2,5-diamino-1,4-benzoquinones nucleus as spacer connecting two aromatic prion recognition motifs, was designed and evaluated against prion infection. Notably, 6-chloro-1,2,3,4-tetrahydroacridine 10 showed an EC(50) of 0.17 μM, which was lower than that displayed by reference compound BiCappa. More importantly, 10 possessed the capability to contrast prion fibril formation and oxidative stress, together with a low cytotoxicity. This study further corroborates the bivalent strategy as a viable approach to the rational design of anti-prion chemical probes.

Discovery of a class of diketopiperazines as antiprion compounds.

Prion diseases are fatal neurodegenerative and infectious disorders for which effective pharmacological tools are not yet available. This unmet challenge and the recently proposed interplay between prion diseases and Alzheimers have led to a more urgent demand for new antiprion agents. Herein, we report the identification of a novel bifunctional diketopiperazine (DKP) derivative 1 d, which exhibits activity in the low micromolar range against prion replication in ScGT1 cells, while showing low cytotoxicity. Supported by properly addressed molecular modeling studies, we hypothesized that a planar conformation is the major determinant for activity in this class of compounds. Moreover, studies aimed at assessing the mechanism‐of‐action at the molecular level showed that 1 d might interact directly with recombinant prion protein (recPrP) to prevent its conversion to the pathogenic misfolded prion protein (PrPSc)‐like form. This investigation suggests that DKP based antiprion compounds can serve as a promising lead scaffold in developing new drugs to combat prion diseases.

Docking Ligands on Protein Surfaces: The Case Study of Prion Protein.

Molecular docking of ligands targeting proteins undergoing fibrillization in neurodegenerative diseases is difficult because of the lack of deep binding sites. Here we extend standard docking methods with free energy simulations in explicit solvent to address this issue in the context of the prion protein surface. We focus on a specific ligand (2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide), which binds to the structured part of the protein as shown by NMR (Kuwata, K. et al. Proc Natl Acad Sci U.S.A. 2007, 104, 11921-11926). The calculated free energy of dissociation (7.8 ± 0.9 kcal/mol) is in good agreement with the value derived by the experimental dissociation constant (Kd = 3.9 μM, corresponding to ΔG(0) = -7.5 kcal/mol). Several binding poses are predicted, including the one reported previously. Our prediction is fully consistent with the presence of multiple binding sites, emerging from NMR measurements. Our molecular simulation-based approach emerges, therefore, as a useful tool to predict poses and affinities of ligand binding to protein surfaces.

Quantification of [(11)C]PIB PET for imaging myelin in the human brain: a test-retest reproducibility study in high-resolution research tomography.

An accurate in vivo measure of myelin content is essential to deepen our insight into the mechanisms underlying demyelinating and dysmyelinating neurological disorders, and to evaluate the effects of emerging remyelinating treatments. Recently [11C]PIB, a positron emission tomography (PET) tracer originally conceived as a beta-amyloid marker, has been shown to be sensitive to myelin changes in preclinical models and humans. In this work, we propose a reference-region methodology for the voxelwise quantification of brain white-matter (WM) binding for [11C]PIB. This methodology consists of a supervised procedure for the automatic extraction of a reference region and the application of the Logan graphical method to generate distribution volume ratio (DVR) maps. This approach was assessed on a test–retest group of 10 healthy volunteers using a high-resolution PET tomograph. The [11C]PIB PET tracer binding was shown to be up to 23% higher in WM compared with gray matter, depending on the image reconstruction. The DVR estimates were characterized by high reliability (outliers < 1%) and reproducibility (intraclass correlation coefficient (ICC) > 0.95). [11C]PIB parametric maps were also found to be significantly correlated (R2 > 0.50) to mRNA expressions of the most represented proteins in the myelin sheath. On the contrary, no correlation was found between [11C]PIB imaging and nonmyelin-associated proteins.

Synthesis and evaluation of a library of 2,5-bisdiamino-benzoquinone derivatives as probes to modulate protein-protein interactions in prions.

A small library combining two different benzoquinone cores with seven (L) amino acid methyl esters (alanine, Nomega-nitro-arginine, Nepsilon-BOC-lysine, isoleucine, methionine, phenylalanine and tryptophan) was prepared and tested for prion replication inhibition in ScGT1 cells. The most potent hit, 6a, displayed an EC(50) value of 0.87 microM, which is very close to that of quinacrine (0.4 microM).

Targeting the Receptor for Advanced Glycation Endproducts (RAGE): A Medicinal Chemistry Perspective

The receptor for advanced glycation endproducts (RAGE) is an ubiquitous, transmembrane, immunoglobulin-like receptor that exists in multiple isoforms and binds to a diverse range of endogenous extracellular ligands and intracellular effectors. Ligand binding at the extracellular domain of RAGE initiates a complex intracellular signaling cascade, resulting in the production of reactive oxygen species (ROS), immunoinflammatory effects, cellular proliferation, or apoptosis with concomitant upregulation of RAGE itself. To date, research has mainly focused on the correlation between RAGE activity and pathological conditions, such as cancer, diabetes, cardiovascular diseases, and neurodegeneration. Because RAGE plays a role in many pathological disorders, it has become an attractive target for the development of inhibitors at the extracellular and intracellular domains. This review describes the role of endogenous RAGE ligands/effectors in normo- and pathophysiological processes, summarizes the current status of exogenous small-molecule inhibitors of RAGE and concludes by identifying key strategies for future therapeutic intervention.

Hybrid Lipoic Acid Derivatives to Attack Prion Disease on Multiple Fronts

Prion diseases or transmissible spongiform encephalopathies are a group of invariably fatal disorders, for which there is neither early diagnosis nor a cure. 2] These maladies are characterized by spongiform brain neurodegeneration caused by a misfolded protein with unique infective properties : prion protein scrapie (PrP). According to the protein-only hypothesis, in the central nervous system of the infected host the cellular prion protein (PrP), PrP, is converted into an abnormal insoluble amyloidogenic isoform, that is, PrP or prion. The latter acts as a template for PrP leading to nascent PrP molecules. The process of conversion is associated with conformational changes of secondary structure from a-helices to b-sheets. 2] While this hypothesis is supported by in vitro conversion of PrP to PrP, the mechanism underlying in vivo conversion, although not yet fully elucidated, seems to be more complex, and possibly involves some molecular chaperones. In recent years, it has been gradually accepted that prion disease pathogenesis involves a complex array of processes that operate simultaneously and synergistically. These include: 1) protein aggregation; 5] 2) oxidative stress (OS) accompanied by lipid and protein oxidation; 3) decreased levels of potent freeradical scavengers such as polyunsaturated fatty acids, a-tocopherol, and glutathione; 11] 4) an imbalance of metal ions; and 5) brain inflammation with activation of astrocytes and microglia. Thus, the failures of drug candidates developed according to the traditional drug discovery paradigm “one molecule, one target” and the current challenge of discovering an efficacious therapy are likely related to the multifactorial nature of these diseases. This is in line with what has been observed in other neurodegenerative diseases such as Alzheimer’s disease (AD). Against this backdrop, a polypharmacological approach consists of a concerted pharmacological intervention against multiple targets, and therefore it can have superior efficacy and safety toward complex neurological disorders. Although this approach is still in its infancy, two different strategies have already been addressed to rationally achieve polypharmacology: drug combination (DC) and the multi-target-directed ligand (MTDL) approach. In the DC approach, multiple drugs (a drug cocktail) are combined in the therapeutic regimen. A disadvantage of DC is the potential for synergistic toxicity, which could be expected if the mechanisms that cause side effects are related to those that mediate efficacy. Another drawback of DC therapy are drug–drug interactions. Conversely, the MTDL approach, in which two pharmacophores with distinct mechanisms of action are chemically merged in a single structure with a single absorption, distribution, metabolism, excretion, and toxicity (ADMET) profile, offers advantages over DC therapy. Notably, this approach, already used for other complex diseases, has been envisaged as optimal for the treatment of prion diseases as well. For prion diseases, the DC strategy has been applied in numerous in vitro and in vivo approaches with the aim of exploiting synergistic effects. The several examples reported in table S2 (Supporting Information) suggest that inhibition of prion replication can be effectively potentiated by DC treatment. As for the MTDL approach, there are reported cases in which the deliberate aim of creating an MTDL has not always been explicitly stated. Instead, the molecular hybridization strategy has been followed, leading to chimeric molecules that are, in principle, capable of modulating multiple targets. The first antiprion chimeric ligand, quinpramine, was designed on the basis of the in vitro synergistic antiprion effects of the drugs quinacrine and imipramine. Quinpramine, obtained by linking quinacrine and imipramine moieties through a piperazine ring, 28] showed improved antiprion activity by as much as 15-fold over quinacrine and 250-fold over imipramine. 28] Recently, our research group reported a new class of antiprion compounds obtained by linking the antioxidant nucleus of 2,5diamino-1,4-benzoquinone to several heterocyclic scaffolds potentially able to perturb protein–protein interactions in prion (9-amino-6-chloro-2-methoxyacridine, or 4-amino-7-chloroquinoline, or 9-amino-6-chloro-1,2,3,4-tetrahydroacridine). These compounds displayed a multi-target profile, effectively con[a] Prof. A. Cavalli, Prof. M. Roberti, Dr. M. Rosini, Prof. M. L. Bolognesi Department of Pharmaceutical Sciences, University of Bologna Via Belmeloro 6, 40126 Bologna (Italy) Fax: (+ 39) 0512099734 E-mail : marialaura.bolognesi@unibo.it [b] S. Bongarzone Statistical and Biological Physics Sector Scuola Internazionale Superiore di Studi Avanzati (SISSA) Via Bonomea 265, 34136 Trieste (Italy) [c] S. Bongarzone, Prof. G. Legname Italian Institute of Technology (IIT), SISSA-Unit Via Beirut 2–4, 34014, Trieste, Italy [d] H. N. A. Tran, Prof. G. Legname Laboratory of Prion Biology, Neurobiology Sector Scuola Internazionale Superiore di Studi Avanzati (SISSA) Via Bonomea 265, 34136 Trieste (Italy) [e] Prof. A. Cavalli Department of Drug Discovery and Development Italian Institute of Technology (IIT) Via Morego 30, 16163 Genova (Italy) [f] Prof. P. Carloni German Research School for Simulation Sciences GmbH Forschungszentrum J lich GmbH RWTH Aachen University Aachen (Germany) [] These authors equally contributed to the experimental work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201100072.

Dominant-negative effects in prion diseases: insights from molecular dynamics simulations on mouse prion protein chimeras

Mutations in the prion protein (PrP) can cause spontaneous prion diseases in humans (Hu) and animals. In transgenic mice, mutations can determine the susceptibility to the infection of different prion strains. Some of these mutations also show a dominant-negative effect, thus halting the replication process by which wild type mouse (Mo) PrP is converted into Mo scrapie. Using all-atom molecular dynamics (MD) simulations, here we studied the structure of HuPrP, MoPrP, 10 Hu/MoPrP chimeras, and 1 Mo/sheepPrP chimera in explicit solvent. Overall, ∼2 μs of MD were collected. Our findings suggest that the interactions between α1 helix and N-terminal of α3 helix are critical in prion propagation, whereas the β2–α2 loop conformation plays a role in the dominant-negative effect. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:4.