Maria Laura Bolognesi

Naphthoquinone Derivatives Exert Their Antitrypanosomal Activity via a Multi-Target Mechanism

Background and Methodology Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED50 of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach. Principal Findings A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC50 values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity. Conclusions and Significance Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands.

Polypharmacology in a single drug: multitarget drugs.

Polypharmacology offers a model for the way drug discovery must evolve to develop therapies most suited to treating currently incurable diseases. It is driven by a worldwide demand for safer, more effective, and affordable medicines against the most complex diseases, and by the failures of modern drug discovery to provide these. Polypharmacology can involve combinations and/or multitarget drugs (MTD). Although not mutually exclusive, my premise is that MTDs have inherent advantages over combinations. This review article focuses on MTDs from a medicinal chemistry perspective. I will explore their use in current clinical practice, their likely application in the future, and the challenges to be overcome to achieve this goal.

Exploiting the lipoic acid structure in the search for novel multitarget ligands against Alzheimer’s disease

Lipoic acid (LA) is a natural antioxidant. Its structure was previously combined with that of the acetylcholinesterase inhibitor tacrine to give lipocrine (1), a lead compound multitargeted against Alzheimers disease (AD). Herein, we further explore LA as a privileged structure for developing multimodal compounds to investigate AD. First, we studied the effect of LA chirality by evaluating the cholinesterase profile of 1s enantiomers. Then, a new series of LA hybrids was designed and synthesized by combining racemic LA with motifs of other known anticholinesterase agents (rivastigmine and memoquin). This afforded 4, which represents a step forward in the search for balanced anticholinesterase and antioxidant capacities.

Multifunctional Tacrine Derivatives in Alzheimer’s Disease

Tacrine (1) was the first acetylcholinesterase inhibitor (AChEI) introduced in therapy for the treatment of Alzheimers disease (AD), but similarly to the most recent approved AChEIs and memantine, an N-methyl-D-aspartate receptor (NMDAR) antagonist, it does not represent an effective drug in halting the progression of AD. The continuous research in this field has contributed to delineate AD as a multifactorial syndrome with several biological targets involved in its etiology. On these bases, the development of new effective therapeutics becomes crucial and the design of molecules that address more than one specific AD target should represent thus a succeeded strategy for AD treatment. This review will focus on and summarize multifunctional 1 derivatives starting from our last paper published on the same topic in 2010. In the last three years, the design and synthesis of 1 homo- and heterodimers, as well as of 1-hybrid structures for AD therapy, was aimed mainly to discover safer drugs, with decreased hepatotoxicity in comparison to 1, taking also into account the multifactorial pathogenesis of the disease. Most of these new hetero/homo-dimers and/or hybrids of 1, although addressed mainly to acetylcholinesterase (AChE) and Aβ aggregation inhibition, are able to hit additional targets relevant to AD, among which, β-secretase (BACE1), reactive oxygen species (ROS), calcium channels, NMDAR and M1- muscarinic receptors.

Conjugation of Quinones with Natural Polyamines: Toward an Expanded Antitrypanosomatid Profile

A combinatorial library of quinone-polyamine conjugates designed to optimize the antitrypanosomatid profile of hit compounds 1 and 2 has been prepared by a solid-phase approach. The conjugates were evaluated against the three most important human trypanosomatid pathogens (Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani), and several showed promising activity. A subset also inhibited trypanothione reductase in vitro and induced oxidase activity of the enzyme. A highly potent analogue (7) was identified with activity against T. brucei as low as 70 nM and a selectivity index of 72. Interestingly, the presence of a cadaverine tail confers to 7 the ability to target mitochondrial function in Leishmania. In fact, in L. donovani promastigotes, we verified for 7 a decrease of cytoplasmic ATP and mitochondrial potential. Therefore, the current results support the suitability of the conjugation approach for the development of novel polyamine conjugates with enhanced therapeutic potential.

A small chemical library of 2-aminoimidazole derivatives as BACE-1 inhibitors: Structure-based design, synthesis, and biological evaluation

In this work, we report a rational structure-based approach aimed at the discovery of new 2-aminoimidazoles as β-secretase inhibitors. Taking advantage of a microwave-assisted synthetic protocol, a small library of derivatives was obtained and biologically evaluated. Two compounds showed promising activities in both enzymatic and cellular assays. Moreover, one of them exhibited the capability to cross the blood-brain barrier as assessed by the parallel artificial membrane permeability assay.

The bivalent ligand approach as a tool for improving the in vitro anti-alzheimer multitarget profile of dimebon

Alzheimer’s disease (AD) is an extremely challenging and often frustrating area of drug discovery. Since the withdrawal of tacrine (Cognex) in 2006, more than 200 AD drug candidates have failed in late-stage clinical trials. The antihistamine drug dimebon (1, Figure 1) belongs to this long list. In 2008, 1 attracted considerable interest within the AD community when it successfully completed a small six-month clinical trial in Russia, in which it showed impressive cognition-enhancing effects in patients suffering from mild to moderate AD. 3] However, these positive outcomes were unconfirmed in a replication trial in the United States. We were particularly interested in 1 because, from initial reports on its in vitro activity profile, it seemed to fulfill the promise of an effective multitarget drug for treating AD. In recent years, multitarget drug development has emerged as an effective approach in the search for disease-modifying drugs against AD. This approach involves the development of single chemical entities that can simultaneously modulate multiple targets critically involved in the neurotoxic pathway. It therefore runs parallel to drug combination in the search for appropriate therapeutic interventions against the complex pathogenesis of AD. 9] Indeed, early research suggested that the clinical benefits of dimebon were related to its ability to simultaneously inhibit two crucial AD molecular targets: acetylcholinesterase (AChE) and N-methyl-d-aspartate receptor (NMDAR). The effectiveness of simultaneous inhibition of both targets was further supported by higher shortand long-term efficacies observed in clinical trials involving co-administration of the NMDAR antagonist memantine and an AChE inhibitor (AChEI). Combining memantine and AChEIs is the current standard of care for AD patients. Furthermore, pre-clinically, the combination was demonstrated to act synergistically, which may explain the observed clinical effects. In this respect, we have already successfully combined the symptomatic relief of AChE inhibition and the neuroprotective action of NMDAR antagonism in a single multitarget molecule. 14] In the case of 1, however, its low in vitro activity against these two key targets (IC50 = 42 mm and 10–70 mm against AChE and NMDAR, respectively) might be one of the causes of its clinical failure. Indeed, Giorgetti et al. demonstrated that the brain concentration reached after acute oral administration of 1 to rats is much lower than that required to significantly affect AChE or NMDA pathways (nanomolar vs. micromolar). Although exposure levels of dimebon in plasma and cerebrospinal fluids in humans have not been published, it has been implied that the same situation might be replicated in the AD patients that were enrolled in the trial. This raised much uncertainty on the real mechanism by which dimebon may beneFigure 1. Design strategy for generating compounds 2–6.

Quinones bearing non-steroidal anti-inflammatory fragments as multitarget ligands for Alzheimer's disease.

The anti-amyloid properties shared by several quinones inspired the design of a new series of hybrids derived from the multi-target drug candidate memoquin (1). The hybrids consist of a central benzoquinone core and a fragment taken from non-steroidal anti-inflammatory drugs, connected through polyamine linkers. The new hybrids retain the potent anti-aggregating activity of the parent 1, while exhibiting micromolar AChE inhibitory activities. Remarkably, 2, 4, (R)-6 and (S)-6 were Aβ aggregation inhibitors even more potent than 1. The balanced amyloid/cholinesterase inhibitory profile is an added value that makes the present series of compounds promising leads against Alzheimers disease.

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.