Carlo Melchiorre

Multi-target-Directed Ligands To Combat Neurodegenerative Diseases

Our understanding of the pathogenesis of diseases has advanced enormously in recent decades. As a consequence, drug discovery has gradually shifted from an entirely humanphenotype-based endeavor to today’s reductionist approach centered on single molecular targets. The focus has shifted from the early animal models to isolated proteins via cellular models. This change has led to a decrease in complexity but also to a decrease in relevance to the human condition. In this context, drug research has become (and still is) aimed mainly at the discovery of small molecules able to modulate the biological function of a single protein target thought to be fully responsible for a certain disease. Much effort has been devoted to achieving selectivity for that given target, and indeed, nowadays, many ligands endowed with outstanding in vitro selectivity are available. This one-molecule, one-target paradigm has led to the discovery of many successful drugs, and it will probably remain a milestone for years to come. However, it should be noted that a highly selective ligand for a given target does not always result in a clinically efficacious drug. This may be because (a) the ligand does not recognize the target in vivo, (b) the ligand does not reach the site of action, or (c) the interaction with the respective target does not have enough impact on the diseased system to restore it effectively. Reasons for the latter might lie in both the multifactorial nature of many diseases and the fact that cells can often find ways to compensate for a protein whose activity is affected by a drug, by taking advantage of the redundancy of the system, i.e., of the existence of parallel pathways. Medicinal chemists are often faced with these frustrating aspects of drug research. Drawbacks a and b can be addressed through the well-established rational ligand modification approaches. But issue c is more problematic and needs to be carefully discussed. This is one of the aims of the present article.

Inhibition of Acetylcholinesterase, β-Amyloid Aggregation, and NMDA Receptors in Alzheimer's Disease : A Promising Direction for the Multi-target-Directed Ligands Gold Rush

Alzheimers disease (AD) is a multifactorial syndrome with several target proteins contributing to its etiology. To confront AD, an innovative strategy is to design single chemical entities able to simultaneously modulate more than one target. Here, we present compounds that inhibit acetylcholinesterase and NMDA receptor activity. Furthermore, these compounds inhibit AChE-induced Abeta aggregation and display antioxidant properties, emerging as lead candidates for treating AD.

Memoquin: A Multi-Target–Directed Ligand as an Innovative Therapeutic Opportunity for Alzheimer's Disease

SummaryAlzheimer’s disease is currently thought to be a complex, multifactorial syndrome, unlikely to arise from a single causal factor; instead, a number of related biological alterations are thought to contribute to its pathogenesis. This may explain why the currently available drugs, developed according to the classic drug discovery paradigm of “one-molecule-one-target,” have turned out to be palliative. In light of this, drug combinations that can act at different levels of the neurotoxic cascade offer new avenues toward curing Alzheimer’s and other neurodegenerative diseases. In parallel, a new strategy is emerging—that of developing a single chemical entity able to modulate multiple targets simultaneously. This has led to a new paradigm in medicinal chemistry, the “multi-target-directed ligand” design strategy, which has already been successfully exploited at both academic and industrial levels. As a case study, we report here on memoquin, a new molecule developed following this strategy. The in vitro and in vivo biological profile of memoquin demonstrates the suitability of the new strategy for obtaining innovative drug candidates for the treatment of neurodegenerative diseases.

Prejunctional muscarinic inhibitory control of acetylcholine release in the human isolated detrusor: involvement of the M4 receptor subtype.

Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response. In detrusor strips preincubated with [3H]‐choline, electrical field stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium outflow and a contractile response. In the presence of 10 μM paraoxon (to prevent ACh degradation) the tritium outflow was characterized by HPLC analysis as [3H]‐ACh (76%) and [3H]‐choline (24%). Electrically‐evoked [3H]‐ACh release was abolished by tetrodotoxin (TTX: 300 nM) and unaffected by hexamethonium (10 μM), indicating a postganglionic event. It was reduced by physostigmine (100 nM) and the muscarinic receptor agonist, muscarone (10 nM–1 μM), and enhanced by atropine (0.1–100 nM). These findings indicate the presence of a muscarinic negative feedback mechanism controlling ACh release. The effects of various subtype‐preferring muscarinic receptor antagonists were evaluated on [3H]‐ACh release and muscle contraction. The rank potency (−log EC50) orders at pre‐ and postjunctional level were: atropine 4‐diphenyl‐acetoxy‐N‐piperidine (4‐DAMP)>mamba toxin 3 (MT‐3)>tripitramine>para‐fluorohexahydrosiladiphenidol (pF‐HHSiD)methoctraminepirenzepine>tripinamide, and atropine4‐DAMP>pF‐HHSiD>>pirenzepine=tripitramine>tripinamide>methoctramine>>MT‐3, respectively. The comparison of pre‐ and post‐junctional potencies and the relationship analysis with the affinity constants at human cloned muscarinic receptor subtypes indicates that the muscarinic autoreceptor inhibiting ACh release in human detrusor is an M4 receptor, while the receptor involved in muscular contraction belongs to the M3 subtype.

Combining Galantamine and Memantine in Multitargeted, New Chemical Entities Potentially Useful in Alzheimer’s Disease

Herein we report on a novel series of multitargeted compounds obtained by linking together galantamine and memantine. The compounds were designed by taking advantage of the crystal structures of acetylcholinesterase (AChE) in complex with galantamine derivatives. Sixteen novel derivatives were synthesized, using spacers of different lengths and chemical composition. The molecules were then tested as inhibitors of AChE and as binders of the N-methyl-d-aspartate (NMDA) receptor (NMDAR). Some of the new compounds were nanomolar inhibitors of AChE and showed micromolar affinities for NMDAR. All compounds were also tested for selectivity toward NMDAR containing the 2B subunit (NR2B). Some of the new derivatives showed a micromolar affinity for NR2B. Finally, selected compounds were tested using a cell-based assay to measure their neuroprotective activity. Three of them showed a remarkable neuroprotective profile, inhibiting the NMDA-induced neurotoxicity at subnanomolar concentrations (e.g., 5, named memagal, IC(50) = 0.28 nM).

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.

Structure-Activity Relationships of Acetylcholinesterase Noncovalent Inhibitors Based on a Polyamine Backbone. 4. Further Investigation on the Inner Spacer

Novel multi-target-directed ligands were designed by replacing the inner dipiperidino function of 3 with less flexible or completely rigid moieties to obtain compounds endowed with multiple biological properties that might be relevant to Alzheimers disease. 15 was the most interesting, inhibiting AChE in the nanomolar range and inhibiting AChE-induced and self-promoted beta-amyloid aggregation in the micromolar range.

Multitargeted drugs discovery: Balancing anti-amyloid and anticholinesterase capacity in a single chemical entity

Memoquin (1) is a lead compound multitargeted against Alzheimers disease (AD). It is an AChE inhibitor, free-radical scavenger, and inhibitor of amyloid-β (Aβ) aggregation. A new series of 1 derivatives was designed and synthesized by linking its 2,5-diamino-benzoquinone core with motifs that are present in the structure of known amyloid binding agents like curcumin, the benzofuran derivative SKF64346, or the benzothiazole bearing compounds KHG21834 and BTA-1. The weaker AChE inhibitory potencies and the concomitant nearly equipotent anti-amyloid activities of the new compounds with respect to 1 resulted in a more balanced biological profile against both targets. Selected compounds turned out to be effective Aβ aggregation inhibitors in a cell-based assay. By properly combining two or more distinct pharmacological properties in a molecule, we can achieve greater effectiveness compared to single-targeted drugs for investigating AD.

Toward a rational design of multitarget-directed antioxidants: merging memoquin and lipoic acid molecular frameworks.

Novel multitargeted antioxidants 3-6 were designed by combining the antioxidant features, namely, a benzoquinone fragment and a lipoyl function, of two multifunctional lead candidates. They were then evaluated to determine their profile against Alzheimers disease. They showed antioxidant activity, improved following enzymatic reduction, in mitochondria and T67 cell line. They also displayed a balanced inhibitory profile against amyloid-beta aggregation and acetylcholinesterase, emerging as promising molecules for neuroprotectant lead discovery.

Binding profile of the selective muscarinic receptor antagonist tripitramine

The binding selectivity of the muscarinic antagonist tripitramine has been tested on the five cloned human muscarinic receptor subtypes (Hm1 to Hm5) expressed in chinese hamster ovary (CHO-K1) cells. The results indicate that tripitramine binds to the muscarinic Hm2 receptor with a Ki value of 0.27 +/- 0.02 nM. Tripitramine distinguishes Hm2 vs. Hm4 by a factor of 24 and vs. Hm3 and Hm5 by a factor of 142 and 125, respectively. A lower affinity ratio, about 6-fold, was found between muscarinic Hm2 and Hm1 receptors. A comparative study with the well-known selective muscarinic M2 receptor antagonist methoctramine indicates that tripitramine has gained both potency and selectivity for the muscarinic Hm2 receptor subtype.