Damijan Knez

Structure-based development of nitroxoline derivatives as potential multifunctional anti-Alzheimer agents.

Tremendous efforts have been dedicated to the development of effective therapeutics against Alzheimers disease, which represents the most common debilitating neurodegenerative disease. Multifunctional agents are molecules designed to have simultaneous effects on different pathological processes. Such compounds represent an emerging strategy for the development of effective treatments against Alzheimers disease. Here, we report on the synthesis and biological evaluation of a series of nitroxoline-based analogs that were designed by merging the scaffold of 8-hydroxyquinoline with that of a known selective butyrylcholinesterase inhibitor that has promising anti-Alzheimer properties. Most strikingly, compound 8g inhibits self-induced aggregation of the amyloid beta peptide (Aβ1-42), inhibits with sub-micromolar potency butyrylcholinesterase (IC50=215 nM), and also selectively complexes Cu(2+). Our study thus designates this compound as a promising multifunctional agent for therapeutic treatment of Alzheimers disease. The crystal structure of human butyrylcholinesterase in complex with compound 8g is also solved, which suggests ways to further optimize compounds featuring the 8-hydroxyquinoline scaffold.

Structure-Activity Relationships of New Cyanothiophene Inhibitors of the Essential Peptidoglycan Biosynthesis Enzyme Murf.

Peptidoglycan is an essential component of the bacterial cell wall, and enzymes involved in its biosynthesis represent validated targets for antibacterial drug discovery. MurF catalyzes the final intracellular peptidoglycan biosynthesis step: the addition of D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc-L-Ala-γ-D-Glu-meso-DAP (or L-Lys). As MurF has no human counterpart, it represents an attractive target for the development of new antibacterial drugs. Using recently published cyanothiophene inhibitors of MurF from Streptococcus pneumoniae as a starting point, we designed and synthesized a series of structurally related derivatives and investigated their inhibition of MurF enzymes from different bacterial species. Systematic structural modifications of the parent compounds resulted in a series of nanomolar inhibitors of MurF from S. pneumoniae and micromolar inhibitors of MurF from Escherichia coli and Staphylococcus aureus. Some of the inhibitors also show antibacterial activity against S. pneumoniae R6. These findings, together with two new co-crystal structures, represent an excellent starting point for further optimization toward effective novel antibacterials.

Nonpeptidic Selective Inhibitors of the Chymotrypsin‐Like (β5 i) Subunit of the Immunoproteasome

Elevated expression of the immunoproteasome has been associated with autoimmune diseases, inflammatory diseases, and various types of cancer. Selective inhibitors of the immunoproteasome are not only scarce, but also almost entirely restricted to peptide-based compounds. Herein, we describe nonpeptidic reversible inhibitors that selectively block the chymotrypsin-like (β5i) subunit of the human immunoproteasome in the low micromolar range. The most potent of the reversibly acting compounds were then converted into covalent, irreversible, nonpeptidic inhibitors that retained selectivity for the β5i subunit. In addition, these inhibitors discriminate between the immunoproteasome and the constitutive proteasome in cell-based assays. Along with their lack of cytotoxicity, these data point to these nonpeptidic compounds being suitable for further investigation as β5i-selective probes for possible application in noncancer diseases related to the immunoproteasome.

Development of an in-vivo active reversible butyrylcholinesterase inhibitor.

Alzheimer’s disease (AD) is characterized by severe basal forebrain cholinergic deficit, which results in progressive and chronic deterioration of memory and cognitive functions. Similar to acetylcholinesterase, butyrylcholinesterase (BChE) contributes to the termination of cholinergic neurotransmission. Its enzymatic activity increases with the disease progression, thus classifying BChE as a viable therapeutic target in advanced AD. Potent, selective and reversible human BChE inhibitors were developed. The solved crystal structure of human BChE in complex with the most potent inhibitor reveals its binding mode and provides the molecular basis of its low nanomolar potency. Additionally, this compound is noncytotoxic and has neuroprotective properties. Furthermore, this inhibitor moderately crosses the blood-brain barrier and improves memory, cognitive functions and learning abilities of mice in a model of the cholinergic deficit that characterizes AD, without producing acute cholinergic adverse effects. Our study provides an advanced lead compound for developing drugs for alleviating symptoms caused by cholinergic hypofunction in advanced AD.

Synthesis, Molecular Modelling and Biological Evaluation of Novel Heterodimeric, Multiple Ligands Targeting Cholinesterases and Amyloid Beta

Cholinesterases and amyloid beta are one of the major biological targets in the search for a new and efficacious treatment of Alzheimer’s disease. The study describes synthesis and pharmacological evaluation of new compounds designed as dual binding site acetylcholinesterase inhibitors. Among the synthesized compounds, two deserve special attention—compounds 42 and 13. The former is a saccharin derivative and the most potent and selective acetylcholinesterase inhibitor (EeAChE IC50 = 70 nM). Isoindoline-1,3-dione derivative 13 displays balanced inhibitory potency against acetyl- and butyrylcholinesterase (BuChE) (EeAChE IC50 = 0.76 μM, EqBuChE IC50 = 0.618 μM), and it inhibits amyloid beta aggregation (35.8% at 10 μM). Kinetic studies show that the developed compounds act as mixed or non-competitive acetylcholinesterase inhibitors. According to molecular modelling studies, they are able to interact with both catalytic and peripheral active sites of the acetylcholinesterase. Their ability to cross the blood-brain barrier (BBB) was confirmed in vitro in the parallel artificial membrane permeability BBB assay. These compounds can be used as a solid starting point for further development of novel multifunctional ligands as potential anti-Alzheimer’s agents.

N-Propargylpiperidines with naphthalene-2-carboxamide or naphthalene-2-sulfonamide moieties: Potential multifunctional anti-Alzheimer's agents.

In the brains of patients with Alzheimers disease, the enzymatic activities of butyrylcholinesterase (BChE) and monoamine oxidase B (MAO-B) are increased. While BChE is a viable therapeutic target for alleviation of symptoms caused by cholinergic hypofunction, MAO-B is a potential therapeutic target for prevention of neurodegeneration in Alzheimers disease. Starting with piperidine-based selective human (h)BChE inhibitors and propargylamine-based MAO inhibitors, we have designed, synthesized and biochemically evaluated a series of N-propargylpiperidines. All of these compounds inhibited hBChE with good selectivity over the related enzyme, acetylcholinesterase, and crossed the blood-brain barrier in a parallel artificial membrane permeation assay. The crystal structure of one of the inhibitors (compound 3) in complex with hBChE revealed its binding mode. Three compounds (4, 5, 6) showed concomitant inhibition of MAO-B. Additionally, the most potent hBChE inhibitor 7 and dual BChE and MAO-B inhibitor 6 were non-cytotoxic and protected neuronal SH-SY5Y cells from toxic amyloid β-peptide species.

LiSiCA: A Software for Ligand-Based Virtual Screening and Its Application for the Discovery of Butyrylcholinesterase Inhibitors.

We developed LiSiCA (ligand similarity using clique algorithm)--ligand-based virtual screening software that uses a fast maximum clique algorithm to find two- and three-dimensional similarities between pairs of molecules and applied it to the discovery of novel potent butyrylcholinesterase inhibitors. LiSiCA, which runs in parallel on multiple processor cores, was successfully tested on the Database of Useful Decoys-Enhanced, to evaluate its ability to discriminate active molecules from decoys. We then applied LiSiCA for the discovery of novel inhibitors of human butyrylcholinesterase, a promising anti-Alzheimer target, using a known inhibitor as the reference compound. We demonstrated that LiSiCA is capable of finding potent nanomolar inhibitors, whose scaffolds differed from the reference compound, thus proving its ability for scaffold hopping and usefulness in drug discovery.

Synthesis and Biological Assessment of Racemic Benzochromenopyrimidinimines as Antioxidant, Cholinesterase, and Aβ1−42 Aggregation Inhibitors for Alzheimer's Disease Therapy

Given the complex nature of Alzheimer′s disease (AD), compounds that are able to simultaneously address two or more AD‐associated targets show greater promise for development into drugs for AD therapy. Herein we report an efficient two‐step synthesis and biological evaluation of new racemic benzochromene derivatives as antioxidants, inhibitors of cholinesterase and β‐amyloid (Aβ1−42) aggregation. Based on the results of the primary screening, we identified 15‐(3‐methoxyphenyl)‐9,11,12,15‐tetrahydro‐10H,14H‐benzo[5,6]chromeno[2,3‐d]pyrido[1,2‐a]pyrimidin‐14‐imine (3 e) and 16‐(3‐methoxyphenyl)‐9,10,11,12,13,16‐hexahydro‐15H‐benzo[5′,6′]chromeno[2′,3′:4,5]pyrimido[1,2‐a]azepin‐15‐imine (3 f) as new potential multitarget‐directed ligands for AD therapy. Further in‐depth biological analysis showed that compound 3 f is a good human acetylcholinesterase inhibitor [IC50=(0.36±0.02) μm], has strong antioxidant activity (3.61 μmol Trolox equivalents), and moderate Aβ1−42 antiaggregating power (40.3 %).

Design, synthesis and evaluation of second generation MurF inhibitors based on a cyanothiophene scaffold.

MurF ligase is a crucial enzyme that catalyses the ultimate intracellular step of bacterial peptidoglycan biosynthesis, and thus represents an attractive target for antibacterial drug discovery. We designed, synthesized and evaluated a new series of cyanothiophene-based inhibitors of MurF enzymes from Streptococcus pneumoniae and Escherichia coli. The target compounds had increased polarity compared to the first generation of inhibitors, with demonstrated enzyme inhibitory potencies in the low micromolar range. Furthermore, the best inhibitors displayed promising antibacterial activities against selected Gram-positive and Gram-negative strains. These results represent an important step towards the development of new antibacterial agents targeting peptidoglycan biosynthesis.

Dual inhibitors of cholinesterases and monoamine oxidases for Alzheimer’s disease

Accumulating evidence indicates a solid relationship between several enzymes and Alzheimers disease. Cholinesterases and monoamine oxidases are closely associated with the disease symptomatology and progression and have been tackled simultaneously using several multifunctional ligands. This design strategy offers great chances to alter the course of Alzheimers disease, in addition to alleviation of the symptoms. More than 15 years of research has led to the identification of various dual cholinesterase/monoamine oxidase inhibitors, while some showing positive outcomes in clinical trials, thus giving rise to additional research efforts in the field. The aim of this review is to provide an update on the novel dual inhibitors identified recently and to shed light on their therapeutic potential.