Peptidomimetics based on ammonium decasubstituted pillar[5]arenes: Influence of the alpha-amino acid residue nature on cholinesterase inhibition.

Cholinesterase inhibitors are a group of medicines that are widely used for the treatment of cognitive impairments accompanying Alzheimer's disease as well as for the treatment of pathological muscle weaknesses syndromes such as myasthenia gravis. The search for novel non-toxic and effective cholinesterase inhibitors for creating neuroprotective and neurotransmitter agents is an urgent interdisciplinary problem. For the first time, the application of water-soluble pillar[5]arenes containing amino acid residues as effective cholinesterase inhibitors was shown. The influence of the nature of aliphatic and aromatic alpha-amino acid residues (glycine, l-alanine, l-phenylalanine and l-tryptophan) on self-assembly, aggregate's stability, cytotoxicity on A549 and LEK cells and cholinesterase inhibition was studied. It was found that the studied compounds with aliphatic amino acid residues showed a low inhibitory ability against cholinesterases. It was established that the pillar[5]arene containing fragments of l-phenylalanine is the most promising inhibitor of butyrylcholinesterase (IC50 = 0.32 ± 0.01 µM), the pillar[5]arene with l-tryptophan residues is the most promising inhibitor of acetylcholinesterase (IC50 = 0.32 ± 0.01 µM). This study has shown a possible application of peptidomimetics based on pillar[5]arenes to inhibit cholinesterase, as well as control the binding affinity to a particular enzyme in a structure-dependent manner.

Novel slow-binding reversible acetylcholinesterase inhibitors based on uracil moieties for possible treatment of myasthenia gravis and protection from organophosphate poisoning.

A series of new compounds in which uracil and 3,6-dimethyluracil moieties are bridged with different spacers were prepared and evaluated in vitro for the acetyl- and butyrylcholinesterase (AChE and BChE) inhibitory activities. These bisuracils are shown to be very effective inhibitors of AChE, inhibiting the enzyme at nano- and lower molar concentrations with extremely high selectivity for AChE vs. BChE. Kinetic analysis showed that the lead compound 2h acts as a slow-binding inhibitor of AChE and possess a long drug-target residence time (τ = 1/koff = 18.6 ± 7.5 min). Moreover, compound 2h ameliorated muscle weakness in myasthenia gravis rat model with a lower effective dose and longer lasting effect than pyridostigmine bromide. Besides, it was shown that compound 2h has an effect of increasing efficiency of antidotal therapy as a pretreatment for poisoning by organophosphates.

Specific inhibition of acetylcholinesterase as an approach to decrease muscarinic side effects during myasthenia gravis treatment.

Non-selective inhibitors of cholinesterases (ChEs) are clinically used for treatment of myasthenia gravis (MG). While being generally safe, they cause numerous adverse effects including induction of hyperactivity of urinary bladder and intestines affecting quality of patients life. In this study we have compared two ChEs inhibitors, a newly synthesized compound C547 and clinically used pyridostigmine bromide, by their efficiency to reduce muscle weakness symptoms and ability to activate contractions of urinary bladder in a rat model of autoimmune MG. We found that at dose effectively reducing MG symptoms, C547 did not affect activity of rat urinary bladder. In contrast, at equipotent dose, pyridostigmine caused a significant increase in tonus and force of spontaneous contractions of bladder wall. We also found that this profile of ChEs inhibitors translates into the preparation of human urinary bladder. The difference in action observed for C547 and pyridostigmine we attribute to a high level of pharmacological selectivity of C547 in inhibiting acetylcholinesterase as compared to butyrylcholinesterase. These results raise reasonable hope that selective acetylcholinesterase inhibitors should show efficacy in treating MG in human patients with a significant reduction in adverse effects related to hyperactivation of smooth muscles.

C-547, a 6-methyluracil derivative with long-lasting binding and rebinding on acetylcholinesterase: Pharmacokinetic and pharmacodynamic studies.

C-547, a potent slow-binding inhibitor of acetylcholinesterase (AChE) was intravenously administered to rat (0.05 mg/kg). Pharmacokinetic profiles were determined in blood and different organs: extensor digitorum longus muscle, heart, liver, lungs and kidneys as a function of time. Pharmacokinetics (PK) was studied using non-compartmental and compartmental analyses. A 3-compartment model describes PK in blood. Most of injected C-547 binds to albumin in the bloodstream. The steady-state volume of distribution (3800 ml/kg) is 15 times larger than the distribution volume, indicating a good tissue distribution. C-547 is slowly eliminated (kel = 0.17 h-1; T1/2 = 4 h) from the bloodstream. Effect of C-547 on animal model of myasthenia gravis persists for more than 72 h, even though the drug is not analytically detectable in the blood. A PK/PD model was built to account for such a pharmacodynamical (PD) effect. Long-lasting effect results from micro-PD mechanisms: the slow-binding nature of inhibition, high affinity for AChE and long residence time on target at neuromuscular junction (NMJ). In addition, NMJ spatial constraints i.e. high concentration of AChE in a small volume, and slow diffusion rate of free C-547 out of NMJ, make possible effective rebinding of ligand. Thus, compared to other cholinesterase inhibitors used for palliative treatment of myasthenia gravis, C-547 is the most selective drug, displays a slow pharmacokinetics, and has the longest duration of action. This makes C-547 a promising drug leader for treatment of myasthenia gravis, and a template for development of other drugs against neurological diseases and for neuroprotection.

Slow-binding inhibition of acetylcholinesterase by an alkylammonium derivative of 6-methyluracil: mechanism and possible advantages for myasthenia gravis treatment.

Inhibition of human AChE (acetylcholinesterase) and BChE (butyrylcholinesterase) by an alkylammonium derivative of 6-methyluracil, C-547, a potential drug for the treatment of MG (myasthenia gravis) was studied. Kinetic analysis of AChE inhibition showed that C-547 is a slow-binding inhibitor of type B, i.e. after formation of the initial enzyme·inhibitor complex (Ki=140 pM), an induced-fit step allows establishment of the final complex (Ki*=22 pM). The estimated koff is low, 0.05 min(-1) On the other hand, reversible inhibition of human BChE is a fast-binding process of mixed-type (Ki=1.77 µM; Ki'=3.17 µM). The crystal structure of mouse AChE complexed with C-547 was solved at 3.13 Å resolution. The complex is stabilized by cation-π, stacking and hydrogen-bonding interactions. Molecular dynamics simulations of the binding/dissociation processes of C-547 and C-35 (a non-charged analogue) to mouse and human AChEs were performed. Molecular modelling on mouse and human AChE showed that the slow step results from an enzyme conformational change that allows C-547 to cross the bottleneck in the active-site gorge, followed by formation of tight complex, as observed in the crystal structure. In contrast, the related non-charged compound C-35 is not a slow-binding inhibitor. It does not cross the bottleneck because it is not sensitive to the electrostatic driving force to reach the bottom of the gorge. Thus C-547 is one of the most potent and selective reversible inhibitors of AChE with a long residence time, τ=20 min, longer than for other reversible inhibitors used in the treatment of MG. This makes C-547 a promising drug for the treatment of this disease.

6-Methyluracil Derivatives as Bifunctional Acetylcholinesterase Inhibitors for the Treatment of Alzheimer's Disease.

Novel 6-methyluracil derivatives with ω-(substituted benzylethylamino)alkyl chains at the nitrogen atoms of the pyrimidine ring were designed and synthesized. The numbers of methylene groups in the alkyl chains were varied along with the electron-withdrawing substituents on the benzyl rings. The compounds are mixed-type reversible inhibitors of cholinesterases, and some of them show remarkable selectivity for human acetylcholinesterase (hAChE), with inhibitory potency in the nanomolar range, more than 10,000-fold higher than that for human butyrylcholinesterase (hBuChE). Molecular modeling studies indicate that these compounds are bifunctional AChE inhibitors, spanning the enzyme active site gorge and binding to its peripheral anionic site (PAS). In vivo experiments show that the 6-methyluracil derivatives are able to penetrate the blood-brain barrier (BBB), inhibiting brain-tissue AChE. The most potent AChE inhibitor, 3 d (1,3-bis[5-(o-nitrobenzylethylamino)pentyl]-6-methyluracil), was found to improve working memory in scopolamine and transgenic APP/PS1 murine models of Alzheimer's disease, and to significantly decrease the number and area of ß-amyloid peptide plaques in the brain.