Structure and function of S-adenosylhomocysteine hydrolase

In mammals, S-adenosylhomocysteine hydrolase (AdoHcyase) is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine (AdoHcy) to homocysteine and adenosine. AdoHcy is the product of all adenosylmethionine (AdoMet)-dependent biological transmethylations. These reactions have a wide range of products, and are common in all facets of biometabolism. As a product inhibitor, elevated levels of AdoHcy suppress AdoMet-dependent transmethylations. Thus, AdoHcyase is a regulator of biological transmethylation in general. The three-dimensional structure of AdoHcyase complexed with reduced nicotinamide adenine dinucleotide phosphate (NADH) and the inhibitor (1′R, 2′S, 3′R)-9-(2′,3′-dihyroxycyclopenten-1-yl)adenine (DHCeA) was solved by a combination of the crystallographic direct methods program, SnB, to determine the selenium atom substructure and by treating the multiwavelength anomalous diffraction data as a special case of multiple isomorphous replacement. The enzyme architecture resembles that observed for NAD-dependent dehydrogenases, with the catalytic domain and the cofactor binding domain each containing a modified Rossmann fold. The two domains form a deep active site cleft containing the cofactor and bound inhibitor molecule. A comparison of the inhibitor complex of the human enzyme and the structure of the rat enzyme, solved without inhibitor, suggests that a 17° rigid body movement of the catalytic domain occurs upon inhibitor/substrate binding.     

Design and synthesis of donepezil analogues as dual AChE and BACE-1 inhibitors

Multi-target-directed ligands (MTDLs) centered on β-secretase 1 (BACE-1) inhibition are emerging as innovative therapeutics in addressing the complexity of neurodegenerative diseases. A new series of donepezil analogues was designed, synthesized and evaluated as MTDLs against neurodegenerative diseases. Profiling of donepezil, a potent acetylcholinesterase (hAChE) inhibitor, into BACE-1 inhibition was achieved through introduction of backbone amide linkers to the designed compounds which are capable of hydrogen-bonding with BACE-1 catalytic site. In vitro assays and molecular modeling studies revealed the dual mode of action of compounds 4–6 against hAChE and BACE-1. Notably, compound 4 displayed potent hAChE inhibition (IC₅₀ value of 4.11 nM) and BACE-1 inhibition (IC₅₀ value of 18.3 nM) in comparison to donepezil (IC₅₀ values of 6.21 and 194 nM against hAChE and BACE-1, respectively). Moreover, 4 revealed potential metal chelating property, low toxicity on SH-SY5Y neuroblastoma cells and ability to cross the blood–brain barrier (BBB) in PAMPA-BBB assay which renders 4 a potential lead for further optimization of novel small ligands for the treatment of Alzheimer's disease.     

Brain penetrant kinase inhibitors: Learning from kinase neuroscience discovery

A recent review of kinase inhibitors in clinical trials for brain cancer noted differences in the properties of these compounds relative to the mean property parameters associated with drugs marketed for CNS-associated conditions. However, many of these kinase drugs arose from opportunistic observations of brain activity, rather than design or flow schemes focused on optimizing CNS penetration. Thus, this digest examines kinase inhibitors that have been developed specifically for neurodegenerative indications such as Alzheimer’s or Parkinson’s disease, and considers design, flow scheme, and the physicochemical properties associated with compounds that have demonstrated brain penetrance.     

Inactivation of S-adenosylhomocysteine hydrolase by 5'-deoxy-5'-methylthioadenosine

In the coupling of ATP pyrophosphorolysis to Ca²⁺ transport in beef heart mitochondria, for each molecule of ATP cleaved, one proton is released and one Ca²⁺ is transported into the interior space. With the use of tritium labelled ATP, it could be shown that ATP is pyrophosphorylyzed into a species equivalent to Pi that moves inward, and a species equivalent to ADP that is extruded into the aqueous space on the exterior of the mitochondrion. The species equivalent to Pi has been identified as a negatively charged form of Pi (PO^?) and the species equivalent to ADP as a positively charged form (ADP⁺). The inward flow of PO^? is coupled to the inward flow of Ca²⁺ in 1:1 stoichiometry—a token that Ca²⁺ must enter in the form of Ca²⁺A^?, where A^? is a monovalent anion. During ATP pyrophosphorolysis protons are released on the I side and taken up on the M side—one proton for each molecule of ATP cleaved. The alkalinization of the matrix space leads to the deposition of Ca₃(PO₄)₂ and to the extrusion of the two species released by this deposition (Pi, K⁺). Two thirds of the PO^? is trapped as Ca₃(PO₄)₂ in the matrix space and one third is extruded into the external space. The extrusion of K⁺ provides a mechanism by which protons can be continuously brought into the matrix space to sustain a high rate of coupled pyrophosphorolysis of ATP. The coupling pattern for Ca²⁺ transport driven by ATP pyrophosphorolysis is identical with the corresponding pattern for Ca²⁺ transport driven by electron transfer. This identity is suggestive that coupling mediated by the Fo-F₁ system and coupling mediated by electron transfer complexes are mechanistically indistinguishable.     

Novel racemic tetrahydrocurcuminoid dihydropyrimidinone analogues as potent acetylcholinesterase inhibitors

The synthesis of racemic tetrahydrocurcumin- (THC-), tetrahydrodemethoxycurcumin- (THDC-) and tetrahydrobisdemethoxycurcumin- (THBDC-) dihydropyrimidinone (DHPM) analogues was achieved by utilizing the multi-component Biginelli reaction in the presence of copper sulphate as a catalyst. The evaluation of acetylcholinesterase inhibitors for Alzheimer’s disease of these compounds showed that they exhibited higher inhibitory activity than their parent analogues. THBDC–DHPM demonstrated the most potent inhibitory activity with an IC₅₀ value of 1.34 ± 0.03 μM which was more active than the approved drug galanthamine (IC₅₀ = 1.45 ± 0.04 μM).     

Design,synthesis and evaluation of new 4-arylthiazole-2-amine derivatives as acetylcholinesterase inhibitors

A series of new 4-arylthiazole-2-amine derivatives as acetylcholinesterase inhibitors (AChEIs) were designed and synthesized, Furthermore, their inhibitory activities against acetylcholinesterase in vitro were tested by Ellman spectrophotometry, and the results of inhibitory activity test showed that most of them had a certain acetylcholinesterase inhibitory activity in vitro. Moreover, the IC₅₀ value of compound 4f was to 0.66 μM, which was higher than that of Rivastigmine and Huperzine-A as reference compounds, and it had a weak inhibitory effect on butyrylcholinesterase. The potential binding mode of compound 4f with AChE was investigated by the molecular docking, and the results showed that 4f was strongly bound up with AChE with the optimal conformation, in addition, their binding energy reached −11.27 Kcal*mol⁻¹. At last, in silico molecular property of the synthesized compounds were predicted by using Molinspiration online servers. It can be concluded that the lead AChEIs compound 4f presented satisfactory drug-like characteristics.     

Carbocyclic pyrimidine nucleosides as inhibitors of S-adenosylhomocysteine hydrolase

The design, synthesis, and unexpected inhibitory activity against S-adenosyl-homocysteine (SAH) hydrolase (SAHase, EC 3.3.1.1) for a series of truncated carbocyclic pyrimidine nucleoside analogues is presented. Of the four nucleosides obtained, 10 was found to be active with a Ki value of 5.0 microM against SAHase.     

Inhibition of S-adenosylhomocysteine hydrolase by acyclic sugar adenosine analogue D-eritadenine. Crystal structure of S-adenosylhomocysteine hydrolase complexed with D-eritadenine

D-eritadenine (DEA) is a potent inhibitor (IC(50) = 7 nm) of S-adenosyl-l-homocysteine hydrolase (AdoHcyase). Unlike cyclic sugar Ado analogue inhibitors, including mechanism-based inhibitors, DEA is an acyclic sugar Ado analogue, and the C2' and C3' have opposite chirality to those of the cyclic sugar Ado inhibitors. Crystal structures of DEA alone and in complex with AdoHcyase have been determined to elucidate the DEA binding scheme to AdoHcyase. The DEA-complexed structure has been analyzed by comparing it with two structures of AdoHcyase complexed with cyclic sugar Ado analogues. The DEA-complexed structure has a closed conformation, and the DEA is located near the bound NAD(+). However, a UV absorption measurement shows that DEA is not oxidized by the bound NAD(+), indicating that the open-closed conformational change of AdoHcyase is due to the substrate/inhibitor binding, not the oxidation state of the bound NAD. The adenine ring of DEA is recognized by four essential hydrogen bonds as observed in the cyclic sugar Ado complexes. The hydrogen bond network around the acyclic sugar moiety indicates that DEA is more tightly connected to the protein than the cyclic sugar Ado analogues. The C3'-H of DEA is pointed toward C4 of the bound NAD(+) (C3'...C4 = 3.7 A), suggesting some interaction between DEA and NAD(+). By placing DEA into the active site of the open structure, the major forces to stabilize the closed conformation of AdoHcyase are identified as the hydrogen bonds between the backbone of His-352 and the adenine ring, and the C3'-H...C4 interaction. DEA has been believed to be an inactivator of AdoHcyase, but this study indicates that DEA is a reversible inhibitor. On the basis of the complexed structure, selective inhibitors of AdoHcyase have been designed.     

BACE1 inhibitors: A head group scan on a series of amides

A series of amides bearing a variety of amidine head groups was investigated as BACE1 inhibitors with respect to inhibitory activity in a BACE1 enzyme as well as a cell-based assay. Determination of their basicity as well as their properties as substrates of P-glycoprotein revealed that a 2-amino-1,3-oxazine head group would be a suitable starting point for further development of brain penetrating compounds for potential Alzheimer’s disease treatment.     

Adenosine dialdehyde and neplanocin A: Potent inhibitors of S-adenosylhomocysteine hydrolase in neuroblastoma N2a cells

S-Adenosylhomocysteine hydrolase (AdoHcy hydrolase, E.C. 3.3.1.1) catalyzes the metabolism of S-adenosylhomocysteine (AdoHcy) to adenosine (Ado) and homocysteine (Hcy) in mouse neuroblastoma N2a cells. AdoHcy hydrolase in N2a cells can be inhibited completely by adenosine dialdehyde (Ado dialdehyde) or neplanocin A. The inhibitory effects of Ado dialdehyde (2.5 μM) and neplanocin A (1 μM) on cellular AdoHcy hydrolase were time-dependent, with total enzyme inhibition occurring after 30 min and 15 min of incubation, respectively. The inhibition of AdoHcy hydrolase produced by Ado dialdehyde and neplanocin A persisted for up to 72 h of incubation, and was paralleled by a time-dependent increase in endogenous AdoHcy levels reaching a maximum 4-fold elevation after 8 h of incubation with Ado dialdehyde and an 11-fold increase in the neplanocin A-treated cells. This increase in AdoHcy levels produced a subsequent inhibition of S-adenosylmethionine (AdoMet)-dependent cellular methylations (e.g. protein carboxylmethylation (PCM), lipid methylation). In addition, neplanocin A was metabolically converted to the corresponding AdoMet analog, S-neplanocylmethionine (NepMet), in neuroblastoma N2a cells. NepMet reached maximum levels after 8 h of incubation of the cells with neplanocin A.     

Selectivity of phenothiazine cholinesterase inhibitors for neurotransmitter systems

Synthetic derivatives of phenothiazine have been used for over a century as well-tolerated drugs against a variety of human ailments from psychosis to cancer. This implies a considerable diversity in the mechanisms of action produced by structural changes to the phenothiazine scaffold. For example, chlorpromazine treatment of psychosis is related to its interaction with dopaminergic receptors. On the other hand, antagonistic action of such drugs on cholinergic receptor systems would be counter-productive for treatment of Alzheimer’s disease. In a search for phenothiazines that are inhibitors of cholinesterases, especially butyrylcholinesterase, with potential to treat Alzheimer’s disease, we wished to ascertain that such molecules could be devoid of neurotransmitter receptor interactions. To that end, a number of our synthetic N-10-carbonyl phenothiazine derivatives, with cholinesterase inhibitory activity, were tested for interaction with a variety of neurotransmitter receptor systems. We demonstrate that phenothiazines can be prepared without significant neurotransmitter receptor interactions while retaining high potency as cholinesterase ligands for treatment of Alzheimer’s disease.     

Structure guided design of a series of selective pyrrolopyrimidinone MARK inhibitors

The initial structure activity relationships around an isoindoline uHTS hit will be described. Information gleaned from ligand co-crystal structures allowed for rapid refinements in both MARK potency and kinase selectivity. These efforts allowed for the identification of a compound with properties suitable for use as an in vitro tool compound for validation studies on MARK as a viable target for Alzheimer’s disease.

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Adenosine metabolism in a rat hippocampal slice preparation: incorporation into S-adenosylhomocysteine

Abstract: The incorporation of [¹⁴C]adenosine into various metabolites was studied in a hippocampal slice preparation in order to assess the extent of adenosine metabolism via synthesis of S -adenosylhomocysteine, a potent inhibitor of transmethylation reactions. Highest incorporation of ¹⁴C occurred into nucleotides, with only a few percent being recovered in inosine + hypoxanthine, S -adenosylhomocysteine, and the free adenosine pool. Labeling of S -adenosylhomocysteine did not significantly increase with higher concentrations of added adenosine despite greater accumulation of free [¹⁴C]adenosine in the tissue. Addition of l -homocysteine significantly increased the labelling of S -adenosylhomocysteine. The results indicate that S -adenosylhomocysteine synthesis is a minor pathway of adenosine metabolism in brain tissue under steady-state conditions. Further, changes in adenosine concentration, without a concomitant change in l -homocysteine availability, are unlikely to lead to a significant accumulation of S -adenosylhomocysteine. S -Adenosylhomocysteine is therefore not likely to play a significant role in mediating the biological effects of adenosine in the CNS via inhibition of transmethylations.     

Synthesis and 2D-QSAR study of dispiropyrrolodinyl-oxindole based alkaloids as cholinesterase inhibitors

In this work, we describe the regioselective synthesis of some new dispiro[indene-2,3′-pyrrolidine-2′,3″-indoline]-1,2″(3H)-dione 4-29 attributable to the previously described methods. All the new chemical entities were assessed in-vitro as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes; while no significant inhibitory activity for the tested compounds were assigned on AChE, compounds 4, 27, 29, 28 and 15 were the most active against BChE enzyme with IC₅₀ = 13.7 µM, 21.8 µM, 22.1 µM, 22.9 µM and 24.9 µM respectively compared to Donepezil (IC₅₀ = 0.72 µM). Compound 4 was found to have a mixed type mode of inhibition, the bioactivity of the new chemical entities (N = 26, n = 5, R² = 0.893, R² cvOO = 0.831, R² cvMO = 0.838, F = 33.32, s² = 0.003) was elucidated via a statistically significant QSAR model utilizing CODESSA-Pro software that validated the observed results.     

Design,synthesis, and evaluation of guanylhydrazones as potential inhibitors or reactivators of acetylcholinesterase

Analogs of pralidoxime, which is a commercial antidote for intoxication from neurotoxic organophosphorus compounds, were designed, synthesized, characterized, and tested as potential inhibitors or reactivators of acetylcholinesterase (AChE) using the Ellman’s test, nuclear magnetic resonance, and molecular modeling. These analogs include 1-methylpyridine-2-carboxaldehyde hydrazone, 1-methylpyridine-2-carboxaldehyde guanylhydrazone, and six other guanylhydrazones obtained from different benzaldehydes. The results indicate that all compounds are weak AChE reactivators but relatively good AChE inhibitors. The most effective AChE inhibitor discovered was the guanylhydrazone derived from 2,4-dinitrobenzaldehyde and was compared with tacrine, displaying similar activity to this reference material. These results indicate that guanylhydrazones as well as future similar derivatives may function as drugs for the treatment of Alzheimer's disease.     

Discovery of amino-1,4-oxazines as potent BACE-1 inhibitors

New amino-1,4-oxazine derived BACE-1 inhibitors were explored and various synthetic routes developed. The binding mode of the inhibitors was elucidated by co-crystallization of 4 with BACE-1 and X-ray analysis. Subsequent optimization led to inhibitors with low double digit nanomolar activity in a biochemical and single digit nanomolar potency in a cellular assays. To assess the inhibitors for their permeation properties and potential to cross the blood-brain-barrier a MDR1-MDCK cell model was successfully applied. Compound 8a confirmed the in vitro results by dose-dependently reducing Aβ levels in mice in an acute treatment regimen.     

Discovery of S-adenosyl-l-homocysteine hydrolase inhibitors based on non-adenosine analogs

High throughput screening using Automated Ligand Identification System (ALIS) resulted in the discovery of a new series of S-adenosyl-l-homocysteine hydrolase inhibitors based on non-adenosine analogs. The optimization campaign led to very potent and competitive compound 39 with a K_i value of 1.5 nM. Compound 39 could be a promising lead compound for research to reduce elevated homocysteine levels.     

Novel biphenyl bis-sulfonamides as acetyl and butyrylcholinesterase inhibitors: Synthesis,biological evaluation and molecular modeling studies

A series of new biphenyl bis-sulfonamide derivatives 2a–3p were synthesized in good to excellent yield (76–98%). The inhibitory potential of the synthesized compounds on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was investigated. Most of the screened compounds showed modest in vitro inhibition for both AChE and BChE. Compared to the reference compound eserine (IC₅₀ 0.04 ± 0.0001 μM for AChE) and (IC₅₀ 0.85 ± 0.0001 μM for BChE), the IC₅₀ values of these compounds were ranged from 2.27 ± 0.01 to 123.11 ± 0.04 μM for AChE and 7.74 ± 0.07 to <400 μM for BuChE. Among the tested compounds, 3p was found to be the most potent against AChE (IC₅₀ 2.27 ± 0.01 μM), whereas 3g exhibited the highest inhibition for BChE (IC₅₀ 7.74 ± 0.07 μM). Structure–activity relationship (SAR) of these compounds was developed and elaborated with the help of molecular docking studies.     

Design,synthesis and docking study of novel coumarin ligands as potential selective acetylcholinesterase inhibitors

New coumaryl-thiazole derivatives with the acetamide moiety as a linker between the alkyl chains and/or the heterocycle nucleus were synthesized and in vitro tested as acetylcholinesterase (AChE) inhibitors. 2-(diethylamino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)acetamide (6c, IC₅₀ value of 43?nM) was the best AChE inhibitor with a selectivity index of 4151.16 over BuChE. Kinetic study of AChE inhibition revealed that 6c was a mixed-type inhibitor. Moreover, the result of H4IIE hepatoma cell toxicity assay for 6c showed negligible cell death. Molecular docking studies were also carried out to clarify the inhibition mode of the more active compounds. Best pose of compound 6c is positioned into the active site with the coumarin ring wedged between the residues of the CAS and catalytic triad of AChE. In addition, the coumarin ring is anchored into the gorge of the enzyme by H-bond with Tyr130.