Inhibitor Report for: Dihydrotanshinone-I

Acetylcholinesterase (AChE) is a critical enzyme that regulates neurotransmission by degrading the neurotransmitter acetylcholine in synapses of the nervous system. It is an important target for both therapeutic drugs that treat Alzheimer's disease and organophosphate (OP) chemical warfare agents that cripple the nervous system and cause death through paralysis. We are exploring a strategy to design compounds that bind tightly at or near a peripheral or P-site near the mouth of the AChE active site gorge and exclude OPs from the active site while interfering minimally with the passage of acetylcholine. However, to target the AChE P-site, much more information must be gathered about the structure-activity relationships of ligands that bind specifically to the P-site. Here, we review our recent reports on two uncharged, natural product inhibitors of AChE, dihydrotanshinone I and territrem B, that have relatively high affinities for the enzyme. We describe an inhibitor competition assay and comment on the structures of these inhibitors in complex with recombinant human acetylcholinesterase as determined by X-ray crystallography. Our results reveal that dihydrotanshinone I binding is specific to only the P-site, while territrem B binding spans the P-site and extends into the acylation or A-site at the base of the gorge.

Cholinergic synaptic transmission often requires extremely rapid hydrolysis of acetylcholine by acetylcholinesterase (AChE). AChE is inactivated by organophosphates (OPs) in chemical warfare nerve agents. The resulting accumulation of acetylcholine disrupts cholinergic synaptic transmission and can lead to death. A potential long-term strategy for preventing AChE inactivation by OPs is based on evidence that OPs must pass through a peripheral site or P-site near the mouth of the AChE active site gorge before reacting with a catalytic serine in an acylation site or A-site at the base of the gorge. An ultimate goal of this strategy is to design compounds that bind tightly at or near the P-site and exclude OPs from the active site while interfering minimally with the passage of acetylcholine. However, to target the AChE P-site with ligands and potential drugs that selectively restrict access, much more information must be gathered about the structure-activity relationships of ligands that bind specifically to the P-site. We apply here an inhibitor competition assay that can correctly determine whether an AChE inhibitor binds to the P-site, the A-site, or both sites. We have used this assay to examine three uncharged, natural product inhibitors of AChE, including aflatoxin B1, dihydrotanshinone I, and territrem B. The first two of these inhibitors are predicted by the competition assay to bind selectively to the P-site, while territrem B is predicted to span both the P- and A-sites. These predictions have recently been confirmed by X-ray crystallography. Dihydrotanshinone I, with an observed binding constant (KI) of 750 nM, provides a good lead compound for the development of high-affinity, uncharged inhibitors with specificity for the P-site.

Acetylcholinesterase is a critical enzyme that regulates neurotransmission by degrading the neurotransmitter acetylcholine in synapses of the nervous system. It is an important target for both therapeutic drugs that treat Alzheimers disease and chemical warfare agents that cripple the nervous system and cause death through paralysis. The enzyme has both catalytic and peripheral sites to which inhibitors may bind. Structures of recombinant human acetylcholinesterase in complex with the natural product inhibitors dihydrotanshinone I and territrem B reveal dihydrotanshinone I binding that is specific to only the peripheral site and territrem B binding that spans both sites and distorts the protein backbone in the peripheral site. These inhibitors may function as important molecular templates for therapeutics used for treatment of disease and protection against nerve agents.

Acetylcholinesterase (AChE) is a critical enzyme that regulates neurotransmission by degrading the neurotransmitter acetylcholine in synapses of the nervous system. It is an important target for both therapeutic drugs that treat Alzheimer's disease and organophosphate (OP) chemical warfare agents that cripple the nervous system and cause death through paralysis. We are exploring a strategy to design compounds that bind tightly at or near a peripheral or P-site near the mouth of the AChE active site gorge and exclude OPs from the active site while interfering minimally with the passage of acetylcholine. However, to target the AChE P-site, much more information must be gathered about the structure-activity relationships of ligands that bind specifically to the P-site. Here, we review our recent reports on two uncharged, natural product inhibitors of AChE, dihydrotanshinone I and territrem B, that have relatively high affinities for the enzyme. We describe an inhibitor competition assay and comment on the structures of these inhibitors in complex with recombinant human acetylcholinesterase as determined by X-ray crystallography. Our results reveal that dihydrotanshinone I binding is specific to only the P-site, while territrem B binding spans the P-site and extends into the acylation or A-site at the base of the gorge.

Cholinergic synaptic transmission often requires extremely rapid hydrolysis of acetylcholine by acetylcholinesterase (AChE). AChE is inactivated by organophosphates (OPs) in chemical warfare nerve agents. The resulting accumulation of acetylcholine disrupts cholinergic synaptic transmission and can lead to death. A potential long-term strategy for preventing AChE inactivation by OPs is based on evidence that OPs must pass through a peripheral site or P-site near the mouth of the AChE active site gorge before reacting with a catalytic serine in an acylation site or A-site at the base of the gorge. An ultimate goal of this strategy is to design compounds that bind tightly at or near the P-site and exclude OPs from the active site while interfering minimally with the passage of acetylcholine. However, to target the AChE P-site with ligands and potential drugs that selectively restrict access, much more information must be gathered about the structure-activity relationships of ligands that bind specifically to the P-site. We apply here an inhibitor competition assay that can correctly determine whether an AChE inhibitor binds to the P-site, the A-site, or both sites. We have used this assay to examine three uncharged, natural product inhibitors of AChE, including aflatoxin B1, dihydrotanshinone I, and territrem B. The first two of these inhibitors are predicted by the competition assay to bind selectively to the P-site, while territrem B is predicted to span both the P- and A-sites. These predictions have recently been confirmed by X-ray crystallography. Dihydrotanshinone I, with an observed binding constant (KI) of 750 nM, provides a good lead compound for the development of high-affinity, uncharged inhibitors with specificity for the P-site.

Acetylcholinesterase is a critical enzyme that regulates neurotransmission by degrading the neurotransmitter acetylcholine in synapses of the nervous system. It is an important target for both therapeutic drugs that treat Alzheimers disease and chemical warfare agents that cripple the nervous system and cause death through paralysis. The enzyme has both catalytic and peripheral sites to which inhibitors may bind. Structures of recombinant human acetylcholinesterase in complex with the natural product inhibitors dihydrotanshinone I and territrem B reveal dihydrotanshinone I binding that is specific to only the peripheral site and territrem B binding that spans both sites and distorts the protein backbone in the peripheral site. These inhibitors may function as important molecular templates for therapeutics used for treatment of disease and protection against nerve agents.

The roots of Salvia miltiorrhiza ("Danshen") are used in traditional Chinese medicine for the treatment of numerous ailments including cardiovascular disease, hypertension, and ischemic stroke. Extracts of S. miltiorrhiza roots in the formulation "Compound Danshen Dripping Pill" are undergoing clinical trials in the United States. To date, the active components of this material have not been conclusively identified. We have determined that S. miltiorrhiza roots contain potent human carboxylesterase (CE) inhibitors, due to the presence of tanshinones. K(i) values in the nM range were determined for inhibition of both the liver and intestinal CEs. As CEs hydrolyze clinically used drugs, the ability of tanshinones and S. miltiorrhiza root extracts to modulate the metabolism of the anticancer prodrug irinotecan (CPT-11) was assessed. Our results indicate that marked inhibition of human CEs occurs following incubation with both pure compounds and crude material and that drug hydrolysis is significantly reduced. Consequently, a reduction in the cytotoxicity of irinotecan is observed following dosing with either purified tanshinones or S. miltiorrhiza root extracts. It is concluded that remedies containing tanshinones should be avoided when individuals are taking esterified agents and that patients should be warned of the potential drug-drug interaction that may occur with this material.

Alzhemier's disease (AD) is a common form of dementia in the ageing population which is characterized by depositions of amyloids and a cholinergic neurotransmission deficit in the brain. Current therapeutic intervention for AD is primarily based on the inhibition of brain acetylcholinesterase (AChE) to restore the brain acetylcholine level. Cryptotanshinone (CT) and dihydrotanshinone (DT) were diterpenoids extracted from Salvia miltiorrhiza Bge. having anti-cholinesterase activity. Here we characterized the inhibition property of these two diterpenoids towards human AChE and butyrylcholinesterase (BChE). Both CT and DT were found to be mixed non-competitive inhibitors for human AChE and an uncompetitive inhibitor for human BChE. The docking analyses of CT and DT into the active sites of both cholinesterases indicate that they interact with the allosteric site inside the active-site gorge mainly by hydrophobic interactions.