Timolol Activates the Enzyme Activities of Human Carbonic Anhydrase I and II

Abstract

Therapy for glaucoma is generally individualized for each glaucoma patient, with several factors, including intraocular pressure (IOP) and the degree of optic nerve damage, given consideration. In the treatment of glaucoma, several types of agents such as beta-blockers, prostaglandin analogues and carbonic anhydrase (CA) inhibitors are clinically used at present in pharmacotherapy.1-2) Beta-blockers or prostaglandin F2-alpha analogues have been shown to be effective ocular hypotensive agents when used alone in ocular hypertensive or open-angle glaucoma patients.3,4) These two types of agents are currently recognized as the first-line medications for treating glaucoma.5) However, mono-therapy with timolol, a beta-blocker, or latanoprost, a prostaglandin analogue, is often insufficient to maintain a desirable IOP over the long term in many patients. 6) In these cases, CA inhibitors are often used as an adjunctive therapy to timolol or to latanoprost in order to achieve a desirable IOP. 7) Although it has been shown that monotherapy with timolol results in a smaller IOP reduction than monotherapy with latanoprost, the combination therapies of either timolol or latanoprost with CA inhibitors show comparable IOP reduction activity in glaucoma patients. 8) These clinical observations suggest that there exist different interactions between CA inhibitors and latanoprost or timolol that affect IOP reduction.In our previous paper, 9) latanoprost was shown in an in vitro study to be a weak noncompetitive inhibitor of human carbonic anhydrase (HCA) I and II and to bind to the zinc ion, which plays an important role in the expression of enzyme activity according to the AutoDocking simulation method.In the present study, to elucidate the interaction mechanism between timolol and CA inhibitors, the reaction mechanism and binding mode of timolol with respect to CA were investigated by enzyme-kinetic studies and an AutoDocking simulation method. MATERIALS AND METHODS MaterialsHuman CA I and II (HCA I and HCA II) were purchased from Sigma Co., Ltd. Since the purities of HCA I and II were shown to be more than 95% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these enzymes were used without further purification. Timolol hemihydrate (formal name:,5-thiadiazol-3-yl}oxy]-2-propanol 1/2H 2 O) was provided by Santen Pharmaceutical Co. Ltd. All other reagents used in this experiment were of analytical grade and were purchased from Wako Pure Chemical Co., Ltd. (Tokyo, Japan).Determination of the Concentration of CO 2 The CO 2 concentration of pure water saturated with CO 2 was measured using a CO 2 electrode (Orion 9502BN) connected to an Orion Ion-meter 720A. The standard solutions of CO 2 were prepared using 0.5-5 ml of 0.1 M NaHCO 3 solutions mixed with 5 ml of 1 M citric acid buffer (pH 4.5) and filled up to 50 ml with pure water.Determination of the Enzyme Activity of Carbonic Anhydrase Determination of the enzyme activity was based on the hydration of CO 2 catalyzed by HCA I and II. The technique used for...

Activation of carbonic anhydrases from human brain by amino alcohol oxime ethers: towards human carbonic anhydrase VII selective activators

“…Interestingly, pharmacological agents born for other medical applications ( Figure 4 ) showed also to possess more or less relevant CA-activating effects, among which psychoactive compounds of the amphetamine and methamphetamine family 54 , the selective serotonin reuptake inhibitors fluoxetine, sertraline and citalopram 55 , the phosphodiesterase IV inhibitor sildenafil 56 , and the β-blocker amino alcohol derivative timolol 57 . Timolol was taken as lead compound in the present study to design a new series of uncommon CAAs, which do not bear imidazole like scaffolds, intesively explored in the field.…”

“…The molecule of histamine has been extensively modified (Figure 3), including substituents on the imidazole C atoms (A) 41,42 , replacing the imidazole ring with other heterocycles, such as 2,4,6-trisubstituted pyridinium (B), 1,3,4-thiadiazole (C) or a combination of these two ring systems (D) 43,44 and functionalising the NH 2 group, as in carboxamides/ureas/thioureas (E) 45 , sulphonamides (F) 46 , arylsulfonylureas (G) 47 , bis-histamine (H) 48,49 , oligopeptides 49,50 , or imidazole/imidazoline derivatives of the alkaloyd spinaceamine 51,52 and drug clonidine 53 (Figure 3) were reported to possess improved CA activatory profile when potency and isoform selectivity are concerned. Interestingly, pharmacological agents born for other medical applications (Figure 4) showed also to possess more or less relevant CA-activating effects, among which psychoactive compounds of the amphetamine and methamphetamine family 54 , the selective serotonin reuptake inhibitors fluoxetine, sertraline and citalopram 55 , the phosphodiesterase IV inhibitor sildenafil 56 , and the b-blocker amino alcohol derivative timolol 57 . Timolol was taken as lead compound in the present study to design a new series of uncommon CAAs, which do not bear imidazole like scaffolds, intesively explored in the field.…”

“…The enzyme kinetic method showed that timolol noncompetitively activates hCA I and II by forming of a ternary complex consisting of the enzyme, the substrate, and timolol. Docking studies were used to point out that timolol also binds at the entrance of the active site cavity nearby the proton shuttle residue His64 57 . Thus, a series of aminoalcohol oxime ether derivatives previously shown to possess b-blocking or analgesic/ antiarrhythmic activity (compounds 1-16) [25][26][27][28][29][30][31][32] , was investigated for the activation of a panel of brain hCAs.…”

Autonomic drugs in the treatment of canine and feline glaucoma – Part II: Medications that lower intraocular pressure by reducing aqueous humour production

“…Na + /K + -ATPase, Mg 2+ -ATPase, and prostaglandin biosynthesis (Watanabe and Chiou 1983); what is more, it has been recently demonstrated that timolol activates the enzyme activities of human CA-II (Sugimoto et al 2010).…”

Investigation of Drug Interactions among Ophthalmic Solutions for Treating Glaucoma and Establishment of Rational Choice on Pharmacotherapy for Cost-Minimization

“…The structure of the timolol-hCA-I or -hCA-II complex8) A: The structure of the timolol-hCA-I complex superimposed onto that of the timolol-hCA-II complex. The rounded ribbon model structures of hCA-I and hCA-II are shown in white and magenta, respectively, and the zinc ions located at the active sites are shown as a green space fill model.…”