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1、Relationships between Inhibition Constants, Types of Inhibition and IC50 Calculation and Comparison on IC50 of Two Model Tyrosinase Inhibitors Hairong Mao1, Jiangang Xie1, Qimeng Zhang2, Xinyu L3, Shubai Li2,* 1 Department of Chemistry, Zhengzhou Teachers College, Zhengzhou, China 2 Department of Ch
2、emical Engineering Technology, Changzhou Institute of Engineering Technology, Changzhou, China 3 Institute of Fine Chemicals, Jiangsu Polytechnic University, Changzhou, China * Corresponding author. E-mail: AbstractCalculating methods for inhibitor concentration (IC50) leading to 50% activity lost
3、(for diphenolase activity) of a novel parabolic competitive inhibitor (the trifluoromethyl-containing 1,2,3-triazole, TF-TA) and a novel mixed inhibitor (E-4-(1-imidazoylmethyl) cinnamic acid (ozagrel) were derived on the basis of kinetics of tyrosinase inhibition. These methods were applied to calc
4、ulate IC50 values of the two tyrosinase inhibiting compounds. The experimental IC50 values of TF-TA and ozagrel were 41.9 M and 3.45 mM, respectiviely. Moreover, the calculated IC50 value of TF-TA and ozagrel were 82.8 M and 3.15 mM, respectively. The calculation result of ozagrel is closer to exper
5、imental data. Keywords-tyrosinase; IC50; trifluoromethyl-containing 1,2,3-triazole (TF-TA); E-4-(1-imidazoylmethyl) cinnamic acid (ozagrel) I. INTRODUCTION The regulation of enzyme activity is of great interest for biochemical research purposes and for a number of applications in medicine, physiolog
6、y, and pharmacology. Inhibition of enzymes from any biological source can be achieved by several agents, such as self-inactivating substrates and reversible or irreversible inhibitors. Tyrosinase (EC 1.14.18.1) is a copper containing enzyme with responsibilities for skin, hair, melanization and enzy
7、matic browning in fruits and vegetables 1-3. It catalyzes the hydroxylation of monophenols (monophenolase activity) and the oxidation of o-diphenols to o-quinones (diphenolase activity), both depending on molecular oxygen 4,5. The browning is responsible for loss in nutritional quality, and therefor
8、e becomes a major problem in the food industry. Therefore, the control of the tyrosinase activity is of importance in preventing the synthesis of melanin in the browning of mushrooms and other vegetables and fruits. Many efforts have been put in the search for feasible and effective tyrosinase inhib
9、itors 6-9. In our search for tyrosinase novel inhibitors, trifluoromethyl-containing 1,2,3-triazoles 10, and ozagrel analogues 11, were found to have obvious inhibitory effects on the monophenolase activity and the diphenolase activity of mushroom tyrosinase. The IC50 (inhibitor concentration leadin
10、g to 50% activity lost) is a measure of the effectiveness of a compound in inhibiting biological or biochemical function. Often, the compound in question is a drug candidate. This quantitative measure indicates how much of a particular drug or other inhibitors needed to inhibit a given biological pr
11、ocess by half. It is commonly used as a measure of antagonist drug potency in pharmacological research. However, it appears not to have been noticed previously that there is a very simple relationship between IC50 with inhibition constants and types of inhibition type. Therefore, the aim of the pres
12、ent work was to develop a method to calculate IC50 of two novel tyrosinase inhibitors. II. CALCULATION AND COMPARISON ON IC50 A. Calculation of IC50 for theParabolic-competitive Inhibitor The trifluoromethyl-containing 1,2,3-triazoles exhibit antimicrobial, antiviral and antitumor activities and hav
13、e a range of important applications in pharmaceutical and agrochemical industries 12. NNNF3COH Fig.1. Structure of the trifluoromethyl-containing 1,2,3-triazole (TF-TA). We found a novel trifluoromethyl-containing 1,2,3-triazole (TF-TA, see structure in Fig.1) can inhibit mushroom tyrosinase activit
14、y (IC50=41.9 M) with its increasing concentrations. The inhibition kinetics, analyzed by Lineweaver-Burk plots, indicated TF-TA to be a parabolic-competitive inhibitor of diphenolase when L-DOPA was used as substrate. It is suggested that two TF-TA molecules can combine with free tyrosinase to form
15、a dead-end complex at enzyme active site. The inhibition constants (Ki1 for inhibitor-enzyme complex and Ki2 for inhibitor-enzyme-inhibitor 978-1-4244-4713-8/10/$25.00 2010 IEEEcomplex) were estimated to be 92.9 mM and 1.36 mM, respectively. The mechanism representing parabolic-competitive inhibitio
16、n for the tyrosinase-catalyzed reaction is as follows: E + S ES E + P k1k2k-1k-3k3k- 4+ I- IEIk4EI2- I+ I E, S, I, P denote enzyme (mushroom tyrosinase), substrate (L-DOPA), inhibitor (TF-TA), and product (dopachrome), respectively; ES, EI, and EI2 are the respective compounds. As it is usually the
17、case that S ET and I ET. In the steady state, the rate of formation and disappearance of the complexes are identical: ES)(ES211kkk+= (1.1) 33IEEIkk= (1.2) EIIEI244= kk (1.3) The expressions for the total enzyme concentration and the reaction rate: EIEIESEE2T+= (1.4) ES2kV = (1.5) Insertion of the ex
18、pressions for E, EI, and EI2 in term of ES, into the expression for the total enzyme concentration gives ES) I 1 ( I S1SE4433121121T+=kkkkkkkkkk (1.6) ) I 1 ( I SSE4433121121T2+=kkkkkkkkkkkV (1.7) Because (k-1+k2)/k1 is the Michaelis constant (Km) for the reaction in the absence of inhibitor, k-3/k3
19、 is the equilibrium constant (Ki1) for the dissociation of the complex EI into E and I, and k-4/k4 is the equilibrium constant (Ki2) for the dissociation of the complex EI2 into EI and I, the rate equation may be written in the form ) I I 1 (SSi2i12i1mmaxKKKKVV+= (1.8) which in reciprocal for become
20、s maxi2i12i1maxm1) I I 1 (S1VKKKVKV+= (1.9) It is evident from Eq. 1.9 that the slope of Lineweaver-Burk lines is influenced by the changing I and I2. According to Eq. 1.8, in the absence of TF-TZ, the kinetic function is SSmmax0+=KVV (1.10) The inhibition ratio: 0%(1) 100ViV= SS) I I 1 (SS1mmaxi2i1
21、2i1mmax+=KVKKKKV (1.11) When i% reaches 50%, I = IC50, so, ) I I 1 (SS21i2i12i1mmKKKKK+= (1.12) The IC50 value (the negative value should be removed) is ()mm2mmi1i1i1i250mi1i24 S-()IC2KKKKKKK KKK K+= (1.13) According to the experiment conditions and Eq. 1.13 (Km = 0.117 mM, Ki1 = 92.9 mM, Ki2 = 1.36
22、 mM, and S = 1.0 mM), so the calculated IC50 value of is to be 82.8 M. Then, according to Fig. 5, the experimental IC50 value is 41.9 M. In this investigation, the calculated IC50 value is about two times that of the experimental one. B. Calculation of IC50 for the Mixed Inhibitor Ozagrel (E-4-(1-im
23、idazoylmethyl) cinnamic acid, see structure in Fig.2), as a selective inhibitor of thromboxane synthase (TXAS) in human platelets especially, is used in the treatment and prevention of various thrombotic diseases 13. We found that ozagrel inhibited mushroom tyrosinase activity. The IC50 value was 3.
24、45 mM. Ozagrel was estimated to be a reversible mixed-type inhibitor of diphenolase activity when L-DOPA was used as substrate with the constants (KS1, KS2, Ki1, and Ki2) determined to be 2.21, 3.89, 0.454, 0.799 mM, repectively. The inhibition kinetics, analyzed by Lineweaver-Burk plots, indicated
25、ozagrel to be a mixed inhibitor of diphenolase when L-DOPA was used as substrate. It demonstrated that ozagrel bound the enzyme at a site distincted from the substrate active site, but it bound to either E or ES. Fig. 2. Structure of (E-4-(1-imidazoylmethyl) cinnamic acid (ozagrel). In this type of
26、reversible inhibition, ozagrel can interact with both the free enzyme (tyrosinase) and the enzyme-substrate complex at a site other than the active site with a classical Michaelis-Menten reaction mechanism: E + S ES E + P kcatEI + S+I+IESIKi1KS1KS2Ki2 Here, E, S, I, P denote enzyme (mushroom tyrosin
27、ase), substrate (L-DOPA), inhibitor (ozagrel), and product (dopachrome), respectively; ES, EI, and ESI are the respective compounds. As it is usually the case that S E0 and I E0. It is assumed that the effect is not only on affinity but also on the rate of the breakdown of the ES complex; thus ozagr
28、el would influence the enzyme activity in two ways, affecting Vm as well as Km. It is formally convenient to regard KS1, KS2, Ki1and Ki2 as the dissociation constants of the respective complexes: ES into E and S, ESI into EI and S, EI into E and I, and ESI into ES and I, respectively. In the Michael
29、is-Menten state, the substrate-binding step and formation of the ES complex are fast relative to the breakdown rate. As is the usually the case, the equilibrium equations of the enzyme, substrate, inhibitor, product, and their respective compounds (ES, EI, ESI) are given: S1ESESK= (2.1) i1EI=EIK (2.
30、2) S2EISESIK= (2.3) i2ESI=ESIK (2.4) According to Eq. 2.1 to Eq. 2.4, the relationship with KS1, KS2, Ki1 and Ki2 is given: S1i1S2i2KKKK= (2.5) The expressions for the total enzyme concentration and the reaction rate: TE EES EIESI=+ (2.6) The overall velocity will be given as follows: catESVk= (2.7)
31、 Insertion of the expressions for E, EI, and ESI in term of ES, into the expression for the total enzyme concentration gives S1S1Ti1i2IIE (1)ESSSKKKK=+ + (2.8) catTS1i1i2E SII(1)S(1)kVKKK=+ (2.9) The rate equation may be written in the form maxS1i1i2SII(1)S(1)VVKKK=+ (2.10) which in reciprocal form
32、becomes S1i1i2maxmaxII(1)(1)11SKKKVVV+=+ (2.11) It is evident from Equation 11 that the mixed-type inhibition affects both Km and Vmax (its special case is noncompetitive inhibition which would affect the Vmax but not the Km). So, the slope and intercept of Equation 11 are given as the following for
33、m: S1i1maxI(1)SlopeKKV+= (2.12) i2maxI(1)InterceptKV+= (2.13) For the steady-state model, substrate binding occurs faster than the breakdown of the ES complex, so the kinetic function in the absence of inhibitor is max0S1SSVVK=+ (2.14) The inhibition ratio: 0%(1) 100ViV= maxS1i1i2maxS1SII(1)S(1)1SSV
34、KKKVK+= + (2.15) When i% reaches 50%, I = IC50, so, S15050S1i1i2S11ICIC2(1)S(1)KKKK+= + (2.16) The IC50 value is S150S1i2i1SICSKKKK+=+ (2.17) According to Eq. 2.17 and KS1, KS2, Ki1, and Ki2, the calculated IC50 is 3.15 mM, it is very close to the experimental data of IC50 (3.45 mM). III. CONCLUSION
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