Linear Relationships between the Ligand Binding Energy and the Activation Energy of Time-dependent Inhibition of Steroid 5alpha -Reductase by Delta 1-4-Azasteroids*

Gaochao TianDagger § and Curt D. Haffner

From the Departments of Dagger  Molecular Biochemistry and  Medicinal Chemistry, GlaxoSmithKline Research and Development, Research Triangle Park, North Carolina 27709

Received for publication, January 29, 2001, and in revised form, February 16, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The inhibition of steroid 5alpha -reductase (5AR) by Delta 1-4-azasteroids is characterized by a two-step time-dependent kinetic mechanism where inhibitor combines with enzyme in a fast equilibrium, defined by the inhibition constant Ki, to form an initial reversible enzyme-inhibitor complex, which subsequently undergoes a time-dependent chemical rearrangement, defined by the rate constant k3, leading to the formation of an apparently irreversible, tight-binding enzyme-inhibitor complex (Tian, G., Mook, R. A., Jr., Moss, M. L., and Frye, S. V. (1995) Biochemistry 34, 13453-13459). A detailed kinetic analysis of this process with a series of Delta 1-4-azasteroids having different C-17 substituents was performed to understand the relationships between the rate of time-dependent inhibition and the affinity of the time-dependent inhibitors for the enzyme. A linear correlation was observed between ln(1/Ki), which is proportional to the ligand binding energy for the formation of the enzyme-inhibitor complex, and ln(1/(k3/Ki)), which is proportional to the activation energy for the inhibition reaction under the second order reaction condition, which leads to the formation of the irreversible, tight-binding enzyme-inhibitor complex. The coefficient of the correlation was -0.88 ± 0.07 for type 1 5AR and -1.0 ± 0.2 for type 2 5AR. In comparison, there was no obvious correlation between ln(1/Ki) and ln(1/k3), which is proportional to the activation energy of the second, time-dependent step of the inhibition reaction. These data are consistent with a model where ligand binding energies provided at C-17 of Delta 1-4-azasteroids is fully expressed to lower the activation energy of k3/Ki with little perturbation of the energy barrier of the second, time-dependent step.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

5AR1 catalyzes the NADPH-dependent reductive conversion of testosterone to dihydrotestosterone. Two isozymes of 5AR, designated types 1 and 2, have been described (1, 2). Although 5AR1 is predominantly expressed in skin and liver, 5AR2 is mainly expressed in prostate, seminal vesicles, liver, and epididymis (3). Both 5AR1 and 5AR2 are implicated in benign prostatic hyperplasia (2), a condition affecting the majority of men over age of 60 (4). Intense efforts made over the past decade to develop drugs against the activity of this enzyme has led to the discovery of potent, time-dependent Delta 1-4-azasteroidal inhibitors of 5AR, including finasteride and GG745 (see Fig. 1).

Finasteride inhibits both 5AR1 and 5AR2 in a time-dependent manner (5-7). The kinetic mechanism of this time-dependent inhibition is characterized by a fast binding step for the formation of an initial enzyme-inhibitor complex (EI), followed by a time-dependent event leading to the formation of an apparently irreversible enzyme-inhibitor complex (EI*). This time-dependent event involves a chemical transformation at the Delta 1 double bond (8). The finding of a NADP-dihydrofinasteride adduct as a product of this time-dependent inhibition reaction suggests that the chemical event is a combination of a nucleophilic attack of the hydride of the enzyme-bound NADPH on the Delta 1 double bond of finasteride and the subsequent capture of the resulting NADP cation by the reduced finasteride (7). This NADP-dihydrofinasteride adduct is a tight-binding inhibitor of both 5AR1 and 5AR2 with a Ki that is less than 1 pM (7).

Although it is an extremely potent dual inhibitor of 5AR thermodynamically in vitro, finasteride does not fully suppress plasma dihydrotestosterone level at doses up to 100 mg. A theoretical analysis of pharmacodynamic effects of time-dependent inhibitors indicates that the in vivo effects of such inhibitors depend upon both the kinetic and thermodynamic potency of the inhibitor (9) and provides a theoretical basis for improving the in vivo efficacy of Delta 1-4-azasteroids by improving their kinetic potency in the inhibition of 5AR (10, 11). Because a smaller Ki value would translate into a greater k3/Ki, the second order rate constant for the time-dependent inhibition, it was reasoned that improving the affinity of Delta 1-4-azasteroids for 5AR would enhance the kinetic potency of the inhibitors against 5AR (10, 11). Frye and co-workers (12-14) had shown that structural variation at C-17 of 6-azasteroids, a class of reversible inhibitors of 5AR, with bulky lipophilic substituents significantly enhances the affinity of the steroid inhibitors for 5AR. Replacing the N-t-butyl substituent at C-17 of finasteride with a much more lipophilic N-(2,5-bis(trifluoromethyl))phenyl group (GG745; see Fig. 1) indeed significantly increased the rate of inhibition of 5AR, supporting the strategy to improve the rate of time-dependent inhibition of 5AR by using ligand binding energies provided at C-17 of Delta 1-4-azasteroids (10, 11).

Although this approach was successful in discovering GG745, whether it was generally possible to use ligand binding energies to optimize the kinetic potency of Delta 1-4-azasteroids remained unclear. In the current study, we synthesized a series of Delta 1-4-azasteroids with different C-17 substituents and evaluated the effect of the ligand binding energies of these Delta 1-4-azasteroids on the rate of time-dependent inhibition of both 5AR1 and 5AR2. Linear relationships were observed between the binding energies of these compounds and the reduction in the activation energy for the inhibition reaction under second order reaction conditions. The fact that coefficients for these linear relationships were close to unity indicated full realization of the binding energies provided with Delta 1-4-azasteroids at C-17 in reducing the energy barrier for the time-dependent inhibition, supporting the notion that systematic optimization of the kinetic potency of Delta 1-4-azasteroids by enhancing ligand binding energies is feasible.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Materials-- [1,2,6,7-3H]Progesterone (95 Ci/mmol) was purchased from PerkinElmer Life Sciences. Progesterone, NADPH, dithiothreitol, glucose 6-phosphate, and glucose-6-phosphate dehydrogenase were purchased from Sigma. All other reagents purchased were of the highest quality possible. Human recombinant 5AR1 and 5AR2 were prepared according to the procedure described previously (6).

Preparation of Azasteroids-- Finasteride was synthesized as described (15). GG745 was synthesized as described (16). Other 4-azasteroids and 6-azasteroids were synthesized according to published procedures (17). The cycloalkylamines used to couple at C-17 were synthesized via a three-step synthetic procedure utilizing the general method outlined by Kalir and Balderman (18). The 2-t-butyl-5-trifluoromethyl aniline was synthesized via a five-step procedure starting with 4-bromotrifluoromethylbenzene whereby the t-butyl group was incorporated utilizing chemistry as described (19).

5AR Activity Assays-- Buffers, solutions, and reaction mixtures were prepared according to the procedure described previously (6) except that in this study, [1,2,6,7-3H]progesterone was used in the place of [1,2,6,7-3H]testosterone and kept at 20 nM. All the assays were performed at pH 7.0, µ = 0.3, and 22 °C unless noted otherwise. These reactions were initiated by addition of enzyme and quenched at desired times with ethanol. The substrate and product were separated by a C-18 reversed phase column (4.6 × 150 mm) with a mobile phase of 35% water and 65% acetonitrile. The amount of product formed was quantitated by an in-line radiodetector (beta -Ram; Tampa, FL). The reactions catalyzed by type 1 5AR was first order in progesterone ([S] = 20 nM compared with Km = 690 nM), and the activity of enzyme was expressed as the first order rate constant (upsilon , min-1) for the loss of substrate. For the reactions with 5AR2, the substrate concentration (20 nM) was higher than its Km (4.9 nM). Under this condition, the activity of enzyme was expressed as the initial rate (upsilon , nM min-1) at 20 nM substrate.

Inhibition Assays of 5AR-- Reactions were performed, and the product was analyzed as described above except that these reactions contained an inhibitor (I) at a defined concentration. The inhibition data were analyzed by non-linear least squares fitting of data to the following equation,


R≅1−<FR><NU>&ugr;<SUB><UP> i</UP></SUB></NU><DE>&ugr;</DE></FR>=<FR><NU>[<UP>I</UP>]</NU><DE>IC<SUB>50</SUB>+[<UP>I</UP>]</DE></FR> (Eq. 1)
where upsilon i is the enzyme activity in the presence of inhibitor, to obtain IC50, the inhibitor concentration, where 50% of the original enzyme activity was inhibited (6).

Progress Curve Analysis-- Reaction mixtures were prepared as described above, and an inhibitor was added at a desired concentration. The volume of a reaction mixture was set to 400 µl. Enzyme, at a concentration in the range of 0.1 to 2 nM, with [I]/[E] being kept at greater than 10, was added to initiate the reaction. At different times, 20-µl aliquots were removed and quenched with 40 µl of ethanol. Substrate (S) remaining or product (P) formed during the reaction was monitored as described above. For 5AR1, data of substrate remaining at different times were fitted to the following equation (6),


<FR><NU>[<UP>S</UP>]</NU><DE>[<UP>S</UP>]<SUB>0</SUB></DE></FR>=<UP>exp</UP><FENCE>(<UP>&ugr;<SUB> i </SUB>/</UP>k<SUB>obsd</SUB>)<FENCE>e<SUP><UP>−</UP>k<SUB>obsd</SUB></SUP>−1</FENCE></FENCE>)] (Eq. 2)
to obtain kobsd, the observed inhibition rate constant, and upsilon i, the enzyme activity at 0 min. For 5AR2, data of product formed at various time points were analyzed by using Equation 3 (20).
[<UP>P</UP>]=<FR><NU>&ugr;<SUB><UP> i</UP></SUB></NU><DE>k<SUB>obsd</SUB></DE></FR><FENCE>1−e<SUP><UP>−</UP>k<SUB>obsd</SUB>t</SUP></FENCE> (Eq. 3)

To obtain Ki, the inhibition constant for the formation of the initial EI complex in a two-step inhibition mechanism (see "Results and Discussion"), and k3, the rate constant for the second, time-dependent step, either of the following two methods were used. In the first method, kobsd values at various inhibitor concentrations were obtained and then reanalyzed against [I] to abstract Ki and k3 by using Equation 4 (6).
k<SUB>obsd</SUB>=<FR><NU>k<SUB>3</SUB>[<UP>I</UP>]</NU><DE>K<SUB>i</SUB><FENCE>1+<FR><NU>[<UP>S</UP>]</NU><DE>K<SUB>m</SUB></DE></FR></FENCE>+[<UP>I</UP>]</DE></FR> (Eq. 4)

This method is necessary for determination of the kinetic mechanism of the time-dependent inhibition and provides good estimates of Ki and k3. For certain inhibitors, the reactions were run at a single [I] to obtain kobsd and upsilon i. These values were then used to calculate Ki and k3, respectively, by using Equations 5 and 6.
K<SUB>i</SUB>=<FR><NU>[<UP>I</UP>]</NU><DE><FENCE><FR><NU>&ugr;<SUB> i</SUB></NU><DE>&ugr;</DE></FR>−1</FENCE><FR><NU>[<UP>S</UP>]</NU><DE>K<SUB>m</SUB></DE></FR>−1</DE></FR> (Eq. 5)

k<SUB>3</SUB>=k<SUB>obsd</SUB><FENCE><FR><NU>K<SUB>i</SUB></NU><DE>[<UP>I</UP>]</DE></FR><FENCE>1+<FR><NU>[<UP>S</UP>]</NU><DE>K<SUB>m</SUB></DE></FR></FENCE>+1</FENCE> (Eq. 6)

This method is much simpler to perform than the method of progress curve analysis at a range of inhibitor concentrations and provides a reasonable estimate of Ki and k3.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The in vivo potency of irreversible, time-dependent inhibitors depends on the kinetic potency of such inhibitors (9). One way to improve the kinetic potency of time-dependent inhibitors is to provide the inhibitors with increased ligand binding energies (10). The primary goal of this study was to investigate the relationship between ligand binding energies and the rate of time-dependent inhibition of 5AR by Delta 1-4-azasteroids. Understanding such relationships could be helpful in the design of strategies for optimizing the kinetic potency of Delta 1-4-azasteroids. Prior to performing such investigations, it is necessary to understand the mechanism of the time-dependent inhibition, which sets the framework upon which the relationships can be investigated.

Kinetic Mechanism of Time-dependent Inhibition by 4-Azasteroids Is Two Steps-- Previously, the kinetics of time-dependent inhibition of 5AR by finasteride and GG745 were shown to involve two steps (6, 10), shown in Equation 7,


E+<UP>I</UP> <LIM><OP><ARROW>⇌</ARROW></OP><UL>K<SUB>i</SUB></UL></LIM> E<UP>I</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>3</SUB></UL></LIM> <UP>EI</UP>* (Eq. 7)
where inhibitor associates with enzyme to form an initial EI, which then undergoes a time-dependent rearrangement to form EI*. To gain further confidence in this two-step mechanism for the structural class of Delta 1-4-azasteroids, we evaluated the inhibition kinetics for four additional Delta 1-4-azasteroids (1-4) (Fig. 1) by using the method of progress curve analysis at a range of inhibitor concentrations. The mechanism of inhibition by all the four Delta 1-4-azasteroids showed a two-step kinetic mechanism as judged from the hyperbolic dependence of kobsd on the inhibitor concentration [I] (data not shown). The kinetic constants obtained by this method are compared with the values obtained previously for finasteride and GG745 in Table I.


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Fig. 1.   Structures of Delta 1-4-azasteroids for which the method of progress curve analysis at a range of inhibitor concentrations was performed to determine the kinetic mechanism of inhibition. Finasteride, 17beta -N-t-butylcarbamoyl-4-aza-5alpha -androstan-1-en-3-one; GG745, 17beta -N-(2,5-bis(trifluoromethyl))phenylcarbamoyl-4-aza-5alpha -androst-1-en-3-one; 1, 17beta -N-1-(3,4-methylenedioxyphenyl)cyclohexylcarbamoyl-4-aza-5alpha -androst-1-en-3-one; 2, 17beta -N-1-(4-methoxyphenyl)cyclohexyl carbamoyl-4-aza-5alpha -androst-1-en-3-one; 3, 17beta -N-1-(4-t-butylphenyl)cyclohexylcarbamoyl-4-aza-5alpha -androst-1-en-3-one; 4, 17beta -N-1-(4-chlorophenyl)cyclopentylcarbamoyl-4-aza-5alpha -androst-1-en-3-one.

                              
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Table I
Summary of kinetic parameters of the time-dependent inhibition of 5AR by Delta 1-4-azasteroids obtained using the method of progress curve analysis at a range of inhibitor concentrations

Methods to Improve the Rate of Time-dependent Inhibition-- Having gained further confidence in the two-step kinetic mechanism of time-dependent inhibition of 5AR by Delta 1-4-azasteroids, we then turned our efforts to developing strategies to improve the kinetic potency of Delta 1-4-azasteroids. Analogous to enzyme catalysis, time-dependent inhibition reactions may be "catalyzed" by the use of binding energies derived from inhibitor-target interaction (10). According to the theory developed for understanding the evolution of maximal catalytic effectiveness of enzymes (21, 22), the binding energies an enzyme can provide during reaction may be used to lower the significant reaction energy barriers via three different mechanisms, "uniform binding", "differential binding," and "catalysis of an elementary step." Uniform binding is a mechanism by which the enzyme lowers an energy barrier of the catalyzed reaction by binding the transition and ground states equally well. Differential binding is the ability of enzyme to differentiate between internal states, which although important for enzyme evolution is irrelevant for enhancing the rate of time-dependent inhibition, because there is only one relevant internal state, that of EI, in a two-step mechanism. The catalysis of an elementary step is the reduction of the activation energy for an individual step along the reaction coordinate and requires the enzyme to differentiate between the transition state and the ground state involving that individual step. Comparing enzyme catalysis and time-dependent inhibition, the effects of uniform binding and catalysis of an elementary step in the evolution of enzyme catalytic effectiveness are equivalent to a "Ki effect" (Fig. 2B) and a "k3 effect" (Fig. 2C), respectively, in the enhancement of the rate of time-dependent inhibition. Obviously, the rate of inhibition can be improved by either lowering the energy barrier for the time-dependent event, a k3 effect, or by enhancing the ligand binding to the ground state, a Ki effect. Although the Ki effect would produce a linear relationship between the ligand binding energy and the energy barrier for the inhibition, the k3 effect may not. Chemically, options to produce a k3 effect by activating the Delta 1 double bond are limited, and this double bond activation could also diminish selectivity. Given the understanding that 5AR tolerates bulky groups at C-17 and the structure-activity relationship previously revealed between such groups and the affinity of 6-azasteroids for the enzyme (12-14), it appeared feasible to improve the rate of time-dependent inhibition of 5AR by exploiting the Ki effect of the C-17 substitution.


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Fig. 2.   Energy profiles for a typical two-step time-dependent inhibition. The rectangular bars represent energies for inhibitor binding or activation energies for conversion of the internal states, EI (A). The energy barrier of k3/Ki may be reduced by a Ki effect (B), analogous to uniform binding for optimizing enzyme catalysis by the nature (see "Results and Discussion") that reduces the transition state and the state of the EI complex equally as indicated by the arrows. The energy barrier of k3/Ki may also be reduced by a k3 effect (C), which, analogous to the catalysis of an elementary step in optimization of enzyme catalysis by the nature (see "Results and Discussion"), only reduces the transition state.

Linear Relationships between the Binding Energy of Delta 1-4-Azasteroids for 5AR and the Activation Energy of Time-dependent Inhibition-- As indicated above, if ligand binding energies have purely a Ki effect in the time-dependent inhibition, one would see a linear correlation between the ligand binding energy and the activation energy for the time-dependent reaction. A series of Delta 1-4-azasteroids with a diversified set of C-17 substituents were chosen to evaluate the effect of ligand binding energies on the rate of time-dependent inhibition of 5AR. The method of progress curve analysis at a single concentration of inhibitor (see "Experimental Procedures") was used to obtain the constants k3, Ki, and k3/Ki. The values obtained for the inhibition of 5AR1 and 5AR2 are listed in Tables II and III, respectively. The value of ln(1/Ki), which is proportional to the ligand binding energy, was then plotted against ln(1/(k3/Ki)), which is proportional to the activation energy of the second order rate constant, k3/Ki. A linear relationship between ln(1/Ki) and ln(1/(k3/Ki)) was evident for the inhibition of both 5AR1 and 5AR2 as shown in Figs. 3A and 4A, respectively. The coefficient of the linear correlation was -0.88 ± 0.07 for type 1 5AR and -1.0 ± 0.2 for type 2 5AR. The fact that these coefficients are close to unity suggests a pure Ki effect of the ligand binding energies on the activation energy for the time-dependent inhibition reaction. Also consistent with this, the ligand binding energies had little effect on the activation energy of 1/k3 as judged by the lack of correlation between ln(1/Ki) and ln(1/k3) (Figs. 3B and 4B).

                              
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Table II
Summary of kinetic parameters of the time-dependent inhibition of 5AR1 by Delta 1-4-azasteroids obtained using the method of progress curve analysis at a single inhibitor concentration
The values of Ki and k3 were calculated using Equations 5 and 6, respectively, with kobsd and upsilon i values obtained by fitting data to Equation 2. The relative errors associated with kobsd and upsilon I ranged from 10 to 35%.

                              
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Table III
Summary of kinetic parameters of the time-dependent inhibition of 5AR2 by Delta 1-4-azasteroids obtained using the method of progress curve analysis at a single inhibitor concentration
The values of Ki and k3 were calculated using Equations 5 and 6, respectively, with kobsd and upsilon i values obtained by fitting data to Equation 3. The relative errors associated with kobsd and upsilon I ranged from 10 to 35%.


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Fig. 3.   Effect of ligand binding energies (ln(1/Ki)) of Delta 1-4-azasteroids on the activation barrier of k3/Ki (ln(1/(k3/Ki))) (A) and k3 (ln(1/(k3))) (B) for 5AR1. The ligand binding energy (ln(1/Ki)) is linearly correlated to the activation energy of k3/Ki (ln(1/(k3/Ki))) (A), with a coefficient of -0.88 ± 0.07. There is no apparent correlation between the ligand binding energy (ln(1/Ki)) and the activation energy of k3/Ki (ln(1/(k3/Ki))) (B).


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Fig. 4.   Effect of ligand binding energies (ln(1/Ki)) of Delta 1-4-azasteroids on the activation barrier of k3/Ki (ln(1/(k3/Ki))) (A) and k3 (ln(1/(k3))) (B) for 5AR2. The ligand binding energy (ln(1/Ki)) is linearly correlated to the activation energy of k3/Ki (ln(1/(k3/Ki))) (A), with a coefficient of -1.0 ± 0.2. There is no apparent correlation between the ligand binding energy (ln(1/Ki)) and the activation energy of k3/Ki (ln(1/(k3/Ki))) (B).

Linear Correlation between the Affinity of Delta 1-4-Azasteroids and the Affinity of 6-Azasteroids for 5AR-- The linear relationship demonstrated above for ligand binding energies and the activation energy for the time-dependent inhibition of 5AR by Delta 1-4-azasteroids suggests that the kinetic potency of Delta 1-4-azasteroids can be optimized systematically by evaluating the binding affinities of these compounds for 5AR. Given the time-dependent nature of Delta 1-4-azasteroids in the inhibition of 5AR and that the binding affinities of these compounds cannot be evaluated precisely by regular, non-time-dependent inhibition kinetics, time-dependent kinetics are needed for determining the binding affinities of Delta 1-4-azasteroids for 5AR. However, the time-consuming nature of performing kinetic studies of time-dependent inhibition precludes fast evaluation of the initial binding of Delta 1-4-azasteroids to 5AR. Because a large number of 6-azasteroids, which are non-time-dependent inhibitors of 5AR, had been synthesized, one way to circumvent this technical inconvenience is to conduct regular inhibition assays of 6-azasteroids and then predict the ligand binding energies that the C-17 substituents could provide with Delta 1-4-azasteroids. This approach requires that the C-17 substituent of non-time-dependent steroids binds 5AR in the same way as does the C-17 substituent of Delta 1-4-azasteroids. To evaluate this, the ligand binding energies of a series of Delta 1-4-azasteroids and 6-azasteroids that bear the same set of C-17 substituents were compared. The Ki values for the Delta 1-4-azasteroids (Table IV) were obtained using the method of progress curve analysis at a single concentration of inhibitor, and IC50 values (Table IV) of the 6-azasteroids were obtained using regular inhibition assays. The plots of ln(1/Ki) versus ln(1/IC50) (Fig. 5) indeed indicate a reasonable linear correlation between the binding affinities of the Delta 1-4-azasteroids and 6-azasteroids,2 supporting the approach of using 6-azasteroids to quickly evaluate the ligand binding energies of various C-17 substituents that can be used to improve the kinetic potency of Delta 1-4-azasteroids. By using this approach, a great number of potent Delta 1-4-azasteroids have been discovered (data not shown), which in turn greatly facilitated the effort to discover drugs that are effective at inhibiting 5AR.

                              
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Table IV
Summary of IC50 and Ki values, respectively, of 6-azasteroids and 4-azasteroids with the same C-17 substituents
The Ki values were calculated using Equation 5 with kobsd and upsilon I values (relative errors in the range of 10 to 35%) obtained using the method of progress curve analysis at a single inhibitor concentration, and the IC50 values were obtained using the regular inhibition method at pH 7.0, 22 °C (see "Experimental Procedures").


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Fig. 5.   Correlation of ligand binding energies (ln(1/Ki)) of Delta 1-4-azasteroids and ligand binding energies (ln(1/IC50)) of 6-azasteroids for 5AR1 (A) and 5AR2 (B).

Conclusions-- A series of Delta 1-4-azasteroids having different C-17 substituents were evaluated for their time-dependent inhibition of 5alpha -reductase to understand the relationships between the rate of time-dependent inhibition and the affinity of the time-dependent inhibitors for the enzyme. The results indicated a linear correlation between the ligand binding energy for the formation of the EI complex and the activation energy for the overall inhibition reaction under the second order reaction condition. There was no obvious correlation between the ligand binding energy and the activation energy for the second, time-dependent step of the inhibition reaction. These data are consistent with a model where ligand binding energies provided at C-17 of Delta 1-4-azasteroids is fully expressed to lower the activation energy of the overall time-dependent inactivation reaction with little perturbation of the energy barrier of the second, time-dependent step. Subsequently, a strategy and procedures to improve rates of time-dependent inhibition by providing inhibitor with ligand binding energies were presented that may be generally useful for developing potent time-dependent inhibitors of pharmaceutical values.

    ACKNOWLEDGEMENTS

We thank Dr. Stephen V. Frye for insights over the entire course of this study and useful discussions during the writing of this manuscript. J. Darren Stuart is acknowledged for preparing recombinant human 5AR1 and 5AR2 proteins used in this study.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 302-886-8138; Fax: 302-886-4983; E-mail: gaochao.tian@astrazeneca.com.: Dept. of Lead Discovery, Astrazeneca, Wilmington, DE 19850.

Published, JBC Papers in Press, March 8, 2001, DOI 10.1074/jbc.M100793200

2 The greater scattering in the values determined for the type 2 5AR may be partly attributed to the time-dependent nature of some 6-azasteroids in the inhibition of type 2 5AR (23).

    ABBREVIATIONS

The abbreviations used are: 5AR, 5alpha -reductase; EI, enzyme-inhibitor complex; EI*, apparently irreversible enzyme-inhibitor complex..

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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