research article docking studies and biological activity...

Research ArticleDocking Studies and Biological Activity of Fosinopril Analogs

Jayant Choudary,1 Suvarna G. Kini,1

Sreedhara Ranganath Pai Karkala,2 and Muhammad Mubeen1

1 Department of Chemistry, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal 576104, India2Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal 576104, India

Correspondence should be addressed to Suvarna G. Kini; [email protected]

Received 30 September 2013; Revised 12 May 2014; Accepted 13 May 2014; Published 6 July 2014

Academic Editor: Maria Cristina Breschi

Copyright © 2014 Jayant Choudary et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

The purpose of the present study was to determine the angiotensin-I converting enzyme inhibitory activity of few novel Fosinoprilderivatives which were predicted to possess better ACE inhibitory activity and lesser side effects than the existing drug molecule.In vitro study was carried out to determine ACE inhibitory activity of six different Fosinopril analogs by spectrophotometric assayprocedure. Analog A2 showed the highest activity compared to other analogs and as well as Fosinopril itself. Docking studies ofthe compounds were done with the help of VLife MDS 3.0 software using GRIP batch docking method to find out which derivativehad a better docking with ACE.The compounds which showed the highest negative score in docking have also exhibited good ACEinhibitory activity.

1. Introduction

Angiotensin converting enzyme (ACE, EC 3.4.15.1) inhibitorswork by inhibiting the action of angiotensin convertingenzymes, thus preventing the conversion of angiotensin toangiotensin-II. ACE inhibitors are widely used in the treat-ment of hypertension as well as heart failure [1, 2]. Fosinoprilis the first and only phosphonate containing ACE inhibitor.The search for ACE inhibitors that lacked the sulfhydrylgroup leads to the investigation of phosphorus containingcompounds. The phosphinic acid is capable of binding toACE in a manner similar to enalapril. The interaction of thezinc atomwith the phosphinic acid is similar to that seenwithsulfhydryl groups [3, 4].

Additionally, this compound is capable of formingthe ionic, hydrogen, and hydrophobic bonds. A featureunique to this compound is the ability of the phosphinicacid to more truly mimic the ionized, tetrahedral inter-mediate of peptide hydrolysis. Structural modifications toinvestigate more hydrophobic, C-terminal ring systems,lead to a 4-cyclohexylproline analog of the original phos-phinic acid. This compound, Fosinoprilat, was more potent

than captopril but less potent than enalaprilat. Fosino-prilat proved to have the same problem as enalaprilatand other carboxylate-containing ACE inhibitors like poorbioavailability. The solution fortunately was very similar—the addition of a hydrophobic side chain to modulatethe ionization characteristics of the molecule—thus Fos-inopril was developed. Fosinopril is administered as aprodrug and is converted in vivo to the active formFosinoprilat (Scheme 1).

2. Results and Discussion

2.1. Chemistry. Fosinopril containing analogs of trans-4-phenyl-L-proline (A1), 1,2,3,4-tetrahydro isoquinoline-3-carboxylic acid (A2), trans-4-cyclohexyl-L-proline (A3),trans-4-cyclohexyl-L-proline benzyl ester (A4), and L-proline (A5 andA6) were used for this particular study.Thesesix Fosinopril analogs were given as gift samples by RanbaxyLab. Ltd.

2.2. In Vitro Angiotensin-I Converting Enzyme InhibitoryActivity. This method is based on the cleavage of the

Hindawi Publishing CorporationInternational Journal of Medicinal ChemistryVolume 2014, Article ID 721834, 5 pageshttp://dx.doi.org/10.1155/2014/721834

2 International Journal of Medicinal Chemistry

0

ACE inhibition studies

ACE

inhi

bitio

n (%

)

20

40

60

80

100

Fosinopril A1

0.2nM0.4nM

0.6nM0.8nM1nM

0.1nM

Figure 1: Percent ACE inhibition by Fosinopril and A1.

A2

ACE inhibition studies

0

ACE

inhi

bitio

n (%

)

20

40

60

80

100

0.01nM0.02nM

0.4nM0.2nM

0.6nM0.8nM1nM

Figure 2: Percent ACE inhibition by A2.

substrate hippuryl-glycyl-glycine by ACE and subsequentreaction with trinitrobenzenesulfonic acid to form 2,4,6-trinitrophenyl-glycyl-glycine [5]. The method relies on spec-trophotometric determination of hippuric acid. It involves acolorimetric reaction of hippuric acid with benzene sulfonylchloride [6].

The assay systemconsisted of 200 𝜇Lof substrate solution,water, and drug solution in a volume of 0.25mL.The reactionwas started by the addition of 40 𝜇L rat lung extract. After 30minutes of incubation at 37∘C, the reaction was stopped by

0

ACE

inhi

bitio

n (%

)

20

40

60

80

100ACE inhibition studies

A3 A4 A5 A6

0.2nM0.4nM0.6nM

0.8nM1nM

Figure 3: Percent ACE inhibition by A3, A4, A5, and A6.

1.0

0.8

0.6

0.4

0.2

0.0

FosinoprilA1

A2

A3

A4

A5

A6

IC50

(nM

)

IC50 (nM) for ACE inhibition studies

Figure 4: Graph of IC50

values of analogs.

adding 2mL of stop solution (1.195 g HEPES and 0.0465 g ofEDTA were dissolved in 40mL distilled water. The pH wasadjusted to 9.0 with 1mol/lit sodium hydroxide and dilutedto 50mL with distilled water). Then 0.5mL distilled waterand 1.0mL colour reagent were added, mixed vigorously,left for 5min, and centrifuged at 3000–4000 rpm for 10minutes at room temperature to remove any precipitate.The corresponding enzyme inhibitors blanks were assayedin the same manner except for adding the stop solutionbefore the lung extract.The absorbance of the yellow productof the reaction between liberated hippurate and cyanuricchloride was measured spectrophotometrically at 405 nm

International Journal of Medicinal Chemistry 3

Figure 5: Hydrogen bond of A2 (green dotted lines).

Figure 6: Hydrophobic bond of A2 (blue dotted lines).

against distilled water as blank [7].Controls without drugwere run simultaneously in all cases. The results of this studyare given in Tables 1, 2, and 3 and Figures 1, 2, and 3 and theIC50values are given in Table 4 and Figure 4. “One inhibitory

unit is the amount of inhibitor that suppressed the ACEactivity by one unit.”

2.3. Docking Simulation. Docking study was done by GRIPbatch docking method with the help of VLife MDS 3.0software. The crystal structure of ACE in complex with anirreversible inhibitor (PDB ID: 1R4L) for antihypertensivedocking studies was obtained from the protein data bank [8].The parameter fixed for docking simulation was like numberof placements: 30, rotation angle: 30∘, and exhaustivemethod.The ligand forming most stable drug-receptor complex is theone which is having minimum dock score. After dockingsimulation, the best docked conformer of each ligand waschecked for various interactions with receptor like hydrogenbonding, hydrophobic bonding, van der Waals, and chargeinteraction. Compound A2 was found with highest negativedock score (Table 5), meaning that it forms most stable drug-receptor complex amongst all the compounds. It was foundto form hydrogen bonding (Figure 5), hydrophobic bonding(Figure 6), and van der Waals interaction. Amongst all theanalogs, A3 was not found to exhibit hydrogen bonding with

Figure 7: Charge interaction of A2 (yellow dotted lines).

P

O

OH

N

O

O OH

XACE

ACEHydrophobic site

Zn++

NH3

H2

Scheme 1: Interaction of Fosinopril with ACE [4].

the receptor through “O” of−OHofPhosphoramidonmoiety.Analogs A1, A2, A4, A5, A6, and Fosinopril form hydrogenbonding with residues Arg273 and Thr371 by “O” atom of−COOH group of proline moiety and “O” −OH group ofphosphoramidon moiety. All the analogs were found to havehydrophobic bonding. Some of the residues involved in thistype of interaction are Leu144, Asp269, Met270, Pro346,Thr347, His345, Ala348,Thr360, Leu370, andThr445.Thr347andThr371 were common residues for hydrophobic bondingof all analogs including Fosinopril. All the analogs includingFosinopril were found to show van der Waals’ interactionwith common residues Arg273, Thr347, Thr371, Tyr510, andTyr515. Only A2 was found to show charge interaction(Figure 7) by interaction of “O” atom of −COOH groupof tetrahydroisoquinoline-3-carboxylic acid with three “N”atoms of Arg514.The ligand plot of all analogs and Fosinoprilis shown in Figure 8 using VLife MDS 3.0.

3. Conclusion

In vitro studies revealed that the analog A2 exhibited high-est ACE inhibition activity than other analogs includingFosinopril. The percent inhibition of ACE activity of dif-ferent analogs is as follows: analog A2 showed highestactivity followed by Fosinopril, A1, A4, A3, A5, and A6,respectively. In docking study analog A2 was found to

4 International Journal of Medicinal Chemistry

3.895

A

4.153

A

O

O

O

OO

O

O

N

ASP350A

HIS 401A

ARG514A1.759 A

4.305 A

4.516 A

3.998

A

3.995

A4.0

72ANa

p

2.2A

ALA348A

TYR510A

ASN394A

HIS378A

ARG514A

THR347A

TRP349A

N

PO

O

O

O

O

OO

4.998A 4

.811 A

4.803

A

4.871A

4.8944A

3.928A

3.928 A

1.499

A

3.588 A

4.957 A

4.2337 A

ASP350A

ASP382A

TYR510A

ASP350A

THR347A

HIS505A

HIS345A

P

O

O

O

O

O

O

O

N

4.998 A4.843 A

4.996 A

3.773 A4.988

A3.598A

4.688A

4.08

1A3.

92A

2.888 A

TRP349AALA348A

THR347A

ASN394A

P

OO

O

O

O O

N

4.89 A 4.89 A

4.764

A

3.382

A4.527 A

4.33

A

8.3 A

1.832

A

PO

O

O

O

O

O

ON

GLU402A

GLU398A

HIS401A

THR347A

ALA348A

TYR515A

2.322 A4.899 A

4.64

A4.756 A 4.

642

A4.33 A

4.398 A4.762A

4.747 A

3.787

A

4.827 A

3.499 A3.886 A3

.922 A

TYR510A

ARG514AASP350A

ASN394A

TRP349A

4.38 A

4.49A

3.765 A

3.379 A3.987 A

4.978

A

4.855

A

4.906

A4.904

A

4.936

A

2.254 A

P

O

O

OO

O

O

O

N

HIS378A

GLU375A THR347A

ALA348A TRP349A

TYR510A

Fosinopril

4.862

A

4.588

A 4.587

A

4.666

A

4.109 A

2.578 A

3.889 A3.889 A3.8

89A

4.703 A

4.776 A3.806 A

4.281A O

O

O O

OO

PN

A1 A2

A3

A4

A5

A6

4.8944 A

3.208 A

3.985 A

GLU375A

Figure 8: Ligplot of six analogs along with Fosinopril showing hydrogen bond indicated by dark dotted lines and hydrophobic bonds by lightdotted lines with respective amino acid residues of the ACE.

International Journal of Medicinal Chemistry 5

Table 1: Percent inhibition of ACE by Fosinopril and A1 at different concentrations.

Analog Percent inhibition0.1 nM 0.2 nM 0.4 nM 0.6 nM 0.8 nM 1 nM

Fosinopril 44.07 ± 4.35 63.03 ± 6.01 68.72 ± 3.99 72.98 ± 4.76 88.15 ± 5.20 89.09 ± 3.82A1 40.45 ± 3.60 50.20 ± 5.83 54.46 ± 7.14 60.04 ± 4.99 67.97 ± 3.84 76.25 ± 4.25

Table 2: Percent inhibition of ACE by A2 at different concentrations.

Analog Percent inhibition0.01 nM 0.02 nM 0.2 nM 0.4 nM 0.6 nM 0.8 nM 1 nM

A2 44.72 ± 3.85 53.17 ± 5.73 75.12 ± 4.66 77.80 ± 3.21 81.74 ± 6.09 83.98 ± 5.11 84.45 ± 4.52

Table 3: Percent ACE inhibition by A3, A4, A5, and A6 at different concentrations.

Analog Percent inhibition0.2 nM 0.4 nM 0.6 nM 0.8 nM 1 nM

A3 43.75 ± 3.05 56.26 ± 5.43 62.50 ± 5.11 65.62 ± 3.96 71.87 ± 4.50A4 41.41 ± 4.88 60.05 ± 5.24 64.27 ± 3.25 65.70 ± 3.89 68.53 ± 3.67A5 45.29 ± 3.65 52.30 ± 4.91 54.05 ± 3.79 56.77 ± 4.14 59.43 ± 5.02A6 37.80 ± 4.76 43.29 ± 3.15 48.64 ± 4.20 51.35 ± 4.73 54.03 ± 3.99

Table 4: IC50 values of analogs.

Analog IC50 (nM)Fosinopril 0.143 ± 0.012A1 0.196 ± 0.015A2 0.017 ± 0.009A3 0.340 ± 0.044A4 0.267 ± 0.028A5 0.389 ± 0.030A6 0.749 ± 0.061

Table 5: Docking scores of the Fosinopril analogs.

Analog Dock scoreA1 −78.9549A2 −81.0598A3 −72.0179A4 −76.0455A5 −68.0202A6 −58.0563Fosinopril −79.0399

have minimum dock score indicating highest binding withthe receptor and in vitro studies also showed the sameanalog having highest ACE inhibitory activity. Hence wecan conclude that analog A2 can be considered as a bet-ter molecule than Fosinopril and should be investigatedfurther.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

Acknowledgments

The authors are thankful to Ranbaxy Lab. Ltd, India, for thesamples of Fosinopril and its analogs and spectral studies.

References

[1] E. K. Jackson, “Drugs acting on Renin angiotensin system,” inGoodman Gilman's Pharmacological Bases of Therapeutics, vol.11, pp. 808–809, 2005.

[2] ACE Inhibitors, healthivillage, http://ww31.heart.health.ivillge.com/bloodpressure/ace%20inhibitors2.cfm.

[3] “Fosinopril—Wikipedia, the free encyclopedia,” http://en.wikipedia.org/wiki/Fosinopril.

[4] M. Harrold, “Angiotensin converting enzyme inhibitors,” Foye’sPrinciples of Medicinal Chemistry, vol. 6, pp. 746–747, 2008.

[5] C. P. Serra, S. F. Cortes, J. A. Lombardi, A. B. de Oliveira,and F. C. Braga, “Validation of a colorimetric assay for the invitro screening of inhibitors of angiotensin-converting enzyme(ACE) from plant extracts,” Phytomedicine, vol. 12, no. 6-7, pp.424–432, 2005.

[6] C. P. Serra, S. F. Cortes, J. A. Lombardi, A. Braga de Oliveira,and F. C. Braga, “Validation of a colorimetric assay for the invitro screening of inhibitors of angiotensin-converting enzyme(ACE) from plant extracts,” Phytomedicine, vol. 12, no. 6-7, pp.424–432, 2005.

[7] M. Hayakari, Y. Kondo, and H. Izumi, “A rapid and simplespectrophotometric assay of angiotensin-converting enzyme,”Analytical Biochemistry, vol. 84, no. 2, pp. 361–369, 1978.

[8] VLife MDS 3.5 documentation, Tutorial, Biopredicta.

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