From Snake Venom to Antihypertensive

Figure 1: The structure and functional groups of captopril.

HISTORY OF ITS DISCOVERY

Captopril sold under the brand name (Capoten^®) is an angiotensin-converting enzyme (ACE) inhibitor. Its indication is to treat hypertension and some types of congestive heart failure. This drug was discovered by a group of scientists led by a professor, John R. Vane who enthusiastically scrutinized the inducement of hypertension (1). Its finding started by studying Bradykinin Potentiating Factor (BPF) in the venom of the Brazilian viper Bothrops jararaca  (1). The initial clinical trial was done by treating 17 patients with essential hypertension, the blood pressure of 14 patients was reduced (1). Captopril was patented in 1976 under American pharmaceutical company, ER Squibb & Sons LLC (now: Bristol Myers Squibb), was approved for medical use in 1980 and later gained FDA approval on April 6, 1981 (2).

NAME, STRUCTURE AND FUNCTIONAL GROUPS

Generic Name            : Captopril

Brand Name               : Capoten^®

Molecular formula     : C₉H₁₅NO₃S

IUPAC Name             : 1-[(2S)-3-mercapto-2-methylpropionyl]-L-proline or (2S)-1-[(2S)-2-methyl-3 sulfanylpropanoyl]pyrrolidine-2-carboxylic acid

Molecular weight       : 217.29 g/mol

The proline nitrogen atom has a partial double bond character controlling the shape of the molecule, as the C–N amide bond is rigid.

Figure 2: Isosteric replacement of proline nitrogen atom with carbon atom.

Figure 3: Isosteric replacement of -SH with -CH₃ (3, 4)

HIT IDENTIFICATION

In absence of 3-D structures of biological targets, Pharmacophore Modelling and Quantitative Structure-Activity Relationship (QSAR) are the methods used to design the novel drug target. Useful information like the nature of target-ligand interactions can be achieved by these tools. Molecular models predicted through these methods are further used for lead discovery and optimization. The design of the novel drug molecule is based on the information about the structure of the active ligand which can interact with biological targets (5).

ACE is a component of the renin-angiotensin system, which controls blood pressure. It is a carboxypeptidase with a cofactor, zinc ion. The discovery and design of captopril, an ACE inhibitor is by computational methods that apply the concept of ligand-based drug-design approach. The similar enzymatic mechanism of ACE and carboxypeptidase A at the carboxyl end of the protein gives a way to design and develop Captopril (5). Similar to carboxypeptidase A, the active site of ACE consists of three important groups that participate in the binding of peptide substrates which are a carboxyl-binding group, a group with an affinity for the C-terminal peptide bond, and bounded zinc ion that could coordinate with the carbonyl of the peptide bond. A succinyl amino acid can interact with these binding groups via its amino acid carboxyl, amide bonds, and succinyl carboxyl. L-benzyl succinic acid was classified as the by-product type of carboxypeptidase A inhibitors but considering it as a model entity, succinyl amino acids could also act as by-product inhibitors of ACE.

Figure 4: Diagrammatic model of the active site of ACE based on the early hypothesis which presumed that ACE and carboxypeptidase A have a lot in common (6).

HIT TO LEAD

Method use                 : Screening natural product, venom of Bothrops jararaca

Lead compound          : Bradykinin Potentiating factor, BPF (one of the teprotide)

Figure 5: The progenies to create the desired ACE inhibitor from the lead compound (7-9)

How it Begin

Vane suggested David W. Cushman, a researcher at Squibb to start a study on poorly characterized lung ACE and it resulted in the development of simple assay methods for measuring ACE activity (10). In 1968, Dr. Y.S Bakhle indicated that BPF from the venom of Brazilian viper Bothrops jararaca can inhibit the activity of ACE in dog lungs (10) (11). Cushman and Ondetti were able to isolate the ACE inhibitory venom peptides using the existing adequate assays developed (10). 

Try and Error

The first peptide, bradykinin-potentiating pentapeptide, BPP-5a was isolated and characterized by Ferreira and Greene. BPP-5a is capable of inhibiting ACE and impermanently lower the blood pressure of animal models. However, it has a short life in hypertensive animal models due to its inclination to enzymatic degradation. Meanwhile, Cushman and Ondetti managed to isolate and synthesize longer ACE inhibitor peptides with a slight sequence difference from BPP-5a. The most potent peptide sequenced, teprotide was a nonpeptide with proline residue as the active part which makes it very stable and considered an effective antihypertensive medication, but its oral activity deficiency made it an unsuitable product. With support from Squibb’s directors, 2000 compounds are tested to find a suitable ACE inhibitor (10).

The Final Solution

On March 13, 1974, Squibb researchers were gathered to talk over a study paper published by Byers and Wolfenden. According to the paper, "L"-benzyl succinic acid was found to inhibit carboxypeptidase A by binding strongly to it. ACE was proposed to be an exopeptidase with an analogous active site like the carboxypeptidase A, with one difference, ACE releases dipeptide residues rather than a single amino acid. Therefore, the team decided to make an analogue of dipeptide Gly-Pro called succinyl-L-proline which combined the enzyme-binding ability of L-benzyl succinic acid with the ACE-inhibition activity of proline (10).

LEAD OPTIMIZATION

Figure 6: Structure of succinyl-L-proline, D-2-methylsuccinyl-L-proline, and captopril.

Even though succinyl-L-proline possessed the characteristics needed for a specific ACE inhibitor like inhibiting the contractile act of angiotensin I and initiating bradykinin properties without impacting the contractile action of angiotensin II, it has inadequate potency for an ACE inhibitor. Structure-activity relationship (SAR) studies that have been carried out on succinyl proline lead to the design of the captopril (12). There are two structural modifications done on succinyl-L-proline, extension of the structure and variation of alkyl substituent.

To optimize the acyl chain length, a methyl group (-CH3)  is added to the succinyl moiety to form D-2-methyl succinyl-L-proline which has more inhibitory capability and more potency to demonstrate its oral activity (10).

Succinyl carboxyl group was replaced by the sulfhydryl group (SH) to form the final product, Captopril. Sulfhydryl group (-SH) has a great affinity to zinc-containing enzymes. It increases binding interaction with zinc ion on the ACE active site and enhances the ACE-inhibitor activity to the optimum (10). 

MECHANISM OF ACTION (PHARMACODYNAMIC)

Flow chart 1: Mechanism of action of captopril

Captopril inhibits the rapid conversion of angiotensin I to angiotensin II and antagonized the RAAS-induced system which leads to a decrease in blood pressure. The inhibition of ACE due to the antagonized effect will result in decreased plasma angiotensin II and increased plasma renin activity (PRA) due to loss of negative feedback on renin release caused by reduction in angiotensin II (14).  During sympathetic stimulation, renin is released from the granular cells of the juxtaglomerular apparatus in the kidneys. Renin would cleave circulating angiotensinogen in the bloodstream to angiotensin I (ATI) and it would subsequently cleave to angiotensin II (ATII) by ACE. Captopril will inhibit the secretion of aldosterone from the adrenal cortex in which it travels to the distal convoluted tubule (DCT) and collecting tubules (CT). It will increase the sodium and water reabsorption by increasing the number of sodium channels and sodium-potassium ATPase on cell membranes. It will also reduce the secretion of vasopressin, which is ADH, an antidiuretic hormone from the posterior pituitary gland. ADH will further stimulate the water reabsorption from the kidneys via the insertion of aquaporin-2 channels on the apical surface of cells of the DCT and CT. Captopril will also inhibit direct vasoconstriction of arterial by ATII. This can be done by inhibition of type 1 ATII receptors on vascular smooth muscle cells which results in myocyte contraction and vasoconstriction. ACE is also involved in the enzymatic degradation of bradykinin. Captopril will interfere with the degradation of the vasodepressor peptide, bradykinin. Thus, concentrations of bradykinin or prostaglandin E will increase and can be significant on the therapeutic effect of captopril (15)

PHARMACOKINETIC

Absorption

The onset time of captopril taken orally and sublingually is within 15-30 minutes and 10-20 minutes, respectively (16). C_max of captopril given orally is around 60-90 minutes and if given sublingually is 60 minutes. The bioavailability of captopril is approximately 65% (17) and is not altered by age or concomitant medications but the co-administration with food, antacids, or probenecid has been shown to reduce its bioavailability by up to 40%. 

Volume of Distribution

This drug is distributed into most body tissues except the central nervous system and is about 25%-40% protein-bound. The volume of distribution and body clearance of Captopril given intravenously was about 0.8L/kg and 0.7L/h/kg, respectively (16).

Metabolism

About 50% of captopril undergo metabolism in the liver and the major metabolites are captopril-cysteine disulfide and disulfide dimer of captopril.

Route of Elimination

Captopril is excreted mainly by renal excretion and the elimination half-life of unchanged Captopril is approximately 2 hours (17). Within 24 hours, over 95% of the drug is eliminated in the urine; 40%-50% of the eliminated drug is unchanged and the rest is captopril metabolites.

Clearance

Within 96 hours (4 days), after intravenous and oral administration, excretion of captopril is accounted for 87% and 61% of the dose, respectively. (18)

REFERENCES

1. Charles G. Smith JRV. The Discovery of Captopril. The FASEB Journal. 2003;17(8).

2. Fischer J, Ganellin CR, Ganesan A, Proudfoot J. Analogue-based drug discovery: Wiley-VCH Hoboken, NJ; 2010.

3. Captopril [Internet]. National Center for Biotechnology Information. PubChem Compound Database. U.S. National Library of Medicine; [cited 2021 1 Jan]. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Captopril.

4. Drug Discovery [Internet]. Drug design principles. [cited 2021 1 Jan]. Available from: https://www.stereoelectronics.org/webDD/DD_04.html.

5. Keshri Kishore Jha RT. Computer-Aided Drug Discovery and Design (CADDD) - A New Approach for the Development of Novel Drugs Human 2017 10(1).

6. Cushman DW, Cheung HS, Sabo EF, Ondetti MA. Design of potent competitive inhibitors of angiotensin-converting enzyme. Carboxyalkanoyl and mercaptoalkanoyl amino acids. Biochemistry. 1977;16(25):5484-91.

7. DRUG: Teprotide [cited 2020 30 Dec]. Available from: https://www.genome.jp/dbget-bin/www_bget?dr:D06076.

8. Venom Peptide BPP5A (Glu-Lys-Phe-Ala-Pro) Chegg Study; [cited 2020 30 Dec]. Available from: https://www.chegg.com/homework-help/questions-and-answers/venom-peptide-bpp5a-glu-lys-phe-ala-pro-natural-ace-inhibitor-served-platform-develop-n-ca-q38121141.

9. Captopril [cited 2020 30 Dec]. Available from: https://www.sigmaaldrich.com/catalog/product/sigma/c4042?lang=en&region=MY.

10. Cushman DW, Ondetti MA. History of the design of captopril and related inhibitors of angiotensin-converting enzyme. Hypertension. 1991;17(4):589-92.

11. Bryan J. From snake venom to ACE inhibitor--The discovery and rise of captopril. Pharmaceutical Journal. 2009;282(7548):455.

12. Roy K, Kar S, Das R. SAR and QSAR in drug discovery and chemical design—some examples. Underst. Basics QSAR Appl. Pharm Sci Risk Assess. 2015.

13. Captopril - Stepwards [Internet]. Stepwards. 2021 [cited 2021 3 Jan]. Available from: https://www.stepwards.com/?page_id=1443.

14. Captopril Tablets, USPRx only [Internet]. Dailymed.nlm.nih.gov. 2021 [cited 2021 3 Jan]. Available from: https://dailymed.nlm.nih.gov/dailymed/fda/fdaDrugXsl.cfm?setid=1395ba5b-2fc9-41ff-9c43-e0740d781dee&type=display.

15. Captopril [Internet]. DrugBank Online. [cited 2021 1 Jan]. Available from: https://go.drugbank.com/drugs/DB01197 .

16. Duchin, K.L., McKinstry, D.N., Cohen, A.I. et al. Pharmacokinetics of Captopril in Healthy Subjects and in Patients with Cardiovascular Diseases. Clin-Pharmacokinet 14, 241–259 (1988). https://doi.org/10.2165/00003088-198814040-00002.

17. Katzung BG. Basic & Clinical Pharmacology. 12th ed. Susan B. Masters AJT, editor. Vol. 12. San Fransisco: Mc Graw Hill; 2010. 175–185 p.

18. Duchin KL, Singhvi SM, Willard DA, Migdalof BH, McKinstry DN. Captopril kinetics. Clin Pharmacol Ther. 1982 Apr;31(4):452-8. doi: 10.1038/clpt.1982.59. PMID: 7037265. https://ascpt.onlinelibrary.wiley.com/doi/abs/10.1038/clpt.1982.59.