ACE inhibitors drug design
Encyclopedia
The discovery of an orally inactive peptide
from snake venom
established the important role of angiotensin converting enzyme (ACE) inhibitors
in regulating blood pressure
. This led to the development
of Captopril
, the first ACE inhibitor
. When the adverse effects of Captopril became apparent new derivates were designed. Then after the discovery of two active site
s of ACE: N-domain and C-domain, the development of domain specific ACE inhibitors began.
-Trp
-Pro
-Arg
-Pro-Gln
-Ile
-Pro-Pro), which was originally isolated from the venom of the Brazilian pit viper “Bothrops jararaca
”, greatly clarified the importance of ACE in hypertension
. However, its lack of oral activity limited its therapeutic utility.
L-benzyl
succinic acid
(2(R)-benzyl-3-carboxypropionic acid) was described to be the most potent
inhibitor of carboxypeptidase A
in the early 1980s. The author
s referred to it as a by-product
analog
and it was proposed to bind to the active site of carboxypeptidase A via succinyl carboxyl group and a carbonyl group. Their findings established that L-benzylsuccinic acid is bound at a single locus at the active site of carboxypeptidase A. The authors discussed but dismissed the suggestion
that the carboxylate function might bind to the catalytically functional zinc
ion
present at the active site. Later however this was found to be the case.
were tested randomly in a guinea pig
ileum
test and succinyl-L-proline was found to have the properties of a specific ACE inhibitor. It showed inhibitory effect of angiotensin I
and bradykinin
without having any effects on angiotensin II
. Then researchers started to search for a model that would explain inhibition on the basis of specific chemical interactions of compounds with the active site of ACE. Previous studies with substrates
and inhibitors of ACE suggested that it was a zinc-containing metalloprotein
and a carboxypeptidase similar to pancreatic carboxypeptidase A. However ACE releases dipeptides rather than single amino acid
s from the C-terminus of the peptide
substrates. And it was assumed that both their mechanism of action
and their active site might be similar. A positively charged
Arg145 at the active site was thought to bind with the negatively charged C-terminal carboxyl group of the peptide substrate. It was also proposed that ACE binds by hydrogen bonding to the terminal, non scissile, peptide bond
of the substrate.
But since ACE is a dipeptide carboxypeptidase, unlike carboxypeptidase A, the distance between the cationic carboxyl-binding site and the zinc atom should be greater, by approximately the length of one amino acid residue. Proline was chosen as the amino acid moiety
because of its presence as the carboxy terminal amino acid residue in teprotide and other ACE inhibitors found in snake venoms. 11 other amino acids were tested but none of them were more inhibitory. So it was proposed that succinyl amino acid derivative should be an ACE inhibitor and succinyl-L-proline was found to be such an inhibitor.
It was also known that the nature of penultimate amino acid residue of a peptide substrate for ACE influences binding to the enzyme. The acyl group of the carboxyalkanoyl amino acid binds the zinc ion of the enzyme and occupies the same position at the active site of ACE as the penultimate. Therefore the substituent
of the acyl group might also influence binding to the enzyme. A 2-methyl
substituent with D configuration was found to enhance the inhibitory potency
by about 15 fold of succinyl-L-proline. Then the search for a better zinc-binding group started. Replacement of the succinyl carboxyl group by nitrogen
-containing functionalities (amine
, amide
or guanidine
) did not enhance inhibitory activity. However a potency breakthrough was achieved by the replacement of the carboxyl group with a sulfhydril function (SH
), a group with greater affinity
for the enzyme bound zinc ion. This yielded a potent inhibitor that was 1000 times more potent than succinyl-L-proline.
The optimal acyl chain length for mercapto
alkanoyl
derivates of proline was found to be 3-mercaptopropanoyl-L-proline, 5 times greater than that of 2-mercaptoalkanoyl derivates and 50 times greater that that of 4-mercaptoalkanoyl derivates. So the D-3-mercapto-2-methylpropanoyl-L-proline or Captopril was the most potent inhibitor. Later, the researchers compared a few mercaptoacyl amino acid inhibitors and concluded that the binding of the inhibitor to the enzyme involved a hydrogen bond between a donor site on the enzyme and the oxygen of the amide carbonyl, much like predicted for the substrates.
rash
and loss of taste
, are the same as caused by mercapto-containing penicillamine
. Therefore a group of researchers aimed at finding potent, selective ACE inhibitors that wouldn’t contain a mercapto (SH) function and would have a weaker chelating
function. They returned to work with carboxyl compounds and started working with substituted N-carboxymethyl-dipeptides as a general structure (R-CHCOOH-A1-A2). According to previous research they assumed that cyclic imino acids would result in good potency if substituted on the carboxyl terminus of the dipeptide. Therefore substituting A2 with proline gave good results. They also noted that according to the enzyme’s specificity imino acids in the position next to the carboxyl terminus would not give a potent compound. Also noticeable is that by substituting R and A1 groups with hydrophobic and basic
residues would give a potent compound. By substituting –NH in the general structure resulted in loss of potency which is consistent to the enzyme’s need for a –NH in corresponding position on the substrates. The results where 2 active inhibitors: Enalaprilat
and Lisinopril
. These compounds both have phenylalanine
in R position which occupies the S1 groove in the enzyme. The result was thus these two new, potent tripeptide analogues with zinc-coordinating carboxyl group: Enalaprilat and Lisinopril.
structure of germinal ACE, which has only one active site that corresponds with C-domain of the somatic
ACE, offers a structural framework for structure-based design approach. Although N- and C-domain have comparable rates in vitro
of ACE hydrolyzing, it seems like that in vivo
the C-domain is mainly responsible for regulating blood pressure. This indicates that C-domain selective inhibitors could have similar profile to that of a current non-selective inhibitors. Angiotensin I is mainly hydrolyzed by the C-domain in vivo but bradykinin is hydrolyzed by both active sites. Thus, by developing a C-domain selective inhibitor would permit some degradation
of bradykinin by the N-domain and this degradation could be enough to prevent accumulation of excess
bradykinin which has been observed during attacks of angioedema
. C-domain selective inhibition could possibly result in specialized control of blood pressure with less vasodilator-related adverse effects. N-domain selective inhibitors on the other hand give the possibility of opening up novel therapeutic areas. Apparently, the N-domain doesn’t have a big role in controlling blood pressure but it seems to be the principal metabolizing
enzyme for AcSDKP, a natural haemoregulatory hormone
.
s could substitute for the amide bond that links Phe and Gly in ACE inhibitors. Keto-ACE, first described in 1980, has emerged as a potential lead compound for C-domain specific ACE inhibitors. Keto-ACE, a tripeptide
analogue of Phe-Gly-Pro, contains a bulky P1 and P2 benzyl
ring and was shown to inhibit the hydrolysis of angiotensin I and bradykinin via the C-domain. The synthesis of keto-ACE analogues with Trp or Phe at the P2’ position led to a marked increase in C-domain selectivity, but the introduction of an aliphatic P2 group conferred N-domain selectivity. Inhibitory potency may further be enhanced by the incorporation of hydrophobic substituent, such as phenyl group at the P1’ position. Also P1’ substituent with S-stereochemistry
has been shown to possess greater inhibitory potency than their R-counterparts.
Keto-ACE was used as the basis for the design of ketomethylene derivates. Its analogues contain a ketomethylene isostere
replacement at the scissile bond that is believed to mimic the tetrahedron
transition state
of the proteolytic
reaction at the active site. The focus was on a simple tripeptide Phe-Ala-Pro, which in earlier enzyme assay
s has shown inhibition activity. Replacement of alanine with glycin gave a tripeptide with 1/14th of the inhibition activity of Phe-Ala-Pro. The benzoylated derivative of Phe-Gly-Pro, Bz-Phe-Gly-Pro, was twice as active. To reduce the peptidic nature of ketomethylene inhibitors the P1’ and P2’ substituent may be cyclized to form a lactam
, where there is a correlation between the inhibitory potency and the ring size. In 2001 it was postulated that a substitution α
to nitrogen and making of 3-methyl-substituted analog of A58365A, a pyridone acid isolated from the fermentation broth
of the bacterium
Streptomyces
chromofuscus with ACE inhibitory activity, might influence the level of biological activity
by steric
or hydrophobic effect
, and/or by preventing reactions at C3. Also it was noticed during the synthetic
work on A58365A that potential precursor
were sensitive to oxidation of the five-membered ring and so the 3-methyl analogue might be more stable in this respect.
and silicone
have similar, but also dissimilar, characteristics triggered the interest in substituting carbon with silanediol as a central, zinc chelating group. Silicone forms a dialkylsilanediol compound that is sufficiently hindered so the formation of a siloxane
polymer
does not occur. Silanediols are more stable than carbon diol
s so they are expected to have longer half-life
. Also silanediols are neutral at physiological pH
(do not ionize
).
Four stereoisomers of Phe-Ala silanediol were compared to ketone-based inhibitors and the silanediol were found to be fourfold less potent than the ketone analogue. This is because silanediols are weaker zinc chelators compared with ketones. Replacement of the silanediol, with a methylsilano group gave little enzyme
inhibition. This confirms that the silanediol group interacts with ACE as a transition state analogue and the interaction is in a manner similar to that of ketone. If the benzyl group of silanediol is replaced by an i-butyl
group it gives a weaker ACE inhibitor. Introduction of a hydrophobic methyl phenyl gives a little more potency than an analogue with a tert-butyl-group at P1. That suggests that methyl phenyl gives a better S1 recognition than a tert-butyl group.
peptides are pseudo-peptides where a phosphinic acid bond (PO2-CH-) has replaced a peptide bond in the peptide analogue sequence. To some extent the chemical structure of phosphinic peptides is similar to that of intermediates
which are produced in hydrolysis
of peptides by proteolytic enzymes. The hypothesis
has been made that these pseudo-peptides mimic the structure of the enzyme substrates in their transition state and crystallography
of zinc proteases in complex with phosphinic peptides supports that hypothesis.
(L)-Pheψ(PO2-CH2)(L)Ala-Ala-NH2 actively inhibited the N-domain and was given the name RXP 407. Structure-function relationship showed that the C-terminus carboxamide group played a crucial role in the selectivity for the N-domain of ACE. Additionally, the N-acetyl group and the aspartic side chain in the P2 position aides in the N-domain selectivity of the inhibitor. These features make the inhibitor inaccessible to the C-domain but give good potency for the N-domain, this leads to a difference in inhibitory potency of the active sites of three orders of magnitude. These results also indicate that the N-domain possess a broader selectivity than the C-domain. Another difference between the older ACE inhibitors and RXP 407 is the molecular size of the compound. The older ACE inhibitors had mostly been interacting with S1’, S2’ and S1 subsites but RXP 407 interacts in addition with the S2 subsite. This also is important for the selectivity of the inhibitor since the aspartic side chain and N-acetyl group are located in the P2 position.
ring in the C-domain but this interaction is much weaker in the N-domain.
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
from snake venom
Snake venom
Snake venom is highly modified saliva that is produced by special glands of certain species of snakes. The glands which secrete the zootoxin are a modification of the parotid salivary gland of other vertebrates, and are usually situated on each side of the head below and behind the eye,...
established the important role of angiotensin converting enzyme (ACE) inhibitors
Enzyme inhibitor
An enzyme inhibitor is a molecule that binds to enzymes and decreases their activity. Since blocking an enzyme's activity can kill a pathogen or correct a metabolic imbalance, many drugs are enzyme inhibitors. They are also used as herbicides and pesticides...
in regulating blood pressure
Blood pressure
Blood pressure is the pressure exerted by circulating blood upon the walls of blood vessels, and is one of the principal vital signs. When used without further specification, "blood pressure" usually refers to the arterial pressure of the systemic circulation. During each heartbeat, BP varies...
. This led to the development
Drug development
Drug development is a blanket term used to define the process of bringing a new drug to the market once a lead compound has been identified through the process of drug discovery...
of Captopril
Captopril
Captopril is an angiotensin-converting enzyme inhibitor used for the treatment of hypertension and some types of congestive heart failure. Captopril was the first ACE inhibitor developed and was considered a breakthrough both because of its novel mechanism of action and also because of the...
, the first ACE inhibitor
ACE inhibitor
ACE inhibitors or angiotensin-converting enzyme inhibitors are a group of drugs used primarily for the treatment of hypertension and congestive heart failure...
. When the adverse effects of Captopril became apparent new derivates were designed. Then after the discovery of two active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
s of ACE: N-domain and C-domain, the development of domain specific ACE inhibitors began.
Development of first generation ACE inhibitors
The development of the nonapeptide teprotide (GluGlutamic acid
Glutamic acid is one of the 20 proteinogenic amino acids, and its codons are GAA and GAG. It is a non-essential amino acid. The carboxylate anions and salts of glutamic acid are known as glutamates...
-Trp
Tryptophan
Tryptophan is one of the 20 standard amino acids, as well as an essential amino acid in the human diet. It is encoded in the standard genetic code as the codon UGG...
-Pro
Proline
Proline is an α-amino acid, one of the twenty DNA-encoded amino acids. Its codons are CCU, CCC, CCA, and CCG. It is not an essential amino acid, which means that the human body can synthesize it. It is unique among the 20 protein-forming amino acids in that the α-amino group is secondary...
-Arg
Arginine
Arginine is an α-amino acid. The L-form is one of the 20 most common natural amino acids. At the level of molecular genetics, in the structure of the messenger ribonucleic acid mRNA, CGU, CGC, CGA, CGG, AGA, and AGG, are the triplets of nucleotide bases or codons that codify for arginine during...
-Pro-Gln
Glutamine
Glutamine is one of the 20 amino acids encoded by the standard genetic code. It is not recognized as an essential amino acid but may become conditionally essential in certain situations, including intensive athletic training or certain gastrointestinal disorders...
-Ile
Isoleucine
Isoleucine is an α-amino acid with the chemical formula HO2CCHCHCH2CH3. It is an essential amino acid, which means that humans cannot synthesize it, so it must be ingested. Its codons are AUU, AUC and AUA....
-Pro-Pro), which was originally isolated from the venom of the Brazilian pit viper “Bothrops jararaca
Bothrops jararaca
Bothrops jararaca is a venomous pit viper species found in southern Brazil, Paraguay and northern Argentina. The species name is derived from the Tupi words yarará and ca, which means "large snake." Within its range it is often abundant and is an important cause of snakebite...
”, greatly clarified the importance of ACE in hypertension
Hypertension
Hypertension or high blood pressure is a cardiac chronic medical condition in which the systemic arterial blood pressure is elevated. What that means is that the heart is having to work harder than it should to pump the blood around the body. Blood pressure involves two measurements, systolic and...
. However, its lack of oral activity limited its therapeutic utility.
L-benzyl
Benzyl
In organic chemistry, benzyl is the term used to describe the substituent or molecular fragment possessing the structure C6H5CH2-. Benzyl features a benzene ring attached to a CH2 group.-Nomenclature:...
succinic acid
Succinic acid
Succinic acid is a dicarboxylic acid. Succinate plays a biochemical role in the citric acid cycle. The name derives from Latin succinum, meaning amber, from which the acid may be obtained....
(2(R)-benzyl-3-carboxypropionic acid) was described to be the most potent
Potency (pharmacology)
In the field of pharmacology, potency is a measure of drug activity expressed in terms of the amount required to produce an effect of given intensity. A highly potent drug evokes a larger response at low concentrations, while a drug of lower potency evokes a small response at low concentrations...
inhibitor of carboxypeptidase A
Carboxypeptidase A
Carboxypeptidase A usually refers to the pancreatic exopeptidase which hydrolyzes peptide bonds of C-terminal residues with aromatic or aliphatic side chains...
in the early 1980s. The author
Author
An author is broadly defined as "the person who originates or gives existence to anything" and that authorship determines responsibility for what is created. Narrowly defined, an author is the originator of any written work.-Legal significance:...
s referred to it as a by-product
By-product
A by-product is a secondary product derived from a manufacturing process or chemical reaction. It is not the primary product or service being produced.A by-product can be useful and marketable or it can be considered waste....
analog
Analog (chemistry)
In chemistry, a structural analog , also known as chemical analog or simply analog, is a compound having a structure similar to that of another one, but differing from it in respect of a certain component. It can differ in one or more atoms, functional groups, or substructures, which are replaced...
and it was proposed to bind to the active site of carboxypeptidase A via succinyl carboxyl group and a carbonyl group. Their findings established that L-benzylsuccinic acid is bound at a single locus at the active site of carboxypeptidase A. The authors discussed but dismissed the suggestion
Suggestion
Suggestion is the psychological process by which one person guides the thoughts, feelings, or behaviour of another. Nineteenth century writers on psychology such as William James used the words "suggest" and "suggestion" in senses close to those they have in common speech—one idea was said to...
that the carboxylate function might bind to the catalytically functional zinc
Zinc
Zinc , or spelter , is a metallic chemical element; it has the symbol Zn and atomic number 30. It is the first element in group 12 of the periodic table. Zinc is, in some respects, chemically similar to magnesium, because its ion is of similar size and its only common oxidation state is +2...
ion
Ion
An ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...
present at the active site. Later however this was found to be the case.
Drug design of Captopril (sulfhydrils)
Over 2000 compoundsChemical compound
A chemical compound is a pure chemical substance consisting of two or more different chemical elements that can be separated into simpler substances by chemical reactions. Chemical compounds have a unique and defined chemical structure; they consist of a fixed ratio of atoms that are held together...
were tested randomly in a guinea pig
Guinea pig
The guinea pig , also called the cavy, is a species of rodent belonging to the family Caviidae and the genus Cavia. Despite their common name, these animals are not in the pig family, nor are they from Guinea...
ileum
Ileum
The ileum is the final section of the small intestine in most higher vertebrates, including mammals, reptiles, and birds. In fish, the divisions of the small intestine are not as clear and the terms posterior intestine or distal intestine may be used instead of ileum.The ileum follows the duodenum...
test and succinyl-L-proline was found to have the properties of a specific ACE inhibitor. It showed inhibitory effect of angiotensin I
Angiotensin
Angiotensin, a peptide hormone, causes blood vessels to constrict, and drives blood pressure up. It is part of the renin-angiotensin system, which is a major target for drugs that lower blood pressure. Angiotensin also stimulates the release of aldosterone, another hormone, from the adrenal cortex...
and bradykinin
Bradykinin
Bradykinin is a peptide that causes blood vessels to dilate , and therefore causes blood pressure to lower. A class of drugs called ACE inhibitors, which are used to lower blood pressure, increase bradykinin further lowering blood pressure...
without having any effects on angiotensin II
Angiotensin
Angiotensin, a peptide hormone, causes blood vessels to constrict, and drives blood pressure up. It is part of the renin-angiotensin system, which is a major target for drugs that lower blood pressure. Angiotensin also stimulates the release of aldosterone, another hormone, from the adrenal cortex...
. Then researchers started to search for a model that would explain inhibition on the basis of specific chemical interactions of compounds with the active site of ACE. Previous studies with substrates
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
and inhibitors of ACE suggested that it was a zinc-containing metalloprotein
Metalloprotein
Metalloprotein is a generic term for a protein that contains a metal ion cofactor. Metalloproteins have many different functions in cells, such as enzymes, transport and storage proteins, and signal transduction proteins. Indeed, about one quarter to one third of all proteins require metals to...
and a carboxypeptidase similar to pancreatic carboxypeptidase A. However ACE releases dipeptides rather than single amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s from the C-terminus of the peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
substrates. And it was assumed that both their mechanism of action
Mechanism of action
In pharmacology, the term mechanism of action refers to the specific biochemical interaction through which a drug substance produces its pharmacological effect...
and their active site might be similar. A positively charged
Electric charge
Electric charge is a physical property of matter that causes it to experience a force when near other electrically charged matter. Electric charge comes in two types, called positive and negative. Two positively charged substances, or objects, experience a mutual repulsive force, as do two...
Arg145 at the active site was thought to bind with the negatively charged C-terminal carboxyl group of the peptide substrate. It was also proposed that ACE binds by hydrogen bonding to the terminal, non scissile, peptide bond
Peptide bond
This article is about the peptide link found within biological molecules, such as proteins. A similar article for synthetic molecules is being created...
of the substrate.
But since ACE is a dipeptide carboxypeptidase, unlike carboxypeptidase A, the distance between the cationic carboxyl-binding site and the zinc atom should be greater, by approximately the length of one amino acid residue. Proline was chosen as the amino acid moiety
Functional group
In organic chemistry, functional groups are specific groups of atoms within molecules that are responsible for the characteristic chemical reactions of those molecules. The same functional group will undergo the same or similar chemical reaction regardless of the size of the molecule it is a part of...
because of its presence as the carboxy terminal amino acid residue in teprotide and other ACE inhibitors found in snake venoms. 11 other amino acids were tested but none of them were more inhibitory. So it was proposed that succinyl amino acid derivative should be an ACE inhibitor and succinyl-L-proline was found to be such an inhibitor.
It was also known that the nature of penultimate amino acid residue of a peptide substrate for ACE influences binding to the enzyme. The acyl group of the carboxyalkanoyl amino acid binds the zinc ion of the enzyme and occupies the same position at the active site of ACE as the penultimate. Therefore the substituent
Substituent
In organic chemistry and biochemistry, a substituent is an atom or group of atoms substituted in place of a hydrogen atom on the parent chain of a hydrocarbon...
of the acyl group might also influence binding to the enzyme. A 2-methyl
Methyl group
Methyl group is a functional group derived from methane, containing one carbon atom bonded to three hydrogen atoms —CH3. The group is often abbreviated Me. Such hydrocarbon groups occur in many organic compounds. The methyl group can be found in three forms: anion, cation and radical. The anion...
substituent with D configuration was found to enhance the inhibitory potency
Potency
Potency may refer to:* Potency , a measure of the activity of a drug in a biological system* Virility* Potency is a measure of the differentiation potential of stem cells...
by about 15 fold of succinyl-L-proline. Then the search for a better zinc-binding group started. Replacement of the succinyl carboxyl group by nitrogen
Nitrogen
Nitrogen is a chemical element that has the symbol N, atomic number of 7 and atomic mass 14.00674 u. Elemental nitrogen is a colorless, odorless, tasteless, and mostly inert diatomic gas at standard conditions, constituting 78.08% by volume of Earth's atmosphere...
-containing functionalities (amine
Amine
Amines are organic compounds and functional groups that contain a basic nitrogen atom with a lone pair. Amines are derivatives of ammonia, wherein one or more hydrogen atoms have been replaced by a substituent such as an alkyl or aryl group. Important amines include amino acids, biogenic amines,...
, amide
Amide
In chemistry, an amide is an organic compound that contains the functional group consisting of a carbonyl group linked to a nitrogen atom . The term refers both to a class of compounds and a functional group within those compounds. The term amide also refers to deprotonated form of ammonia or an...
or guanidine
Guanidine
Guanidine is a crystalline compound of strong alkalinity formed by the oxidation of guanine. It is used in the manufacture of plastics and explosives. It is found in urine as a normal product of protein metabolism. The molecule was first synthesized in 1861 by the oxidative degradation of an...
) did not enhance inhibitory activity. However a potency breakthrough was achieved by the replacement of the carboxyl group with a sulfhydril function (SH
Thiol
In organic chemistry, a thiol is an organosulfur compound that contains a carbon-bonded sulfhydryl group...
), a group with greater affinity
Dissociation constant
In chemistry, biochemistry, and pharmacology, a dissociation constant is a specific type of equilibrium constant that measures the propensity of a larger object to separate reversibly into smaller components, as when a complex falls apart into its component molecules, or when a salt splits up into...
for the enzyme bound zinc ion. This yielded a potent inhibitor that was 1000 times more potent than succinyl-L-proline.
The optimal acyl chain length for mercapto
Thiol
In organic chemistry, a thiol is an organosulfur compound that contains a carbon-bonded sulfhydryl group...
alkanoyl
Acyl
An acyl group is a functional group derived by the removal of one or more hydroxyl groups from an oxoacid, including inorganic acids.In organic chemistry, the acyl group is usually derived from a carboxylic acid . Therefore, it has the formula RCO-, where R represents an alkyl group that is...
derivates of proline was found to be 3-mercaptopropanoyl-L-proline, 5 times greater than that of 2-mercaptoalkanoyl derivates and 50 times greater that that of 4-mercaptoalkanoyl derivates. So the D-3-mercapto-2-methylpropanoyl-L-proline or Captopril was the most potent inhibitor. Later, the researchers compared a few mercaptoacyl amino acid inhibitors and concluded that the binding of the inhibitor to the enzyme involved a hydrogen bond between a donor site on the enzyme and the oxygen of the amide carbonyl, much like predicted for the substrates.
Drug design of other first generation ACE inhibitors
The most common adverse effects of Captopril, skinSkin
-Dermis:The dermis is the layer of skin beneath the epidermis that consists of connective tissue and cushions the body from stress and strain. The dermis is tightly connected to the epidermis by a basement membrane. It also harbors many Mechanoreceptors that provide the sense of touch and heat...
rash
Rash
A rash is a change of the skin which affects its color, appearance or texture. A rash may be localized in one part of the body, or affect all the skin. Rashes may cause the skin to change color, itch, become warm, bumpy, chapped, dry, cracked or blistered, swell and may be painful. The causes, and...
and loss of taste
Taste
Taste is one of the traditional five senses. It refers to the ability to detect the flavor of substances such as food, certain minerals, and poisons, etc....
, are the same as caused by mercapto-containing penicillamine
Penicillamine
Penicillamine is a pharmaceutical of the chelator class. It is sold under the trade names of Cuprimine and Depen. The pharmaceutical form is D-penicillamine, as L-penicillamine is toxic...
. Therefore a group of researchers aimed at finding potent, selective ACE inhibitors that wouldn’t contain a mercapto (SH) function and would have a weaker chelating
Chelation
Chelation is the formation or presence of two or more separate coordinate bonds between apolydentate ligand and a single central atom....
function. They returned to work with carboxyl compounds and started working with substituted N-carboxymethyl-dipeptides as a general structure (R-CHCOOH-A1-A2). According to previous research they assumed that cyclic imino acids would result in good potency if substituted on the carboxyl terminus of the dipeptide. Therefore substituting A2 with proline gave good results. They also noted that according to the enzyme’s specificity imino acids in the position next to the carboxyl terminus would not give a potent compound. Also noticeable is that by substituting R and A1 groups with hydrophobic and basic
Base (chemistry)
For the term in genetics, see base A base in chemistry is a substance that can accept hydrogen ions or more generally, donate electron pairs. A soluble base is referred to as an alkali if it contains and releases hydroxide ions quantitatively...
residues would give a potent compound. By substituting –NH in the general structure resulted in loss of potency which is consistent to the enzyme’s need for a –NH in corresponding position on the substrates. The results where 2 active inhibitors: Enalaprilat
Enalaprilat
Enalaprilat is the active metabolite of enalapril. It is the first dicarboxylate-containing ACE inhibitor and was developed partly to overcome these limitations of captopril...
and Lisinopril
Lisinopril
Lisinopril is a drug of the angiotensin-converting enzyme inhibitor class that is primarily used in treatment of hypertension, congestive heart failure, and heart attacks and also in preventing renal and retinal complications of diabetes. Its indications, contraindications and side effects are as...
. These compounds both have phenylalanine
Phenylalanine
Phenylalanine is an α-amino acid with the formula C6H5CH2CHCOOH. This essential amino acid is classified as nonpolar because of the hydrophobic nature of the benzyl side chain. L-Phenylalanine is an electrically neutral amino acid, one of the twenty common amino acids used to biochemically form...
in R position which occupies the S1 groove in the enzyme. The result was thus these two new, potent tripeptide analogues with zinc-coordinating carboxyl group: Enalaprilat and Lisinopril.
Discovery of 2 active sites: C-domain and N-domain
Most of the ACE inhibitors on the market today are non-selective towards the two active sites of ACE because their binding to the enzyme is based mostly on the strong interaction between the zinc atom in the enzyme and the strong chelating group on the inhibitor. The resolution of the 3D3D modeling
In 3D computer graphics, 3D modeling is the process of developing a mathematical representation of any three-dimensional surface of object via specialized software. The product is called a 3D model...
structure of germinal ACE, which has only one active site that corresponds with C-domain of the somatic
Somatic
The term somatic means 'of the body',, relating to the body. In medicine, somatic illness is bodily, not mental, illness. The term is often used in biology to refer to the cells of the body in contrast to the germ line cells which usually give rise to the gametes...
ACE, offers a structural framework for structure-based design approach. Although N- and C-domain have comparable rates in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
of ACE hydrolyzing, it seems like that in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
the C-domain is mainly responsible for regulating blood pressure. This indicates that C-domain selective inhibitors could have similar profile to that of a current non-selective inhibitors. Angiotensin I is mainly hydrolyzed by the C-domain in vivo but bradykinin is hydrolyzed by both active sites. Thus, by developing a C-domain selective inhibitor would permit some degradation
Chemical decomposition
Chemical decomposition, analysis or breakdown is the separation of a chemical compound into elements or simpler compounds. It is sometimes defined as the exact opposite of a chemical synthesis. Chemical decomposition is often an undesired chemical reaction...
of bradykinin by the N-domain and this degradation could be enough to prevent accumulation of excess
Excess
Excess may refer to:* Angle excess, in spherical trigonometry, quantity, used to calculate the area of polygon on a sphere* Excess, in insurance, similar to deductible* Excess, in chemistry, describing any reagent that is not the limiting reagent...
bradykinin which has been observed during attacks of angioedema
Angioedema
Angioedema or Quincke's edema is the rapid swelling of the dermis, subcutaneous tissue, mucosa and submucosal tissues. It is very similar to urticaria, but urticaria, commonly known as hives, occurs in the upper dermis...
. C-domain selective inhibition could possibly result in specialized control of blood pressure with less vasodilator-related adverse effects. N-domain selective inhibitors on the other hand give the possibility of opening up novel therapeutic areas. Apparently, the N-domain doesn’t have a big role in controlling blood pressure but it seems to be the principal metabolizing
Metabolism
Metabolism is the set of chemical reactions that happen in the cells of living organisms to sustain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories...
enzyme for AcSDKP, a natural haemoregulatory hormone
Hormone
A hormone is a chemical released by a cell or a gland in one part of the body that sends out messages that affect cells in other parts of the organism. Only a small amount of hormone is required to alter cell metabolism. In essence, it is a chemical messenger that transports a signal from one...
.
Drug design of Keto-ACE and its ketomethylene derivatives.
It was found that other carbonyl-containing groups such as ketoneKetone
In organic chemistry, a ketone is an organic compound with the structure RCR', where R and R' can be a variety of atoms and groups of atoms. It features a carbonyl group bonded to two other carbon atoms. Many ketones are known and many are of great importance in industry and in biology...
s could substitute for the amide bond that links Phe and Gly in ACE inhibitors. Keto-ACE, first described in 1980, has emerged as a potential lead compound for C-domain specific ACE inhibitors. Keto-ACE, a tripeptide
Tripeptide
A tripeptide is a peptide consisting of three amino acids joined by peptide bonds.Examples of tripeptides are:*Eisenin is a peptide with immunological activity that is isolated from the Japanese marine alga, Eisenia bicyclis, which more commonly is known as, Arame*GHK-Cu is a human copper binding...
analogue of Phe-Gly-Pro, contains a bulky P1 and P2 benzyl
Benzyl
In organic chemistry, benzyl is the term used to describe the substituent or molecular fragment possessing the structure C6H5CH2-. Benzyl features a benzene ring attached to a CH2 group.-Nomenclature:...
ring and was shown to inhibit the hydrolysis of angiotensin I and bradykinin via the C-domain. The synthesis of keto-ACE analogues with Trp or Phe at the P2’ position led to a marked increase in C-domain selectivity, but the introduction of an aliphatic P2 group conferred N-domain selectivity. Inhibitory potency may further be enhanced by the incorporation of hydrophobic substituent, such as phenyl group at the P1’ position. Also P1’ substituent with S-stereochemistry
Stereochemistry
Stereochemistry, a subdiscipline of chemistry, involves the study of the relative spatial arrangement of atoms within molecules. An important branch of stereochemistry is the study of chiral molecules....
has been shown to possess greater inhibitory potency than their R-counterparts.
Keto-ACE was used as the basis for the design of ketomethylene derivates. Its analogues contain a ketomethylene isostere
Isostere
Isosteres are molecules or ions with the same number of atoms and the same number of valence electrons.Some examples of isoteres include:*Sodium and hydrogen*Carbon dioxide and nitrous oxide *Silicon and carbon...
replacement at the scissile bond that is believed to mimic the tetrahedron
Tetrahedron
In geometry, a tetrahedron is a polyhedron composed of four triangular faces, three of which meet at each vertex. A regular tetrahedron is one in which the four triangles are regular, or "equilateral", and is one of the Platonic solids...
transition state
Transition state
The transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest energy along this reaction coordinate. At this point, assuming a perfectly irreversible reaction, colliding reactant molecules will always...
of the proteolytic
Proteolysis
Proteolysis is the directed degradation of proteins by cellular enzymes called proteases or by intramolecular digestion.-Purposes:Proteolysis is used by the cell for several purposes...
reaction at the active site. The focus was on a simple tripeptide Phe-Ala-Pro, which in earlier enzyme assay
Enzyme assay
Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition.-Enzyme units:...
s has shown inhibition activity. Replacement of alanine with glycin gave a tripeptide with 1/14th of the inhibition activity of Phe-Ala-Pro. The benzoylated derivative of Phe-Gly-Pro, Bz-Phe-Gly-Pro, was twice as active. To reduce the peptidic nature of ketomethylene inhibitors the P1’ and P2’ substituent may be cyclized to form a lactam
Lactam
A lactam is a cyclic amide. Prefixes indicate how many carbon atoms are present in the ring: β-lactam , γ-lactam , δ-lactam...
, where there is a correlation between the inhibitory potency and the ring size. In 2001 it was postulated that a substitution α
Alpha carbon
The alpha carbon in organic chemistry refers to the first carbon that attaches to a functional group . By extension, the second carbon is the beta carbon, and so on....
to nitrogen and making of 3-methyl-substituted analog of A58365A, a pyridone acid isolated from the fermentation broth
Broth
Broth is a liquid food preparation, typically consisting of either water or an already flavored stock, in which bones, meat, fish, cereal grains, or vegetables have been simmered. Broth is used as a basis for other edible liquids such as soup, gravy, or sauce. It can be eaten alone or with garnish...
of the bacterium
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
Streptomyces
Streptomyces
Streptomyces is the largest genus of Actinobacteria and the type genus of the family Streptomycetaceae. Over 500 species of Streptomyces bacteria have been described. As with the other Actinobacteria, streptomycetes are gram-positive, and have genomes with high guanine and cytosine content...
chromofuscus with ACE inhibitory activity, might influence the level of biological activity
Biological activity
In pharmacology, biological activity or pharmacological activity describes the beneficial or adverse effects of a drug on living matter. When a drug is a complex chemical mixture, this activity is exerted by the substance's active ingredient or pharmacophore but can be modified by the other...
by steric
Steric effects
Steric effects arise from the fact that each atom within a molecule occupies a certain amount of space. If atoms are brought too close together, there is an associated cost in energy due to overlapping electron clouds , and this may affect the molecule's preferred shape and reactivity.-Steric...
or hydrophobic effect
Hydrophobic effect
The hydrophobic effect is the observed tendency of nonpolar substances to aggregate in aqueous solution and exclude water molecules. The name, literally meaning "water-fearing," describes the segregation and apparent repulsion between water and nonpolar substances...
, and/or by preventing reactions at C3. Also it was noticed during the synthetic
Chemical synthesis
In chemistry, chemical synthesis is purposeful execution of chemical reactions to get a product, or several products. This happens by physical and chemical manipulations usually involving one or more reactions...
work on A58365A that potential precursor
Precursor (chemistry)
In chemistry, a precursor is a compound that participates in the chemical reaction that produces another compound. In biochemistry, the term "precursor" is used more specifically to refer to a chemical compound preceding another in a metabolic pathway....
were sensitive to oxidation of the five-membered ring and so the 3-methyl analogue might be more stable in this respect.
Drug design of silanediol
The fact that carbonCarbon
Carbon is the chemical element with symbol C and atomic number 6. As a member of group 14 on the periodic table, it is nonmetallic and tetravalent—making four electrons available to form covalent chemical bonds...
and silicone
Silicone
Silicones are inert, synthetic compounds with a variety of forms and uses. Typically heat-resistant and rubber-like, they are used in sealants, adhesives, lubricants, medical applications , cookware, and insulation....
have similar, but also dissimilar, characteristics triggered the interest in substituting carbon with silanediol as a central, zinc chelating group. Silicone forms a dialkylsilanediol compound that is sufficiently hindered so the formation of a siloxane
Siloxane
A siloxane is any chemical compound composed of units of the form R2SiO, where R is a hydrogen atom or a hydrocarbon group. They belong to the wider class of organosilicon compounds....
polymer
Polymer
A polymer is a large molecule composed of repeating structural units. These subunits are typically connected by covalent chemical bonds...
does not occur. Silanediols are more stable than carbon diol
Diol
A diol or glycol is a chemical compound containing two hydroxyl groups A geminal diol has two hydroxyl groups bonded to the same atom...
s so they are expected to have longer half-life
Half-life
Half-life, abbreviated t½, is the period of time it takes for the amount of a substance undergoing decay to decrease by half. The name was originally used to describe a characteristic of unstable atoms , but it may apply to any quantity which follows a set-rate decay.The original term, dating to...
. Also silanediols are neutral at physiological pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
(do not ionize
Ionization
Ionization is the process of converting an atom or molecule into an ion by adding or removing charged particles such as electrons or other ions. This is often confused with dissociation. A substance may dissociate without necessarily producing ions. As an example, the molecules of table sugar...
).
Four stereoisomers of Phe-Ala silanediol were compared to ketone-based inhibitors and the silanediol were found to be fourfold less potent than the ketone analogue. This is because silanediols are weaker zinc chelators compared with ketones. Replacement of the silanediol, with a methylsilano group gave little enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
inhibition. This confirms that the silanediol group interacts with ACE as a transition state analogue and the interaction is in a manner similar to that of ketone. If the benzyl group of silanediol is replaced by an i-butyl
Butyl
In organic chemistry, butyl is a four-carbon alkyl radical or substituent group with general chemical formula -C4H9, derived from either of the two isomers of butane....
group it gives a weaker ACE inhibitor. Introduction of a hydrophobic methyl phenyl gives a little more potency than an analogue with a tert-butyl-group at P1. That suggests that methyl phenyl gives a better S1 recognition than a tert-butyl group.
Phosphinic peptides
PhosphinicPhosphine
Phosphine is the compound with the chemical formula PH3. It is a colorless, flammable, toxic gas. Pure phosphine is odourless, but technical grade samples have a highly unpleasant odor like garlic or rotting fish, due to the presence of substituted phosphine and diphosphine...
peptides are pseudo-peptides where a phosphinic acid bond (PO2-CH-) has replaced a peptide bond in the peptide analogue sequence. To some extent the chemical structure of phosphinic peptides is similar to that of intermediates
Reaction intermediate
A reaction intermediate or an intermediate is a molecular entity that is formed from the reactants and reacts further to give the directly observed products of a chemical reaction. Most chemical reactions are stepwise, that is they take more than one elementary step to complete...
which are produced in hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
of peptides by proteolytic enzymes. The hypothesis
Hypothesis
A hypothesis is a proposed explanation for a phenomenon. The term derives from the Greek, ὑποτιθέναι – hypotithenai meaning "to put under" or "to suppose". For a hypothesis to be put forward as a scientific hypothesis, the scientific method requires that one can test it...
has been made that these pseudo-peptides mimic the structure of the enzyme substrates in their transition state and crystallography
Crystallography
Crystallography is the experimental science of the arrangement of atoms in solids. The word "crystallography" derives from the Greek words crystallon = cold drop / frozen drop, with its meaning extending to all solids with some degree of transparency, and grapho = write.Before the development of...
of zinc proteases in complex with phosphinic peptides supports that hypothesis.
Drug design of RXP 407
RXP 407 is the first N-domain selective phosphinic peptide and was discovered by screening phosphinic peptides libraries. Before the discovery of RXP 407 it had long been claimed that the free C-terminal carboxylate group in P2’ position was essential to the potency of ACE inhibitor so it can be reasoned that this has postponed the discovery of N-domain selective ACE inhibitors. When RXP 407 was discovered researchers looked in to phosphinic peptides with 3 different general formula, each containing 2 unidentified amino acids, only 1 of these general formula showed potent inhibition (Ac-Yaa-Pheψ(PO2-CH2)Ala-Yaa’-NH2). Peptide mixtures were made, substituting Yaa and Yaa’ with different amino acids, trying to establish if there would be a potent inhibitor that could inhibit either the N-domain or the C-domain of the enzyme. The result was that the compound Ac-AspAspartic acid
Aspartic acid is an α-amino acid with the chemical formula HOOCCHCH2COOH. The carboxylate anion, salt, or ester of aspartic acid is known as aspartate. The L-isomer of aspartate is one of the 20 proteinogenic amino acids, i.e., the building blocks of proteins...
(L)-Pheψ(PO2-CH2)(L)Ala-Ala-NH2 actively inhibited the N-domain and was given the name RXP 407. Structure-function relationship showed that the C-terminus carboxamide group played a crucial role in the selectivity for the N-domain of ACE. Additionally, the N-acetyl group and the aspartic side chain in the P2 position aides in the N-domain selectivity of the inhibitor. These features make the inhibitor inaccessible to the C-domain but give good potency for the N-domain, this leads to a difference in inhibitory potency of the active sites of three orders of magnitude. These results also indicate that the N-domain possess a broader selectivity than the C-domain. Another difference between the older ACE inhibitors and RXP 407 is the molecular size of the compound. The older ACE inhibitors had mostly been interacting with S1’, S2’ and S1 subsites but RXP 407 interacts in addition with the S2 subsite. This also is important for the selectivity of the inhibitor since the aspartic side chain and N-acetyl group are located in the P2 position.
Drug design of RXPA 380
RXPA380 was the first inhibitor that was highly selective of the C-domain of ACE, it has the formula Phe-Phe-Pro-Trp. The development of this compound was built on researches that showed that some bradykinin-potentiating peptides showed selectivity for the C-domain and all had several prolines in their structure. These observations lead the researchers to synthesize phosphinic peptides containing a proline residue in the P1’ position and evaluating these compounds led to the discovery of RXPA380. To study the roles of the residues on RXPA380 the researchers made 7 analogues of RXPA380. All of the compounds made were obtained as a mixture of either 2 or 4 diastereoisomers but all of them were easily resolved and only one of them was potent. This is consistent with the initial modeling studies of RXPA380 which showed that only one diastereomer could accommodate in the active site of germinal ACE. Analogues where pseudo-proline or tryptophan residues had been substituted showed less selectivity than RXPA380. This is probably because these two analogues have more potency toward the N-domain than RXPA380 does. Substituting both of these residues gives great potency but none selectivity. This shows that pseudo-proline and tryptophan residues accommodate well in the C-domain but not in the N-domain. 2 more analogues with both pseudo-proline and tryptophan but missing the pseudo-phenylalanine residue in P1 position showed low potency for N-domain, similar to RXPA380. This supports the significant role of these two residues in the selectivity for C-domain. These two analogues also have less potency for the C-domain which shows that the C-domain prefers pseudo-phenylalanine group in P1 position. Modeling of RXPA380-ACE complex showed that the pseudo-proline residue of the inhibitor was surrounded by amino acids similar to that of the N-domain thus interactions with S2’ domain might not be responsible for the selectivity of RXPA380. 7 of 12 amino acids surrounding tryptophan are the same in C- and N-domain, the biggest difference is that 2 bulky and hydrophobic amino acids in the C-domain have been replaced with 2 smaller and polar amino acids in the N-domain. This indicates that low potency of RXPA380 for N-domain is not because the S2’ cavity does not accommodate the tryptophan side chain but rather that important interactions are missing between the tryptophan side chain and the amino acids of the C-domain.Also based on the proximity between the tryptophan side chain and Asp1029 there is a possible hydrogen bond between the carboxylate of Asp1029 and the NH indoleIndole
Indole is an aromatic heterocyclic organic compound. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring. Indole is a popular component of fragrances and the precursor to many pharmaceuticals. Compounds that contain an...
ring in the C-domain but this interaction is much weaker in the N-domain.