Microsomal oxidation increases the reactivity of molecules. P450 cytochromes Cytochrome 450 inhibitors

Cytochrome p450 (CYP 450) is the name of a large family of universal enzymes of the human body, responsible for the metabolism of most drugs and other foreign organic compounds (xenobiotics).

The metabolism of many classes of drugs (antihistamines, retroviral protease inhibitors, benzodiazepines, calcium channel blockers, etc.) occurs with the participation of cytochromes.

In addition, cytochromes mediate various physiological processes, including the biosynthesis of steroids and cholesterol, fatty acid metabolism, and calcium metabolism (hydroxylation of vitamin D3, which is the first step in the formation of calcitriol).

History of cytochrome p450

Cytochrome P450 was discovered in the late 50s of the twentieth century by M. Klingenberg and D. Garfinkel. The term “cytochrome” (cito – cell; c hromos – color) appeared in 1962 as a temporary name for a colored substance found in cells.

As it turned out, various types of cytochrome P450 are widely distributed in the cells of microorganisms, plants and mammals. These enzymes are absent only in anaerobic bacteria.

Scientists suggest that all the genes encoding different types of CYP450 descended from a single precursor gene that existed two billion years ago. The function of this “original” gene was to utilize energy. At the moment, more than 1000 different types of cytochrome CYP 450 have been found in nature.

Diversity of cytochromes

To date, about 55 different types of cytochromes have been discovered in mammals, and more than 100 in plants.

Thanks to the successes of genetic engineering, it was possible to establish that enzymes of the cytochrome family perform various functions, which leads to their division into three main classes:

• involved in the metabolism of drugs and xenobiotics;
• involved in the synthesis of steroids;
• participating in other important endogenous processes occurring in the body.

Classification of cytochromes

All cytochromes and genes encoding their synthesis are named in accordance with the following recommendations:

• the name of the cytochrome must include the root CYP;
• the name of the gene encoding the synthesis of the corresponding cytochrome is also present CYP , but written in italics;
• cytochromes are divided into families (indicated by numbers), subfamilies (indicated by letters) and isoforms (indicated by numbers reflecting the number of the coding gene).

For example, CYP 2 D 6 belongs to the 2nd family, subfamily D, encoded by gene 6. The name of the gene itself looks like CYP 2 D 6.

Basic cytochromes

Despite the diversity of cytochromes in the human body, drug metabolism occurs with the participation of a predominantly limited amount of CYP 450. The most common representatives of this group are: CYP 1A2, CYP 2C9, CYP 2C19, CYP 2 D 6, CYP 2E1, CYP 3A4.

These enzymes catalyze a wide range of metabolic reactions:

• one cytochrome can metabolize several drugs with different chemical structures;
• the same drug can be affected by different CYP 450 in different organs and systems of the human body.

Duality of the nature of cytochromes P450

In most cases, fat-soluble drugs and other chemical substances are transformed into water-soluble metabolites, which are more easily eliminated from the body. The introduction of hydroxyl groups (thanks to cytochrome P450) increases the polarity of molecules and their solubility, which also contributes to their removal from the body. Almost all xenobiotics entering the liver are oxidized by some isoform of cytochrome p450.

However, the same enzymes that catalyze the “purification” processes can activate inert chemical molecules to a highly reactive state. Such intermediary molecules can interact with proteins and DNA.

Thus, the effect of cytochrome p450s can occur through one of two competitive pathways: metabolic detoxification or activation.

Variability of the action of cytochromes

Each person has his own metabolism of medicinal substances, different from that of other people. Individual characteristics depend on genetic factors, the patient’s age, gender, health status, diet, concomitant pharmacotherapy, etc.

Genetic variability in drug metabolism was discovered by chance: standard doses of drugs unexpectedly caused unusual reactions in different individuals.

Enzyme activity is of two (sometimes three) main types: intense and weak (medium), respectively, the metabolism of medicinal substances can occur quickly and slowly.

Cytochromes and drug metabolism

Cytochrome CYP 1A2 is involved in the metabolism of many drugs, including aminophylline and caffeine. The activity of this enzyme increases under the influence of chemicals that enter the human body during smoking.

Cytochrome CYP 2A6 plays an important role in the metabolism of coumarin (an indirect anticoagulant) and nicotine.

Cytochrome CYP 2S9 involved in the metabolism of phenytoin, tolbutamide, warfarin. If at least one amino acid changes in the structure of the gene encoding the synthesis of this cytochrome, then its enzymatic activity is disrupted. Enzyme deficiency of this cytochrome causes a congenital predisposition to phenytoin intoxication and complications resulting from warfarin therapy.

Cytochrome CYP 2S19 participates in the metabolism of omeprazole, diazepam, imipramine. However, the clinical significance of this enzyme polymorphism remains controversial. The effective doses of many drugs metabolized by CYP 2C9 are so far from toxic that potential deviations in the activity of cytochrome CYP 2C9 do not play a significant role.

Cytochrome CYP 2 D 6 is an example of genotypic differences among different ethnic groups. In the 70s of the last century, the pharmacokinetics of the antihypertensive drug debrisoquine and the antiarrhythmic drug sparteine ​​were studied. The following results were obtained: with a general tendency towards ultra-fast metabolism of debrisoquine, among Caucasians, slow metabolism was observed in 5–10% of cases, among Japanese this figure was less than 1%.

Drugs metabolized by CYP2D6 (b-blockers, antiarrhythmics, psychoanaleptics, antidepressants and narcotic analgesics) have a narrow therapeutic index, i.e. There is little difference between the dose required to achieve a therapeutic effect and the toxic dose. In such a situation, individual deviations in the metabolism of drugs can play a dramatic role: an increase in the concentration of the drug to a toxic level, or a decrease to the point of loss of effectiveness.

The history of the use of perhexiline (Australia) clearly demonstrated the enormous significance of the CYP2D6 polymorphism. After the first experience of prescriptions, the drug was withdrawn from the arsenal of drugs for the treatment of angina pectoris due to high hepato- and nephrotoxicity. But currently, perhexiline is being used again and is recognized as a highly effective agent, since it is toxic only for patients with poor CYP2D6 metabolism. The safety of prescribing perhexiline is ensured by preliminary determination of the individual level of this cytochrome.

Cytochrome CYP 3A4 supposedly metabolizes about 60% of all drugs. This is the main cytochrome of the liver and intestines (it makes up 60% of the total number of cytochromes). Its activity may increase under the influence of rifampicin, phenobarbital, macrolides and steroids.

Inhibition of drug metabolism

Inhibition of drug metabolism is the most common cause of clinically significant drug interactions, resulting in undesirable increases in drug concentrations in the blood. This most often occurs when two different drugs compete to be bound to the same enzyme. A drug that “loses” in this competitive “struggle” loses the ability to be adequately metabolized and accumulates excessively in the body. It is gratifying that there are not many drugs that have the characteristics of a pronounced inhibitor. Typical inhibitors are cimetidine, erythromycin, ketoconazole and quinidine. Among newer drugs, selective serotonin reuptake inhibitors and protease inhibitors have potential inhibitory properties.

The rate of inhibition depends on the pharmacokinetic properties of the “conflicting” drugs. If both the inhibitor and the substrate drug have a short half-life (for example, cimetidine and the inhibitor of its metabolism, theophylline), the interaction will be maximum on days 2-4. The same amount of time will be required for the interaction effect to cease.

In the case of simultaneous use of warfarin and amiodarone, it will take 1 month or more to stop the inhibitory effect, which is associated with the long half-life of the latter.

Despite the fact that inhibition of cytochrome-mediated metabolism is a big problem, in clinical practice conditions are sometimes created that allow targeted use of this phenomenon. The antiviral drug saquinavir has very low bioavailability, which is associated with its extensive metabolism by cytochrome CYP 3A4. The bioavailability of the drug when taken orally is only 4%. Coadministration of the related drug ritinavir, which inhibits cytochrome activity, results in a 50-fold increase in plasma concentrations of saquinavir, which allows for a therapeutic effect.

Induction of drug metabolism

Induction of metabolism occurs when a drug stimulates the synthesis of enzymes involved in the metabolism of another drug (or reduces the natural breakdown of these enzymes).

The most well-known cytochrome inducer is rifampicin, which increases the levels of CYP 3A4 and CYP 2C in the liver, resulting in intensified metabolism of a number of drugs (table).

It is quite reasonable to assume that cytochrome inducers reduce the effectiveness of drug substrates. However, there is another side to this phenomenon. Abrupt discontinuation of an inducer drug (or cessation of environmental exposure to an inducer) may unexpectedly result in a large increase in plasma concentrations of a drug that was previously extensively metabolized. An example is a situation where smokers, accustomed to constantly drinking coffee, suddenly decide to quit smoking, as a result of which the activity of CYP 1A2 decreases and the concentration of caffeine in the blood plasma increases. This can aggravate the severity of withdrawal symptoms: headache and agitation.

Interaction of cytochromes with food

A 1991 study found that one glass of grapefruit juice caused a threefold increase in felodipine plasma levels. However, other juices did not cause a similar effect. It is assumed that the components of grapefruit - flavonoids or furanocoumarin - suppress the metabolism of felodepine in the intestine, mediated by cytochrome CYP 3A4.

Pharmacogenomics and its promising directions

The science that studies the body's genetically determined response to drugs has recently come to be called pharmacogenomics. The development of this science will make it possible to accurately predict the body's individual response to a specific treatment, as well as identify patients at high risk of developing toxic reactions.

Table. The main types of cytochrome p450 in humans

Cytochrome	Substrates that are affected	Inhibitor	Inductor
	Amitriptyline, caffeine, clomipramine, imipramine, clozapine, mexiletine, estradiol, paracetamol, propranolol, tacrine, theophylline, R-warfarin	Cimetidine, fluvoxamine, fluoroquinolone antibiotics (ciprofloxacin, norfloxacin), grapefruit juice	Omeprazole, phenobarbital, phenytoin, polycyclic aromatic bicarbonates (eg kebab), cigarette smoking
	Diclofenac, indomethacin, losartan, naproxen, phenytoin, piroxicam, tolbutamide, S-warfarin	Amiodarone, chloramphenicol, cimetidine, fluconazole, fluoxetine, isoniazid, omeprazole, sertraline, sulfinpyrazone	Rifampicin
	Clomipramine, clozapine, diazepam, imipramine, lansoprazole, omeprazole, phenytoin, propranolol	Fluoxetine, fluvoxamine, isoniazid, omeprazole, sertraline	Rifampicin
	Amitriptyline, chlorpromazine, clomipramine, clozapine, codeine, desipramine, dextromethorphan, doxepin, fluoxetine, haloperidol, imipramine, labetalol, methadone, metoprolol, procainamide, promethazine, propafenone, propranolol, thioridazine, timolol	Amiodarone, cimetidine, haloperidol, mibefradil, quinidine, propafenone, all serotonin reuptake inhibitors
	Caffeine, ethanol, paracetamol, theophylline	Cimetidine, disulfiram	Ethanol, isoniazid
	Amiodarone, amitriptyline, atorvastatin, buprenorphine, carbamazepine, clarithromycin, clomipramine, clonazepam, cocaine, cortisol, cyclophosphamide, cyclosporine, dexamethasone, digitoxin, diltiazem, diazepam, doxorubicin, erythromycin, felodipine, fentanyl, imipramine, ketoconazole, , miconazole, midazolam, nifedipine, estradiol, omeprazole, propafenone, quinidine, simvastatin, theophylline, verapamil, vincristine, warfarin	Amiodarone, cannabinoids, cimetidine, clarithromycin, clotrimazole, diltiazem, erythromycin, grapefruit juice, ketoconazole, metronidazole, miconazole	Carbamazepine, glucocorticoids, phenytoin, rifampicin, sulfadimidine

Microsomal oxidation is a sequence of reactions involving oxygenases And NADPH, leading to the introduction of an oxygen atom into the composition of a non-polar molecule and the appearance of its hydrophilicity and increases its reactivity.

Reactions microsomal oxidation carried out by several enzymes located on the membranes of the endoplasmic reticulum (in the case in vitro they are called microsomal membranes). Enzymes organize short chains that end with cytochrome P 450.

Microsomal oxidation reactions include to phase 1 reactions and are intended to impart polar properties to a hydrophobic molecule and/or to increase its hydrophilicity, enhancing the reactivity of molecules to participate in phase 2 reactions. In oxidation reactions, the formation or release of hydroxyl, carboxyl, thiol and amino groups occurs, which are hydrophilic.

Microsomal oxidation enzymes are located in the smooth endoplasmic reticulum and are mixed function oxidases(monooxygenases).

Cytochrome P450

The main protein of microsomal oxidation is hemoprotein - cytochrome P 450. In nature, there are up to 150 isoforms of this protein, which oxidize about 3000 different substrates. The ratio of different cytochrome P450 isoforms varies due to genetic characteristics. It is believed that some isoforms are involved in the biotransformation of xenobiotics, while others metabolize endogenous compounds (steroid hormones, prostaglandins, fatty acids, etc.).

Cytochrome P450 interacts with molecular oxygen and includes one oxygen atom in the substrate molecule, contributing to the appearance (increasing) of its hydrophilicity, and the other - in the water molecule. Its main reactions are:

• oxidative dealkylation, accompanied by the oxidation of the alkyl group (at the N, O or S atoms) to the aldehyde and its elimination,
• oxidation (hydroxylation) of non-polar compounds with aliphatic or aromatic rings,
• oxidation of alcohols to the corresponding aldehydes.

The work of cytochrome P 450 is ensured by two enzymes:

• NADH-cytochrome b 5 oxidoreductase, contains FAD,
• NADPH-cytochrome P 450 oxidoreductase, contains FMN And FAD.

Scheme of the relative positions of microsomal oxidation enzymes and their functions

Both oxidoreductases receive electrons from the corresponding reduced equivalents and transfer them to cytochrome P 450. This protein, having previously attached a molecule of the reduced substrate, binds to an oxygen molecule. Having received another electron, cytochrome P 450 incorporates the first oxygen atom into the hydrophobic substrate (substrate oxidation). At the same time, the reduction of the second oxygen atom to water occurs.

Sequence of reactions of hydroxylation of substrates with the participation of cytochrome P450

An essential feature of microsomal oxidation is the ability to induce or inhibit, i.e. to a change in process power.

Inducers are substances that activate the synthesis of cytochrome P 450 and the transcription of the corresponding mRNA. They are

1. Wide spectrum actions that have the ability to stimulate the synthesis of cytochrome P 450, NADPH-cytochrome P 450 oxidoreductase and glucuronyl transferase. The classic representatives are barbituric acid derivatives - barbiturates, This group also includes diazepam, carbamazepine, rifampicin and etc.

2. Narrow spectrum and actions, i.e. stimulate one of the forms of cytochrome P 450 - aromatic polycyclic hydrocarbons ( methylcholanthrene, spironolactone), ethanol.

For example, ethanol stimulates the synthesis of the P 450 2E1 isoform (alcohol oxidase), which is involved in the metabolism of ethanol, nitrosamines, paracetamol, etc.
Glucocorticoids induce the P 450 3A isoform.

Inhibitors of microsomal oxidation bind to the protein part of cytochrome or heme iron. They are divided into:

1. Reversible

• directactions- carbon monoxide ( CO), antioxidants,
• indirectactions, i.e. influence through intermediate products of their metabolism, which form complexes with cytochrome P 450 - erythromycin.

2. Irreversible inhibitors – allopurinol, aminazine, progesterone, oral contraceptives, teturam, fluorouracil,

Evaluation of phase 1 reactions

Microsomal oxidation can be assessed in the following ways:

• determination of microsomal enzyme activity after biopsy,
• on the pharmacokinetics of drugs,
• using metabolic markers ( antipyrine test).

Antipyrine test

The subject takes it in the morning on an empty stomach amidopyrine at the rate of 6 mg/kg body weight. 4 portions of urine are collected at intervals from 1 to 6 hours, 6-12, 12-24 and 45-48 hours, respectively. The volume of urine is measured. No later than 24 hours later, the urine is centrifuged or filtered. Next, the concentration of 4-aminoantipyrine and its metabolite N-acetyl-4-aminoantipyrine in urine is examined.

Cytochromes P 450 (EC 1. 14. 1) are a family of heme-containing monooxygenases that metabolize xenobiotics, including drugs. Localized in the smooth endoplasmic reticulum of the cell, open - D. Garfinkel, M. Klingenberg, 1958.

Cytochrome P 450 (English Cytochrome P 450, CYP) The name indicates that it is colored (P - from English Pigment). Cytochrome P 450, bound to carbon monoxide, has a light absorption maximum at a wavelength of 450 nm, which determined its name (T. Omura and R. Sato in 1964). CO has nothing to do with the function of P 450. It is added to facilitate the determination of P 450 content by the intensity of the absorption spectrum.

The use of the term “cytochrome” in relation to hemoproteins of the P 450 class cannot be considered successful, since the function of cytochromes is electron transfer, and not catalysis of monooxygenase reactions. Cytochrome P-450 belongs to type b cytochromes. The precursor of heme is protoporphyrin IX.

Molecular weight of various cit. R 450 ranges from 44 to 60 k. Yes. Hemoprotein monomers consist of one polypeptide chain containing from 45 to 55% non-polar amino acid residues. The complete amino acid sequence has been established for more than 150 cit. P 450. Using X-ray crystallography, the three-dimensional structure of cit. P 450 from P. putida. The protein contains 414 amino acid residues, M. m. - 47 k. Yes, it is an asymmetric prism with a base of 3.0 nm and sides of 5.5 and 6.0 nm.

Quote P 450 from P. putida contains 4 antiparallel helical regions, a mixture of helices and disordered structures interspersed with parallel beta structures. The heme is located between two parallel helices. The residues Arg-112, Arg-229 and His-335 interact with the propionic groups of heme. The other amino acids surrounding heme are non-polar. Heme does not reach the surface of the molecule. The shortest distance from the surface to the heme is about 0.8 nm.

All membrane cytochromes P 450 on the N-terminal fragment of the peptide chain have a short hydrophobic region containing from 12 to 21 amino acid residues. It acts as an anchor peptide and contains a signal sequence responsible for the insertion of the protein into the membrane. Behind it is a stop signal sequence that stops the incorporation of the peptide into the phospholipid bilayer.

Cytochromes P 450 are absent only in obligate anaerobic organisms. At least 11,500 described? proteins of the system Cyt. P 450 bacteria and archaea are dissolved in the cytoplasm. The transition to eukaryotic systems was accompanied by the incorporation of P450 into the membrane. All cit. P 450 of higher organisms are membrane enzymes. In evolutionary terms, bacterial monooxygenase is the most ancient.

System cit. P 450 is involved in the oxidation of numerous compounds, both endogenous and exogenous. Quote P450-dependent monooxygenases catalyze the breakdown of various substances with the participation of an electron donor and molecular oxygen. In this reaction, one oxygen atom is added to the substrate and the second is reduced to water. The oxygen binding center is highly specific, the binding center of the converted substrate is relative.

The enzymes of the cytochrome P 450 family are diverse and differ: in functions, types of enzymatic activity, activity regulators (inhibitors, inducers). Individual isoforms (isoenzymes) cyt. P-450s have a certain specificity and each of them is involved in the metabolism of a relatively small number of substances.

Cytochrome P 450, along with monooxygenase activity, can exhibit oxidase activity (A.I. Archakov et al.), i.e., catalyze the removal of hydrogen from the substrate, using oxygen as a hydrogen acceptor and reducing it to water, or generating active forms of oxygen in the form of superoxide and hydroxyl radicals, hydrogen peroxide. P 450 exhibits peroxidase activity using organic peroxides or hydrogen peroxide as cosubstrates in oxidation reactions instead of NADPH. There is evidence that P 450 can catalyze dioxygenase reactions and introduce two oxygen atoms into the oxidized substance. Thus, a characteristic feature of P 450 is its multiplicity of functions, but the main one is monooxygenase.

Quote P-450s are encoded by a superfamily of genes. In a person's system cit. P-450 identified 57 genes and more than 59 pseudogenes (non-functional analogues of structural genes that have lost the ability to encode protein and are not expressed in the cell. The term “pseudogene” was first proposed in 1977). Nebert (1987) developed a classification cit. P-450, based on divergent evolution and homology of nucleotide/amino acid sequences. The superfamily is divided into 18 families and 43 subfamilies. Gene nomenclature cit. The human P-450 is described in detail.

Currently, thousands of isoforms (isoenzymes) of cyt are known. R-450. Isoforms with more than 40% of the total amino acid composition are grouped into families and are designated by Arabic numerals (CYP 1, CYP 2, CYP 3, etc.). Subfamilies are designated by Latin letters and combine isoforms with amino acid composition identity of more than 55% (CYP 2 D, CYP 3 A, etc.) In the subfamily, individual isoforms are designated by Arabic numerals following the Latin letters (CYP 1 A 2, CYP 2 D 6 , CYP 3 A 4). A xenobiotic can be a substrate of two or more isoforms. Different isoforms are able to metabolize one substance in different parts of its molecule

Quote P 450 catalyze the ω-oxidation of saturated fatty acids (FA), peroxidation of unsaturated fatty acids. k., hydroxylation of steroid hormones, bile acids and cholesterol, biosynthesis of prostaglandins (localized in mitochondria, on the nuclear membrane). Cytochromes P 450 microsomes are involved in the metabolic biotransformation of xenobiotics (medicines, poisons, narcotic substances). Isoforms of families I, II and III take part in drug metabolism, of which the main ones are IA 1, 1 A 2, 2 A 6, 2 B 6, 2 D 6, 2 C 9, 2 C 19, 2 E 1, 3A 4 .

Common inducers Enzymes Phenobarbitol inducer Heavy metals l Cytochrome P 450 system Inducer Antitumor drugs Methylcholanthrene Cytochrome P 448 system Epoxide hydrolases Glutathione and UDPglucuronyltransferases GSH synthesis Metallothioneins P-glycoprotein Inducer

Phenobarbital activates the synthesis of cyt. P 450, UDPglucuronyltransferase and epoxide hydrolase. For example, in animals that were administered the inducer phenobarbital, the area of ​​the ER membranes increases, which reaches 90% of all membrane structures of the cell, and, as a result, an increase in the number of enzymes involved in the neutralization of xenobiotics or toxic substances of endogenous origin. Simultaneous use of phenobarbital and certain drugs that metabolize with the participation of cit. P 450, leads to a decrease in the effectiveness of the latter due to the transformation of the molecule in the process of biotransformation or their rapid removal from the body.

Currently, more than 250 chemical compounds that cause the induction of microsomal enzymes have been described. Inducers of monooxygenase systems are divided into two classes. Representatives of the first class (insecticides, ethanol, etc.) cause pronounced proliferation of the smooth endoplasmic reticulum in hepatocytes and an increase in the activity of cytochrome P 450. Stimulation of metabolism caused by inducers of the second class (PAHs - polycyclic aromatic hydrocarbons: tetrachlorodibenzodioxin, 3-methylcholanthrene, benz(a) pyrene, etc., is not accompanied by proliferation of the smooth endoplasmic reticulum, but the activity of many biotransformation enzymes increases significantly. Increased metabolism of most xenobiotics leads to a decrease in toxicity. At the same time, the toxicity of some xenobiotics increases significantly under the influence of inducers. For example, the toxicity of carbon tetrachloride increases. bromobenzene, mustard gas, etc.

There are chemicals that can inhibit both enzymes of the 1st phase of biotransformation (cytochrome P-450 isoenzymes) and 2nd phase of biotransformation (N-acetyltransferase, etc.), as well as 3rd phase transporters (PATPases). With a decrease in the activity of metabolic enzymes, the development of side effects associated with prolonged circulation of these compounds in the body is possible. Inhibition of transporters, as well as their induction, can lead to various changes (mainly an increase) in the concentration of xenobiotics in the blood plasma, depending on the functions of a given transporter.

Electron transport chains ER 1 chain includes: 1) cytochrome P 450, has binding centers for O 2 and a hydrophobic substrate; 2) NADPH-cytochrome P 450 reductase containing coenzymes FAD and FMN; 3) NADPH+H+ is a donor of ē and H+ in this electron transport chain; 4) O 2. 2 chain includes: 1) cytochrome P 450; 2) the enzyme NADH-cytochrome b 5 reductase, the coenzyme of which is FAD; 3) cytochrome b 5 – hemoprotein that transfers ē from NADH-cytochrome b 5 reductase to cytochrome P 450; 4) NADH + H+ – donor of ē and H+; 5) O 2. Cytochrome P 450 includes one O 2 atom in the substrate molecule, and the 2nd is reduced to form H 2 O due to the transfer of ē and H+ from NADPH+H+ with the participation of cytochrome P 450 reductase (or from NADH+ H+ via cytochrome b 5 reductase and cytochrome b 5).

Another diagram of the organization of the electron transport chain of the ER. The source of electrons and protons in the chain is NADPH + H +, which is formed in the reactions of the pentose phosphate pathway of glucose oxidation. The intermediate acceptor of H+ and e- is a flavoprotein containing the coenzyme FAD (cytochrome P 450 reductase). The final link in the microsomal oxidation chain is cytochrome P 450, which reduces oxygen to water. Operation of the system cit. R-450 is associated with the operation of the cit. b-5, the source of electrons and protons in which is NADH + H +, formed in glycolysis. The intermediate acceptor of H+ and e- is a flavoprotein containing the coenzyme FAD (cytochrome b-5 reductase).

Example RH – substrate cyt. R-450; arrows indicate electron transfer reactions. Restored form cit. -b 5 oxidizes stearoyl-Co. A-monooxygenase, which transfers electrons to O₂. One O₂ atom takes 2 e¯ and goes into the O²¯ form. The electron donor is NADPH, which is oxidized by NADPH-cyt. P-450 reductase. O²¯ interacts with protons to form O²¯ + 2 Н⁺ → Н₂О The second act of oxygen is included in the substrate RH, forming the hydroxyl group of the substance R-OH.

NADP-H-cytochrome P-450 reductase – flavoprotein. One mole of the enzyme contains one mole each of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Since cytochrome C can serve as an electron acceptor (used in model systems), this enzyme is often called NADP-cytochrome reductase.

The mechanism of substrate hydroxylation with the participation of cytochrome P-450 Conventionally, 5 stages can be distinguished: 1. the oxidized substance (S) forms a complex with the oxidized form of cytochrome P-450; 2. this complex is restored by an electron from NADPH; 3. the reduced complex combines with an O2 molecule; 4. O 2 as part of the complex attaches another electron to NADPH; 5. the complex decomposes to form a H2O molecule, an oxidized form of cytochrome P-450 and a hydroxylated substrate (S-OH).

Unlike the mitochondrial respiratory chain, the monooxygenase chain does not accumulate energy in the form of ATP during electron transfer. Microsomal oxidation is free oxidation. In most cases, hydroxylation of foreign substances reduces their toxicity. However, products with cytotoxic, mutagenic and carcinogenic properties may be formed.

Cytochromes P-450 are membrane proteins and the study of their catalytic activity requires a complex reconstruction of the monooxygenase system using redox partners and phospholipids. In addition, isoenzymes cyt. P-450s are quickly inactivated. The electrochemical method of analysis has significantly simplified the determination of cyt activity. R-450. The first works devoted to the use of an electrode as an electron donor for catalysis of cyt. R-450 (CYP 3 A 4): Kuznetsov B. A., Mestechkina N. M., Izotov M. V., Karuzina I. I., Karyakin A. V., Archakov A. I. (1979) Biochemistry, 44, 1569 -1574. Archakov A. I., Kuznetsov B. A., Izotov M. V., Karuzina I. I. (1981) Biophysics, 26, 352 -354.

Currently, electrochemical biosensor systems based on cytochromes P-450 immobilized on an electrode have been developed. The electrocatalytic reaction is initiated by electrons from the electrode. This eliminates the need to use redox partners of the monooxygenase system and reducing equivalents of NADPH. Determination of the catalytic activity of immobilized cyt. R-450 is carried out by recording the catalytic current that occurs when a substrate is introduced into the electrochemical system. Registration of the catalytic current is carried out using voltammetry or amperometry methods and allows one to calculate many characteristics of the enzymatic process: the Michaelis-Menten constant, the electrochemical catalytic constant.

Dependence of changes in catalytic current during ampermetric titration cit. P-450 3 A 4 (CYP 3 A 4) with testosterone at a controlled voltage E = -0.5 V (vs. Ag/Ag. Cl). The inset shows the calculation of electrochemical Km [testosterone]. (V.V. Shumyantseva et al., 2015)

The development of biosensors based on electrochemical cytochrome P 450-containing systems makes it possible to identify substrates (xenobiotics), study the effects of drugs on the catalytic activity of specific isoforms of cytochrome P 450. The goal is to create sensor devices suitable for use in personalized medicine, to conduct experiments to study the effect of drugs drugs for the activity of CYP in electrode/cytochrome P 450 systems.

Advantages of the electrochemical method for studying the cytochrome P 450 monooxygenase system 1) the electrochemical system does not require the use of expensive and unstable reducing equivalents of NADPH and NADH, since an alternative source of electrons is used - an electrode; 2) does not require complete reconstruction of the monooxygenase system (use of all components of the microsomal system and redox partner proteins of the catalytic cycle of cytochrome P 450); 3) the method is highly sensitive and allows the use of a minimal amount of expensive enzyme (up to 10 -12 µmol protein/electrode); 4) electrocatalysis and controllability of the enzymatic process using electric current is highly effective; 5) it is possible to prevent the inactivation of intact P450 isoforms by using various synthetic electrode surface modifiers.

Methods for assessing the state of the xenobiotic biotransformation system: 1) High-performance liquid chromatography (HPLC). Makes it possible to study analytes in urine, blood, saliva, and other biological material after administration of a xenobiotic (medicinal substance). Kinetic analysis can be performed to determine the half-life of the test drug, apparent volume of distribution, elimination clearance, and other parameters. An analyte is a component or characteristic of a sample to be measured. This concept includes any element: ion, compound, substance, factor, infectious agent, cell, organelle, activity (enzymatic, hormonal, immunological) or sign: presence or absence, concentration, activity, intensity or other characteristics that need to be determined . The concept was formulated by the US National Committee for Clinical Laboratory Standards (NCCLS, document NRSCL 8 -A). Close to the terms we use “laboratory indicator”, “parameter”, “test”, etc.

2) PCR-RFLP analysis of polymorphism and mutant forms of cyt genes. R-450. W; Results of restriction fragment length polymorphism analysis (RFLP analysis) of the CYP 1 A 2 gene: 1 – molecular weight marker; 2, 4, 6, 7, 8, 10 and 11 – mutant genotype – M (mutation in the restriction site – restriction does not occur); 3 – no mutation – wild type genotype – 5 and 9 – heterozygous genotype – all fragments are present – ​​H; 12 – negative control.

3) DNA chips Allow you to simultaneously determine a very large number of polymorphisms in one sample. A large number of oligonucleotide probes are placed in the form of individual spots on a very small solid chip, each of which provides specific hybridization with normal and mutant alleles of many different genes. Before hybridization, nonspecific fluorescent labeling of the DNA under study is performed. If the sample DNA binds to the probe on the chip, a fluorescent signal from the corresponding section of the chip is detected [Ivanov, Tereshin, Shcherbak, 2010]. Knowing which allele is responsible for the synthesis of one or another cyt isoenzyme. P-450, it is possible to determine which xenobiotics will be biotransformed and in what way.

4) Computer programs for modeling the interaction of ligands with cytochromes P 450 To study the interaction of the substrate and the enzyme, molecular docking and molecular dynamics methods are used. Molecular docking (or molecular docking) is a molecular modeling method that allows one to predict the orientation and position of one molecule relative to another that is most favorable for the formation of a stable complex. Using scoring functions, the most energetically favorable conformations of the ligand in the active center are determined. Molecular dynamics is a method in which the time evolution of a system of interacting atoms or particles is tracked by integrating their equations of motion. Plays an important role (along with crystallography and NMR) in determining the structure of a protein and clarifying its properties. The most popular software packages for modeling the dynamics of biological molecules are: AMBER, CHARMM (and the commercial version CHARMMm), GROMACS, GROMOS, Lammhs, HOOMD-blue, NAMD.

When describing the interaction cit. P-450 and ligands, the role of spatial and energy factors is assessed, since the contribution of these factors for various cit. The R 450 is different. For a comprehensive description of the interaction of low molecular weight ligand and cyt. P-450 in silico and predictions of possible biotransformations must take into account: § the reactivity of the enzyme, § the structure of the active center of the enzyme, § the location and conformation of the ligand in the active center of the enzyme, § the possibility of multiple ways of binding the substrate in the active center of the enzyme (binding can occur indirectly through molecules water), § regiospecific reactivity inherent in the substrate itself (it can vary depending on the conformation adopted by the substrate), § the affinity of the product that must be released from the active site of the enzyme.

R-tion called regiospecific, if as a unit. product (within the limits of error) one of two or more possible regio-isomers is formed. Regioisomers are isomers formed as a result of the transformation of one of several possible reaction centers present in the substrate molecule. If one isomer only predominates in the products of a solution, such a solution is called. regioselective. Eg. , the addition of unsymmetrical electrophilic HBr to unsymmetrical styrene C 6 H 5 CH=CH 2 occurs regiospecifically: only one of two possible addition products is formed - C 6 H 5 CHBr. CH 3, but not C 6 H 5 CH 2 Br.

QSAR models. Quantitative structure-activity relationships (QSAR - English abbreviation for Quantitative Structure-Activity Relationships) make it possible to predict their properties by describing the structure of chemical compounds, including establishing the interaction with cytochromes of low-molecular compounds and their biotransformation. For example: 1) to classify the substrates of various cytochromes, the following are used: the support vector machine method, the K-nearest neighbors method, the decision tree method, etc. 2) to assess the interaction of ligands (substrates and inhibitors) with the active center of the cytochrome, three-dimensional QSAR is used (3 -D QSAR) methods.

Superfamily cit. P-450 catalyzes a large number of reactions that occur via different mechanisms, so classical QSAR methods cannot be applied correctly for substances belonging to different classes. For each individual cytochrome that metabolizes a xenobiotic, it is necessary to build a special QSAR model using different descriptors and different mathematical methods.

The main cytochromes P 450, responsible for the metabolism of drugs in the human body, studied in silico, are the cytochrome subfamily. P-450 3 A. Cytochrome P 450 3A 4 is a membrane-bound protein located in the endoplasmic reticulum. Molecular weight 57299 D, the primary structure contains 502 amino acid residues. The CYP 3 A 4 gene is located on the long arm of the seventh chromosome (7 q 22. 1). The ZA subfamily is the most expressed in the liver and intestine. Approximately 2/3 of liver cytochromes belong to this subfamily. Two cit. R 450 ZA 4 and 3 A 5 are described in detail in the literature. Quote P 450 ZA 5 is more common in adolescents, polymorphically expressed and not induced by glucocorticoids, in contrast to ZA 4. There is another embryonically expressed isoform - ZA 7 (accounts for 50% of fetal cytochromes P 450).

Cytochrome b 5 is a hemoprotein that participates in a variety of biochemical redox reactions as an electron carrier. The microsomal cytochrome b 5 molecule consists of two domains - hydrophilic and hydrophobic. The hydrophilic N-terminal region is located on the surface of the ER membrane, is formed by amino acid residues from 1 to 88, and contains heme, which is part of the active center. Schematic representation of the location of the cytochrome b 5 molecule in the membrane.

The hydrophobic domain of cytochrome b 5 is anchored in the lipid bilayer (ER or mitochondrial), spiralized, formed by amino acid residues at the C-terminal end of the protein molecule (amino acid residues 89-133). Using computer modeling, it was shown that the C-terminal region of the cytochrome b 5 molecule forms a loop and penetrates the lipid membrane through. The greatest hydrophobicity is observed in the middle part of the loop, which is immersed in the membrane. The C-terminal part of the enzyme molecule plays an important role in insertion into the membrane, orientation of the enzyme in the lipid bilayer, and ensuring functional activity.

Cytochrome b 5 of the outer membrane of mitochondria, compared to microsomal, has a lower redox potential, the molecule is more resistant to chemical and thermal denaturation, the bond between apoprotein and heme is much stronger. Two hydrophobic regions have been identified in the cytochrome b 5 molecule of mitochondria. The first is formed by the residues of alanine-18, isoleucine-32, leucine-36 and leucine-47. The second is isoleucine 25, phenylalanine-58, leucine-71 and heme. Using mutant forms of the molecule, it was shown that both hydrophobic regions are of great importance in maintaining the stability of the molecule. In the absence or replacement of amino acid residues in them, the interaction of apoprotein with heme is reduced.

The role of cytochrome b 5 in reactions catalyzed by isoforms of the cytochrome P-450 system. Possible mechanisms of stimulating influence cit. b 5 for isoforms cyt. P-450: direct electron transfer in a monooxygenase reaction, without the mediation of NADP cytochrome P-450 reductase; in the case of using the second electron from cytochrome b 5 in the monooxygenase cycle, the formation of more active oxygen radicals occurs, which, in turn, is accompanied by a more rapid formation of the metabolite;

cit. b 5 interacts with cit. P-450 with the formation of a complex of two hemoproteins and the subsequent transfer of two electrons from NADPH to cytochrome P-450 reductase. This increases the rate of active oxygen formation and eliminates the need for repeated cyt interaction. P-450 and NADPH cytochrome P-450 reductase; cit. b 5 can carry out allosteric stimulation of cyt. P-450 without electron transfer, for example in the second stage of the catalytic cycle; cytochrome b 5 may have a protective effect on terminal oxygenase molecules, which is not associated with the reactions of the redox cycle, which prevents its destruction.

Impact cit. b 5 to change the reaction rate, the spectrum of metabolites and the formation of reactive oxygen species in the reactions of the cit system. R-450. in the presence of cit. b 5 the metabolic rate of most endogenous compounds and xenobiotics most often increases; influence cit. b 5 on the biotransformation of the same compound, for example androstenedione, varies in different animal species. In rabbits in the presence of cyt b 5, the rate of metabolism of the steroid cyt. P-450 2 B 5 increases, and in dogs - cit. R-450 2 V 11 is reduced;

cit. b 5 in different species (human and hamster) may not change the rate of oxidation of the compound (nitrosamine) or have a stimulating effect; presence of cit. b 5 changes the spectrum of metabolites formed during the biotransformation of a compound by the same cyt isoform. P-450, for example tetrachlorobiphenyl cit. R-450 2 V 1; in the presence of cit. b 5 the formation of reactive oxygen species is reduced, the overproduction of which has a negative effect on the vital activity of the body’s cells; metabolism of biologically active compounds (arachidonic acid, leukotrienes) occurs only in the presence of cit. b 5.

The influence of cytochrome b 5 on changes in the reaction rate, the spectrum of metabolites and the formation of reactive oxygen species in reactions involving various cytochrome isoforms. P-450 (fragment) Isoform of cytochrome P-450 Substrate Change in reaction rate P-450 1 A 1 tetrachlorobiphenyl P P-450 2 A 1 P-450 2 B 1 Androstenedione P - II - P P-450 2 B 2 2 -chlor -1, 1 difluoroethine tetrachlorobiphenyl (2, 2", 5, 5" - and 2, 3", 4", 5 -) tetrachlorobiphenyl (2, 3, 4, 4 -) 9 - anthraldehyde R-450 2 B 4 Tetranitromethane N - II - Aminopyrine P Androstenedione P Testosterone does not change Formation of reactive oxygen species ↓ methanol, 7-ethoxycoumarin 9 - anthraldehyde Change in the spectrum of metabolites P-450 1 A 2 - II - P-450 2 B 5 - II - ↓ P P changes ↓ changes P changes

NADH cytochrome b 5 reductase is a flavoprotein. This is a two-domain protein, the globular cytosolic domain binds FAD, the hydrophobic domain (single “tail”) anchors the protein in the membrane.

Cytochromes P450. Structure and function

Among phase 1 enzymes, the leading place is occupied by the cytochrome P450 (P450 or CYP) system in terms of catalytic activity towards a huge number of xenobiotics. The highest concentration of cytochrome P450 is found in the endoplasmic reticulum of hepatocytes (microsomes). Hepatic microsomal cytochromes P450 play a critical role in determining the intensity and time of action of foreign compounds and a key role in the detoxification of xenobiotics, as well as in their activation to toxic and/or carcinogenic metabolites. Cytochrome P450-dependent monooxygenases are a multienzyme electron transport system. All cytochromes P450 are heme-containing proteins. Heme iron is usually in an oxidized state (Fe3+). By being reduced to the Fe2+ state, cytochrome P450 is able to bind ligands such as oxygen or carbon monoxide. The complex of reduced cytochrome P450 with CO has an absorption maximum at 450 nm, which was the basis for

names of these enzymes. The main reaction catalyzed by cytochromes P450 is a monooxygenase reaction, in which one oxygen atom interacts with the substrate (RH) and the other is reduced to H2O. NADPH participates as a reducing agent in the reaction:

RH (substrate) + O2 + NADPH + H+ --> ROH (product) + H2O + NADP+

The mechanism by which cytochrome receives an electron from NADPH depends on the intracellular localization of cytochrome P450. In the ER, where most of the hemoproteins involved in the biotransformation of xenobiotics are located, the electron is transferred through a flavoprotein called NADPH-P450 reductase. One reductase molecule can deliver electrons to several different P450 molecules. In mitochondria, where P450 itochromes involved in the biosynthesis of steroid hormones and vitamin D metabolism are located, the electron is transferred using 2 proteins: ferrodoxin or ferrodoxin reductase.

In Fig. Figure 1 shows the catalytic cycle of cytochrome P450. The 1st part of the cycle involves the activation of oxygen, the 2nd – the oxidation of the substrate. The mechanism of action of the microsomal monooxygenase system was first described by Estabrook et al., and has now been confirmed by many researchers. This scheme is as follows: the first stage consists of the interaction of the substrate with the oxidized form of P450. When P450 binds to substrates

There is a transition of heme iron from a low-spin to a high-spin state. The second stage consists of the reduction of the resulting enzyme-substrate complex with the first electron, which comes from the NADPH-specific transfer chain from NADPH through

flavoprotein I (NADPH-cytochrome P450 reductase). The third stage consists of the formation of a ternary complex: reduced cytochrome P450-substrate-oxygen. Fourth stage

represents the reduction of a ternary complex by a second electron, which, as

believed to come from the NADH-specific electron transport chain, consisting of NADH-

cytochrome b5 reductase or flavoprotein II and cytochrome b5. The fifth stage consists of several processes, including intramolecular transformations of the reduced ternary complex and its decomposition with the formation of a hydroxylated product and water. At this stage, cytochrome P450 transforms into its original oxidized form.

Cytochromes P450 catalyze the following types of reactions: hydroxylation of an aliphatic or aromatic carbon atom; epoxidation of double bond;

oxidation of atom (S, N, I) or N-hydroxylation; transfer of oxidized group;

destruction of etheric communication; dehydrogenation. Some reactions catalyzed

cytochrome P450 are shown in Fig. 2 and 3. Several classes of reagents are good

The last carbon in the chain is hydroxylated, so-called omega-hydroxylation. So

internal hydroxylation occurs in several positions (positions -1,- 2).

This results in many different product variations even with a simple alkane such as hexane. Note that cyclic hydrocarbons also undergo hydroxylation. In the hydroxylation reaction, a hemiacetal is first formed, which is then converted into an alcohol and an aldehyde. When alkenes are oxidized by cytochrome P450, diatomic oxides are formed. They vary in their stability and can be highly reactive. For example, vinyl chloride is metabolically converted into an oxide, which then turns into chloroacetaldehyde, a mutagen that acts directly on DNA. These studies led to a ban on the use of vinyl chloride in nebulizers. The vinyl group of sterol (vinylbenzene) is known for its carcinogenic properties, but the human body is able to neutralize it by converting the oxide into a diol using the enzyme epoxyhydrolase. But epoxyhydrolase does not always help. For example, cytochrome P450 synthesizes Aflotoxin B1 epoxide in vivo. This compound is a highly reactive electrophile, is unstable and quickly forms an adduct with DNA. In addition, the diol formed from the epoxide is also unstable and highly reactive. Oxidation of aromatic compounds with cytochrome P450 also produces epoxides, but they quickly turn into phenol. As a result of hydroxylation of benzene, the resulting phenol can be hydroxylated again, turning into catechol or hydroquinone. Note that catechol and hydroquinone can react with oxygen, inhibiting similar reactions with quinones and superoxides, which are toxins. Such a well-known compound as 2,3,7,8-tetrachlorodibenzenedioxin (TCDD) is not susceptible to hydroxylation and is stable (half-life in the human body is a year or more).