Study Protein Kinetics To Understand Biological Mechanism Of Molecular Imprints Therapeutics

Homeopaths should learn to perceive LIFE in terms of interactions of complex biological molecules, DISEASE in terms of molecular errors in biochemical processes, SYMPTOMS as expressions of underlying pathological molecular errors, DRUGS in terms of constituent molecules, CURE in terms of removal of bio-molecular errors and POTENTIZED DRUGS in terms of constituent molecular imprints. Then only they can understand the scientific explanation of homeopathy as MOLECULAR IMPRINTS THERAPEUTICS.

Molecular mechanism of removal of pathological inhibitions using molecular imprints as therapeutic agents cannot be comprehended without a clear understanding of protein chemistry such as RECEPTOR KINETICS, ENZYME KINETICS,  ANTIBODY KINETICS and related topics of AGONISM, ANTAGONISM, INHIBITION and ACTIVATION of PROTEIN MOLECULES.

Binding of any ligand –natural ligands, drug molecules as well as pathogenic molecules- with complex biological molecules of PROTEIN nature such as receptor, enzyme or immune bodies are determined by conformational affinity between them, as well as chemical affinity or bond strength.

There exist small specific interactive groups on the periphery of protein molecules, known as binding sites, active sites and allosteric sites.

Binding sites are often an important component of the functional characterization of bio-molecules, especially of proteins. For example, the characterization of the binding site of a substrate to an enzyme is essential to model the reaction mechanism responsible for the chemical change from substrate to product. Binding sites of proteins exhibit conformational specificity, a measure of the types of ligands that will bond, and chemical affinity, which is a measure of the strength of the chemical bond. In biochemistry, a binding site is a region on a protein, DNA, or RNA to which specific other molecules and ions—in this context collectively called ligands—form a chemical bond. Binding sites also exist on antibodies as specifically coded regions that bind antigens based upon their structure.

Active site is the small portion of an enzyme or other protein molecules where ligand molecules bind and undergo a chemical reaction. This chemical reaction occurs when a ligand collides with and slots into the active site of a protein molecule. The active site is usually found in a 3-D groove or pocket of the protein 3D structure, lined with amino acid residues. These residues are involved in recognition of the ligands. Residues that directly participate in the reaction mechanism are called active site residues. After an active site has been involved in a reaction, it can be used again.

Usually, a protein molecule has only one active site, and the active site fits with one specific type of ligand. Proteins can be denatured by high temperatures or extreme pH values, meaning that the active site changes shape and does not fit its ligand molecules.  A tighter fit between an active site and the ligand molecule is believed to increase efficiency of a biochemical reaction.

Identification of active sites of biological molecules is crucial in the process of drug discovery. The 3-D structure of the enzyme is analyzed to identify active sites and design drugs which can fit into them. Proteolytic enzymes are targets for some drugs, such as protease inhibitors, which include drugs against AIDS and hypertension.  These protease inhibitors bind to an enzyme’s active site and block interaction with natural substrates. An important factor in drug design is the strength of binding between the active site and an enzyme inhibitor.

Active sites can be mapped to aid design of new drugs such as enzyme inhibitors or receptor blockers. This involves description of the size of an active site and the number and properties of sub-sites, such as details of the binding interaction. Modern database technology called CPASS (Comparison of Protein Active Site Structures) however allows us to compare active sites in more detail and to look at structural similarity using software.

An allosteric site is a site on a protein molecule, unrelated to its active site, which can bind an effector molecule. This interaction is another mechanism characteristic of protein regulation. Allosteric modification usually happens in proteins with more than one subunit. Allosteric interactions are often present in metabolic pathways and are beneficial in that they allow one step of a reaction to regulate another step. They allow an enzyme to have a range of molecular interactions, other than the highly specific active site.

In biochemistry, allosteric regulation is the regulation of an enzyme or other protein by binding an effector molecule at the protein’s allosteric site (that is, a site other than the protein’s active site). Effectors that enhance the protein’s activity are referred to as allosteric activators, whereas those that decrease the protein’s activity are called allosteric inhibitors. The term allostery comes from the fact that the regulatory site of an allosteric protein is physically distinct from its active site. Allosteric regulations are a natural example of control loops, such as feedback from downstream products or feedforward from upstream substrates. Long-range allostery is especially important in cell signaling. Co-factors such as vitamins and metal ions mostly act as allosteric effectors of proteins. Enzymes can use cofactors as ‘helper molecules’. Coenzymes are one example of cofactors. Coenzymes bind to the enzyme temporarily and are released after the reaction has occurred. Metal ions are another type of cofactor.

There are two proposed models of how proteins fit to their specific ligands: the lock and key model and the induced fit model. Emil Fischer’s lock and key model assumes that the active site is a perfect fit for a specific ligand and that once the ligand binds to the protein no further modification occurs. Daniel Koshland’s theory of enzyme-substrate binding is that the active site and the binding portion of the substrate are not exactly complementary. The induced fit model is a development of the lock-and-key model and assumes that an active site is flexible and it changes shape until the substrate is completely bound. The substrate is thought to induce a change in the shape of the active site. The hypothesis also predicts that the presence of certain residues (amino acids) in the active site will encourage the enzyme to locate the correct substrate. Conformational changes may then occur as the substrate is bound. After the products of the reaction move away from the enzyme, the active site returns to its initial shape.

Substrates bind to the active site of the enzyme through hydrogen bonds, hydrophobic interactions, temporary covalent interactions (van der Waals) or a combination of all of these to form the enzyme-substrate complex. Residues of the active site will act as donors or acceptors of protons or other groups on the substrate to facilitate the reaction. In other words, the active site modifies the reaction mechanism in order to change the activation energy of the reaction. An enzyme binding to a substrate will lower the energy barrier that normally stops the reaction from happening. The product is usually unstable in the active site due to steric hindrances that force it to be released and return the enzyme to its initial unbound state.

Inhibitors are chemical molecules of endogenous or exogenous origin that can  disrupt the interaction between enzyme and substrate- in broader sense, between ligands and proteins- slowing down the rate of a reaction. There are different types of inhibitor, including both reversible and irreversible forms. Reversible inhibitors can be competitive or non-competitive. Drugs and various other pathogenic molecules belong to the class of exogenous inhibitors. Metabolic byproducts, hormones and various biological molecules can act as endogenous inhibitors at off-target sites.

Competitive reversible inhibitors have a similar shape to the ligands and bind to the protein’s active site temporarily, blocking entry of the actual ligand into the active site.

Non-competitive reversible inhibitors bind to the enzyme however not in the active site. Despite not interacting with the active site, non-competitive inhibitors do reduce the rate of the reaction because they cause the protein molecules to change shape.

Irreversible inhibitors bind permanently to the protein, blocking access to active sites and therefore reducing the rate of the reaction. Heavy metal toxicity is an example of ‘irreversible’ inhibitions.

An protein inhibitor is a molecule, which binds to proteins and decreases their activity. Since blocking aprotein’s activity can kill a pathogen or correct a metabolic imbalance, many drugs are protein inhibitors. They are also used as herbicides and pesticides. Not all molecules that bind to proteins are inhibitors; there are enzyme activators that bind to enzymes and increase their enzymatic activity, while enzyme substrates bind and are converted to products in the normal catalytic cycle of the enzyme.

The binding of an inhibitor can stop a ligand from entering the protein’s active site and/or hinder the protein from executing its function. Inhibitor binding is either reversible or irreversible. Irreversible inhibitors usually react with the protein and change it chemically (e.g. via covalent bond formation). These inhibitors modify key amino acid residues needed for protein activity. In contrast, reversible inhibitors bind non-covalently and different types of inhibition are produced depending on whether these inhibitors bind to the protein, the protein-ligand complex, or both.

Many drug molecules are protein inhibitors, so their discovery and improvement is an active area of research in biochemistry and pharmacology. A medicinal protein inhibitor is often judged by its specificity (its lack of binding to other proteins) and its potency (its dissociation constant, which indicates the concentration needed to inhibit the protein). A high specificity and potency ensure that a drug will have few side effects and thus low toxicity.

Protein inhibitors also occur naturally and are involved in the regulation of metabolism. For example, enzymes in a metabolic pathway can be inhibited by downstream products. This type of negative feedback slows the production line when products begin to build up and is an important way to maintain homeostasis in a cell. Other cellular enzyme inhibitors are proteins that specifically bind to and inhibit an enzyme target. This can help control enzymes that may be damaging to a cell, like proteases or nucleases. A well-characterised example of this is the ribonuclease inhibitor, which binds to ribonucleases in one of the tightest known protein–protein interactions. Natural enzyme inhibitors can also be poisons and are used as defenses against predators or as ways of killing prey.

Reversible inhibitors bind to proteins with non-covalent interactions such as hydrogen bonds, hydrophobic interactions and ionic bonds. Multiple weak bonds between the inhibitor and the active site combine to produce strong and specific binding. In contrast to ligands and irreversible inhibitors, reversible inhibitors generally do not undergo chemical reactions when bound to the enzyme and can be easily removed by dilution or dialysis.

There are four kinds of reversible protein inhibitors. They are classified according to the effect of varying the concentration of the protein’s ligand on the inhibitor.

In competitive inhibition, the ligand and inhibitor cannot bind to the protein at the same time. This usually results from the inhibitor having an affinity for the active site of a protein  where the ligand also binds; the ligand and inhibitor compete for access to the protein’s active site. This type of inhibition can be overcome by sufficiently high concentrations of ligands  i.e., by out-competing the inhibitor. Competitive inhibitors are often similar in structure to the real ligand.

In uncompetitive inhibition, the inhibitor binds only to the ligand-protein complex, it should not be confused with non-competitive inhibitors.

In mixed inhibition, the inhibitor can bind to the protein at the same time as the ligand. However, the binding of the inhibitor affects the binding of the ligand, and vice versa. This type of inhibition can be reduced, but not overcome by increasing concentrations of ligand. Although it is possible for mixed-type inhibitors to bind in the active site, this type of inhibition generally results from an allosteric effect where the inhibitor binds to a different site on a protein. Inhibitor binding to this allosteric site changes the conformation or tertiary structure (three-dimensional shape) of the protein,  so that the affinity of the ligand for the active site is reduced.

Non-competitive inhibition is a form of mixed inhibition where the binding of the inhibitor to the protein  reduces its activity but does not affect the binding of ligand. As a result, the extent of inhibition depends only on the concentration of the inhibitor

As noted above, a protein  inhibitor is characterised by its two dissociation constants,  to the protein  and to the protein-ligand  complex, respectively. The protein-inhibitor constant can be measured directly by various methods; one extremely accurate method is isothermal titration calorimetry, in which the inhibitor is titrated into a solution of protein and the heat released or absorbed is measured.

Traditionally, reversible protein inhibitors have been classified as competitive, uncompetitive, or non-competitive. The binding of an inhibitor and its effect on the protein activity are two distinctly different things. In noncompetitive inhibition the binding of the inhibitor results in 100% inhibition of  the protein  only, and fails to consider the possibility of anything in between. The common form of the inhibitory term also obscures the relationship between the inhibitor binding to the protein and its relationship to any other binding term.

The dissociation constant is commonly used to describe the affinity between a ligand  such as a drug and a protein  i.e. how tightly a ligand binds to a particular protein. Ligand-protein affinities are influenced by non-covalent intermolecular interactions between the two molecules such as hydrogen bonding, electrostatic interactions, hydrophobic and Van der Waals forces. They can also be affected by high concentrations of other macromolecules, which cause macromolecular crowding.

The dissociation constant has molar units, which correspond to the concentration of ligand at which the binding site on a particular protein is half occupied, i.e. the concentration of ligand, at which the concentration of protein with ligand bound, equals the concentration of protein with no ligand bound.  The smaller the dissociation constant, the more tightly bound the ligand is, or the higher the affinity between ligand and protein. For example, a ligand with a nanomolar dissociation constant binds more tightly to a particular protein than a ligand with a micromolar dissociation constant.

The dissociation constant for a particular ligand-protein interaction can change significantly with solution conditions (e.g. temperature, pH and salt concentration). The effect of different solution conditions is to effectively modify the strength of any intermolecular interactions holding a particular ligand-protein complex together.

Drugs can produce harmful side effects through interactions with proteins for which they were not meant to or designed to interact. Therefore much pharmaceutical research is aimed at designing drugs that bind to only their target proteins (Negative Design) with high affinity or at improving the affinity between a particular drug and its in-vivo protein target (Positive Design).

Receptor-ligand interactions form a major class of biochemical interactions involving protein participation, and similar to enzyme-substrate interactions, are most important from pharmacological point of view. In biochemistry, ‘receptor-ligand kinetics’ is a branch of Protein Kinetics  in which the molecular interactions  are defined by different non-covalent bindings and/or conformations of the molecules involved, which are called  as receptors and  ligands.

Biochemical receptors are large protein molecules that can be activated by the binding of a ligand (such as a hormone or drug).  Receptors can be membrane-bound, occurring on the cell membrane of cells, or intracellular, such as on the nucleus or mitochondrion. Binding occurs as a result of noncovalent interaction between the receptor and its ligand, at locations called the binding site on the receptor. A receptor may contain one or more binding sites for different ligands. Binding to the active site on the receptor regulates receptor activation directly. The activity of receptors can also be regulated by the binding of a ligand to other sites on the receptor, as in allosteric binding sites.  Antagonists mediate their effects through receptor interactions by preventing agonist-induced responses. This may be accomplished by binding to the active site or the allosteric site. In addition, antagonists may interact at unique binding sites not normally involved in the biological regulation of the receptor’s activity to exert their effects

The current accepted definition of receptor antagonist is based on the receptor occupancy model. It narrows the definition of antagonism to consider only those compounds with opposing activities at a single receptor. Agonists were thought to turn “on” a single cellular response by binding to the receptor, thus initiating a biochemical mechanism for change within a cell. Antagonists were thought to turn “off” that response by ‘blocking’ the receptor from the agonist. This definition also remains in use for physiological antagonists, substances that have opposing physiological actions, but act at different receptors. For example, histamine lowers arterial pressure through vasodilation at the histamine H1 receptor, while adrenaline raises arterial pressure through vasoconstriction mediated by β-adrenergic receptor activation.

Our understanding of the mechanism of drug-induced receptor activation and receptor theory and the biochemical definition of a receptor antagonist continues to evolve. The two-state model of receptor activation has given way to multistate models with intermediate conformational states. The discovery of functional selectivity and that ligand-specific receptor conformations occur and can affect interaction of receptors with different second messenger systems may mean that drugs can be designed to activate some of the downstream functions of a receptor but not others. This means efficacy may actually depend on where that receptor is expressed, altering the view that efficacy at a receptor is receptor-independent property of a drug.

A receptor antagonist is a type of receptor ligand or drug that does not provoke a biological response itself upon binding to a receptor, but blocks or dampens agonist-mediated responses. In pharmacology, antagonists have affinity but no efficacy for their cognate receptors, and binding will disrupt the interaction and inhibit the function of an agonist or inverse agonist at receptors. Antagonists mediate their effects by binding to the active site or to allosteric sites on receptors, or they may interact at unique binding sites not normally involved in the biological regulation of the receptor’s activity. Antagonist activity may be reversible or irreversible depending on the longevity of the antagonist–receptor complex, which, in turn, depends on the nature of antagonist receptor binding. The majority of drug antagonists achieve their potency by competing with endogenous ligands or substrates at structurally defined binding sites on receptors.

By definition, antagonists display no efficacy to activate the receptors they bind. Antagonists do not maintain the ability to activate a receptor. Once bound, however, antagonists inhibit the function of agonists, inverse agonists, and partial agonists. The potency of an antagonist is usually calculated  by determining the concentration of antagonist needed to elicit half inhibition of the maximum biological response of an agonist.  The greater the potency of the antagonist, and the lower the concentration of drug that is required to inhibit the maximum biological response. Lower concentrations of drugs may be associated with fewer side-effects.

The affinity of an antagonist for its binding site, i.e. its ability to bind to a receptor, will determine the duration of inhibition of agonist activity. The effects of receptor desensitization on reaching equilibrium must also be taken into account.

Competitive antagonists reversibly bind to receptors at the same binding site (active site) as the endogenous ligand or agonist, but without activating the receptor. Agonists and antagonists “compete” for the same binding site on the receptor. Once bound, an antagonist will block agonist binding. The level of activity of the receptor will be determined by the relative affinity of each molecule for the site and their relative concentrations. High concentrations of a competitive agonist will increase the proportion of receptors that the agonist occupies, higher concentrations of the antagonist will be required to obtain the same degree of binding site occupancy.

The interleukin-1 receptor antagonist, IL-1Ra is an example of a competitive antagonist.The effects of a competitive antagonist may be overcome by increasing the concentration of agonist. Often (though not always) these antagonists possess a very similar chemical structure to that of the agonist.

The term “non-competitive antagonism” can be used to describe two distinct phenomena: one in which the antagonist binds to the active site of the receptor, and one in which the antagonist binds to an allosteric site of the receptor. While the mechanism of antagonism is different in both of these phenomena, they are both called “non-competitive” because the end-results of each are functionally very similar. Unlike competitive antagonists, which affect the amount of agonist necessary to achieve a maximal response but do not affect the magnitude of that maximal response, non-competitive antagonists reduce the magnitude of the maximum response that can be attained by any amount of agonist. This property earns them the name “non-competitive” because their effects cannot be negated, no matter how much agonist is present.

An antagonist that binds to the active site of a receptor is said to be “non-competitive” if the bond between the active site and the antagonist is irreversible or nearly so. This usage of the term “non-competitive” may not be ideal, however, since the term “irreversible competitive antagonism” may also be used to describe the same phenomenon without the potential for confusion with the second meaning of “non-competitive antagonism” discussed below.

The second form of “non-competitive antagonists” act at an allosteric site. These antagonists bind to a distinctly separate binding site from the agonist, exerting their action to that receptor via the other binding site. They do not compete with agonists for binding at the active site. The bound antagonists may prevent conformational changes in the receptor required for receptor activation after the agonist binds. Cyclothiazide has been shown to act as a reversible non-competitive antagonist of mGluR1 receptor.

Uncompetitive antagonists differ from non-competitive antagonists in that they require receptor activation by an agonist before they can bind to a separate allosteric binding site. This type of antagonism produces a kinetic profile in which “the same amount of antagonist blocks higher concentrations of agonist better than lower concentrations of agonist”. Memantine, used in the treatment of Alzheimer’s disease, is an uncompetitive antagonist of the NMDA receptor.

Silent antagonists are competitive receptor antagonists that have zero intrinsic activity for activating a receptor. They are true antagonists, so to speak. The term was created to distinguish fully inactive antagonists from weak partial agonists or inverse agonists.

Partial agonists are defined as drugs that, at a given receptor, might differ in the amplitude of the functional response that they elicit after maximal receptor occupancy. Although they are agonists, partial agonists can act as a competitive antagonist in the presence of a full agonist, as it competes with the full agonist for receptor occupancy, thereby producing a net decrease in the receptor activation as compared to that observed with the full agonist alone. Clinically, their usefulness is derived from their ability to enhance deficient systems while simultaneously blocking excessive activity. Exposing a receptor to a high level of a partial agonist will ensure that it has a constant, weak level of activity, whether its normal agonist is present at high or low levels. In addition, it has been suggested that partial agonism prevents the adaptive regulatory mechanisms that frequently develop after repeated exposure to potent full agonists or antagonists. Buprenorphine, a partial agonist of the μ-opioid receptor, binds with weak morphine-like activity and is used clinically as an analgesic in pain management and as an alternative to methadone in the treatment of opioid dependence.

An inverse agonist can have effects similar to those of an antagonist, but causes a distinct set of downstream biological responses. Constitutively active receptors that exhibit intrinsic or basal activity can have inverse agonists, which not only block the effects of binding agonists like a classical antagonist but also inhibit the basal activity of the receptor. Many drugs previously classified as antagonists are now beginning to be reclassified as inverse agonists because of the discovery of constitutive active receptors. Antihistamines, originally classified as antagonists of histamine H1 receptors have been reclassified as inverse agonists.

In the field of pharmacology, an inverse agonist is an agent that binds to the same receptor as an agonist but induces a pharmacological response opposite to that agonist.

A prerequisite for an inverse agonist response is that the receptor must have a constitutive level activity in the absence of any ligand. An agonist increases the activity of a receptor above its basal level while an inverse agonist decreases the activity below the basal level. A neutral antagonist has no activity in the absence of an agonist or inverse agonist but can block the activity of either. The efficacy of a full agonist is by definition 100%, a neutral antagonist has 0%, while an inverse agonist has < 0% (i.e., negative) efficacy. An example of a receptor that possesses basal activity and for which inverse agonists have been identified is the GABAA receptor. Agonists for the GABAA receptor (such as the benzodiazepines alprazolam and diazepam) elicit a sedative effect while inverse agonists have anxiogenic (for example, Ro15-4513) or even convulsive effects (certain beta-carbolines).

Two known endogenous inverse agonists are the agouti related peptide (AgRP) and its associated peptide Agouti signaling peptide (ASIP) both are expressed in humans and each bind melanocortin receptors 4 and 1 (Mc4R and Mc1R) respectively with nanomolar affinities.

Many antagonists are reversible antagonists that, like most agonists, will bind and unbind a receptor at rates determined by receptor-ligand kinetics.

Irreversible antagonists covalently bind to the receptor target and, in general, cannot be removed; inactivating the receptor for the duration of the antagonist effects is determined by the rate of receptor turnover, the rate of synthesis of new receptors. Phenoxybenzamine is an example of an irreversible alpha blocker—it permanently binds to α adrenergic receptors, preventing adrenaline and noradrenaline from binding. Inactivation of receptors normally results in a depression of the maximal response of agonist dose-response curves and a right shift in the curve occurs where there is a receptor reserve similar to non-competitive antagonists. A washout step in the assay will usually distinguish between non-competitive and irreversible antagonist drugs, as effects of non-competitive antagonists are reversible and activity of agonist will be restored.

Irreversible competitive antagonists also involve competition between the agonist and antagonist of the receptor, but the rate of covalent bonding differs and depends on affinity and reactivity of the antagonist. For some antagonist, there may be a distinct period during which they behave competitively,  and freely associate to and dissociate from the receptor, determined by receptor-ligand kinetics. But, once irreversible bonding has taken place, the receptor is deactivated and degraded.

From the above discussions, it is obvious that most drug substances and pathogenic molecules act on the organism as ANTAGONISTS or INHIBITORS of various PROTEINS such as ENZYMES, RECEPTORS, IMMUNE BODIES, TRANSPORT MOLECULES etc. This understanding is essential to comprehend the MIT model of therapeutic actions of potentized homeopathic drugs.

Diseases are caused by diverse types of pathogenic molecules of exogenous or endogenous origin binding to various types of PROTEIN molecules, there by BLOCKING the receptors and INHIBITING the enzymes.

Cure involves removal of these molecular blocks or inhibitions. Homeopathy uses potentized drugs as therapeutic agents.

According to MIT concepts, active principles of potentized drugs are MOLECULAR IMPRINTS of constituent molecules of drug substances used for potentization. These molecular imprints bind to the pathogenic molecules, thereby relieving the biological molecules from pathological inhibitions.


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