PHYSICO-CHEMICAL INVESTIGATIONS ON THE BINDING OF SUBSTANCES OF PHYSIOLOGICAL AND MEDICINAL IMPORTANCE WITH MICRO-ORGANISMS AND MACROMOLECULES

Abstract

Mankind has used drugs for thousands of years and we have come to rely upon a vast horde of chemical compounds as the answers to some of our illnesses. These drugs influence the human system in a variety of ways. Their action may just be limited to the killing of certain micro-organisms or to the accompaniment of such changes as direct interaction with the amino acids and the proteins, changing the catalytic activity of the enzymes and influencing the transport system of the body. Many physiologists, biochemists, chemists and men of medicine are grossly engaged in investigating the exact mode by which the drugs react but the complete answer to even the simplest system is not yet known. Obviously then the behaviour of a living system where the complexities are beyond compre hension cannot be explained with the existing scientific means at our disposal. Most drugs are small molecules. They would interact with other molecules whatever be the size of the other molecule. This interaction would influence the behaviour of the other molecule and this is the first event in the action of any drug. The behaviour of the other molecuie( sometimes referred to as bioreceptor) then produces alterations in the behaviour of the cell to which it belongs and the modified behaviour of the cell then may influence the metabolism of the whole organism itself. Thus we see that the first event assumes fundamental importance. It may 2 not be possible to work out the entire sequence of events summarised commonly as the "action of a drug", but it is possible to study in fundamental terms the first event referred to above. As an illustration let us take the case of narcosis. Narcotics have been used for a long time for various purposes. The gross effect is known for a considerable time without any information on the mechanism of action. The inhibition of general metabolism of an animal by a little amount of a chemical agent is certainly an extraordinary event. It is known that the effect can be attributed to the interaction of the drug molecules with the molecules of the cell membranes, i.e., a drug whose molecular structure is anticomplimentary to the structure of the membrane molecules would be a convulsant and a drug whose molecular structure is complimentary to the membrane molecules would be a narcotic(l). The action is explainable in terms of increase or decrease in the interstitial space (space between two membrane molecules) causing difference in the flow of electrolyte through the membrane. It is now possible to understand the relative efficacy of a series of narcotic compounds in terms of the thermodynamic parameters and predict the activity of a particular compound in terms of its molar attraction constant(2). We have said above that drugs will interact with molecules and this interaction is the first event in drug action. The first molecule that comes into mind 3 is protein. The importance of protein in living systems need not be emphasized. They are structural units, functional units and take part literally in almost all the life processes. It is believed that most bioreceptors are proteins (enzymes?). It is also note worthy that on equal molecular size basis, proteins contain ten times as much information as the nucleic acids (20 amino acids versus 4 or 5 nucleic acid bases) though the latter alone are responsible for storage and transfer of genetic information. Thus it seems reasonable to assume that most drug interactions will involve proteins (an exception is the postulated carcinogen-nucleic acid interaction for which the evidence is still quite uncertain). There are two transport systems in the body, the lymph and the blood. Since all the metabolic processes are carried out in the aqueous phase, even the nonpolar substances taken care of by the lymph system, at some stage have to come in the aqueous phase and are subjected to the binding etc. by the proteins. The proteins, because of a very wide variety of charges in the molecule would bind, again, a very wide variety of chemical substances. The transport of most metabolic substances is carried out by proteins. The binding of these substances with the protein determines their availability (one could say very nearly in the thermodynamic sense). Thus these substances which bind loosely would be in the higher activity state and those that bind strongly would be in the low activity state. Those bound strongly would have better chance of being transported farther and vice versa. It has been seen 4 in the case of sulfa drugs that only the fraction of the drug unbound with plasma proteins is available for biological activity. The following, in brief, shows the extensive involvement of plasma proteins in drug action(3). It is known that only fractions of unbound drugs can spread from blood. For instance, only unbound penicillin diffuses from blood into either lymph(4) or milk(5), only unbound sulfonamide can spread into the cerebrospinal fluid(6) and only unbound thyroid harmones are available for entry into the body cells(7). On the other hand, it is well known that there is no apparent correlation between sulfonamide levels in plasma and in aqueous humour(8). This is probably due to the fact that a large quantity of sulfonamides is bound to serum proteins and it is reasonable to assume that the aqueous humour concentration of sulfonamides depends on its unbound serum concentration. The proteins have wide variety of surface charges and are dissolved in solutions (plasma, interstitial fluids) which also have a wide variety of molecules of different sizes, shapes and charges. Thus there are always equilibrium factors coming into play and one molecule competes with another for binding sometimes for the same site. Thus penicillins can be displaced from serum macromolecules by various oxazole derivatives, by salicylates and benzoic acid derivatives, by various sulfonamides and by many miscellaneous compounds, (e.g., phenyl butazone, ethacrynic acid etc.) (9-12). Cloxacillin can be displaced 5 by sodium salicylate, ^resorcylic acid, phenyl butazone, sulfadimidine and sulfa methoxy pyridazine(l3), sulfonamides displace tetracyclines( 14). As regards sulfonamides this phenomenon takes place with bilirubin(l5), phenylbutazone^, 17), tripirafen(l8), caprylate, 2,4-dichloro phenoxyacetate, 2,6-dihydroxy benzoate, p-iodobenzoate, novobiocin, probenecid salicylate sulfin pyrazone, tolbutamide(17) alkylating agents( 19), tetracyclines( 14) and pentobarbital (20). Furthermore the binding of thyroxin is prevented by salicylate derivatives(21) • The fraction of the drug which is bound with the protein may act as a reservoir from which the drug would be released as the unbound drug is metabolized. Thus the rates of drug action and its half life in vivo would depend upon its binding characteristics with plasma proteins. It is, therefore, evident that a knowledge of the binding of various chemical species to proteins is of great importance in understanding the mechanism of drug action and a quantitative study of the binding of some simple molecules chemically and structurally akin to drugs to well characterised proteins can very much help in having a proper understanding the mechanism of drug action. As can be imagined this would be a very vast field and one would have to confine oneself to one or two classes of proteins only. Among the drugs one may take a particular class of chemical species and the results would then be applicable to atleast that chemical species.