Published on August 9, 2014

1. Concepts:
   1. Pharmacology: is the branch of science that studies the actions and mechanisms of action of drugs.
   2. Drugs: is the substance of well-defined chemical structure used for therapeutic purposes.
   3. Isosteres: are constituents or groups that have the same size or volume.
   4. Bioisosteres: are constituents or groups that do not necessarily have the same size or volume, but have similarity in chemical and physical properties and produce similar biological properties.
   5. Pharmacophore: is the spatial arrangement of the functional groups of a molecule that interact with the receptor. It considers the relative positions of these groups, allowed areas of space (exclusion volumes and steric effect).
   6. Molecular pharmacology: is the branch of pharmacology that studies the pharmacological action of drugs at the molecular level in an attempt to define the site of action of the drug, the receptor, or possibly the specific enzyme with which it interferes.

2. Molecular pharmacology

The goal of molecular pharmacology is to elucidate the sequence of chemical and biological events resulting from drug-receptor interaction. These biochemical studies are much less susceptible to the variability problem found in animal model experimentation and therefore the structure-activity relationship can be determined more consistently.

Molecular pharmacology considers molecules as fundamental functional units. It seeks to explain the pharmacological effects of biologically active compounds at the molecular level, that is, based on molecular interactions and in terms of molecular structures and physicochemical properties. It ultimately aims to determine and interpret the relationship that exists between chemical structure and biological activity. By understanding what type of interaction occurs between the chemical compound and the molecules of the organism's cells, it is possible to explain its mechanism of action at the molecular level.

3. Relationship between structure and activity

Considering the way they exert their biological action, drugs can be divided into two major classes: structurally nonspecific and structurally specific drugs.

a) Structurally nonspecific drugs: are those in which the biological action is not directly subordinate to the chemical structure, but only to the extent to which it affects the physicochemical properties, which are responsible for the pharmacological effect they produce. Main characteristics: a) they act in relatively high doses.

b) they have very varied chemical structures but cause a similar biological reaction.

c) small variations in their chemical structure do not result in marked changes in biological action.

Example: phenol, ethanol, antacids, mannitol, etc.

d) Structurally specific drugs: are those whose biological action results essentially from their chemical structure, which must adapt to the three-dimensional chemical structure of the receptors in the body, forming a complex with them. In these drugs, the size, stereochemical arrangement of the molecule, distribution of functional groups, inductive effects, electronic distribution, possible indications with functional groups of the receptor, among others, play a decisive role. Main characteristics:

e) they are efficient at lower concentrations than structurally nonspecific drugs.

f) they have certain structural characteristics in common.

g) small variations in the chemical structure can result in substantial changes in pharmacological activity.

Both the purpose of the drug and its intrinsic activity are determined by its chemical structure. Small modifications in its structure can cause major changes in its pharmacological properties. The exploration of this structure/activity relationship has led to the development of important drugs in therapeutics and often with a different indication from that used by the prototype drug. Not necessarily every modification of the molecular structure of the compound needs to alter all its action and effect in a homogeneous way. It basically aims to develop a congener that presents a more favorable therapeutic effect X toxic relationship; increase selectivity between different cells of the tissues, or alter the pharmacokinetic characteristics. For example, some hormone and neurotransmitter antagonists have been developed by modifying the chemical structure of physiological agonists.

Having in hand the molecular structure and pharmacological activity information of a large number of congeners, it is possible to identify those properties that are required for optimal activity at the receptor: size and shape of the molecule, position and orientation of the groups with charge or capable of making hydrogen bonds, etc.

Accurate and quantitative correlation between the pharmacological activity of various drugs with their molecular structures (shape, location and orientation of chemically interactive groups on their surface) makes it possible to develop an accurate model of the structure of the binding site on the receptor. These detailed models allow rationalizing the de novo synthesis of new compounds or creating congeners with greater efficacy, selectivity, affinity or regulatory effects, or even improving their pharmacokinetic properties.

Currently, the use of X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy are fundamental for the rational development of new drugs, allowing the determination of the three-dimensional structure of receptors, drugs and drug-receptor complexes.

In cases where the structure of the receptor is completely unknown, it is always possible to determine the conformation of the bound drug (and if possible of other congeners) and from there generate a probable image of the receptor's binding site.

4. Receptors

4.1. Receptor Isolation

The primary challenge in isolating a specific receptor from its membrane is the ability to identify and separate a particular protein from all other proteins present. Chromatographic techniques have been fundamental to the success of these studies. The initial step involves removing a complex mixture of proteins from a membrane with minimal damage. This is relatively straightforward when the protein is weakly associated (peripheral membrane proteins), and typically, the removal of cations such as Ca2+ and Mg2+ from the medium through washing or chelation suffices. In these instances, the protein is usually released into the solution virtually free of lipid fragments.

However, this is not the case with integral proteins. These proteins span the membrane and possess three distinct regions: an extracellular region that projects into the external environment surrounding the membrane; a transmembrane region that traverses the membrane; and an intracellular region that projects into the cell's interior. Being integral components of the cell membrane, their tertiary structure and adopted conformation can be directed by their presence within the membrane. The isolation of such proteins can severely affect their conformation and, consequently, their ligand-binding ability. More stringent conditions are required to release these proteins, and the use of detergents (Triton X-100, Lubrol, sarkosyl), urea, sodium thiocyanate, and medium-chain alcohols is extensively employed to disrupt the membrane and release the protein of interest.

4.2. Receptor Purification

The isolation of the target protein from the obtained complex mixture necessitates the use of sophisticated chromatographic separation techniques. Among these, affinity chromatography is the most common. Affinity chromatography operates on the principle that the receptor binds strongly to its ligand, such that this interaction can be used to select the protein of interest. Thus, if the ligand is covalently attached to a solid support in a manner that does not significantly affect its binding capacity to the protein of interest, when a solution of various peptides passes through the column, the receptor of interest will be preferentially retained. After washing away the other proteins, the receptor can be released by passing a concentrated solution of the ligand through the column. The protein obtained here is then subjected to further purification techniques to eliminate contaminating proteins.

More recent techniques include high-performance liquid chromatography (HPLC); particularly size-exclusion, ion-exchange, and reversed-phase HPLC, have significantly contributed to the isolation of receptor proteins. Other techniques may be based on immunoaffinity chromatography or immunoprecipitation techniques, where monoclonal or polyclonal antibodies that provide the high selectivity of interaction necessary for the separation of proteins of interest are utilized. These methodologies, however, are somewhat complex and require technical skill to avoid the appearance of artifacts and questionable results. The primary evidence that the appropriate receptor has been obtained is provided by its high affinity for its ligand.

4.3. Use of Molecular Biology

The technical difficulties in isolating receptors are primarily due to their limited quantity present in any membrane, as well as the practical difficulties inherent in determining their protein membrane. The use of molecular biology techniques provides an alternative method to overcome these limitations, such as obtaining large quantities of protein, predicting the primary amino acid sequence, expressing receptors in cell cultures, etc.

5. Planning of a new drug

The basic elements of the development of a new drug have remained the same for many years. There is a need for a hypothesis about the probable cause of a pathological state, a lead compound, and an optimized process to produce the molecule with the best activity profile. The process starts from the identification of a lead compound that has some elements of the desired therapeutic profile and its hypothetical mechanism of action. A search in databases of structures, preferably three-dimensional, can reveal new molecules similar to that of the lead structure. Interaction studies with the target receptor can provide a model to be used to search a large number of molecules and identify the possible pharmacophoric group. "Computer-aided drug design" (CADD) programs are used to design entirely new structures based either on the base structure or the binding site on the receptor.

5.1 Sources of lead structures

The main sources of base (or lead) compounds for the research and development of new drugs are: observation of clinical or pharmacological adverse effects, random research, natural products, and rational de novo design.

a) Adverse drug reactions

Often the unexpected adverse effects of many drugs can be the basis for the development of compounds or new therapeutic indications. For example, isoniazid, whose isopropyl derivative, iproniazid, showed pronounced central effects, in particular the ability to soften the mood of tuberculosis patients with severe depression. This led to the use of iproniazid as an antidepressant and the synthesis of other structural analogs.

b) Random search

The search for a large number of different compounds in vivo consumes time, material, and animals and has been criticized by the scientific community. However, in vitro tests use much less material, are fast, and have been an integral part of the research of new drugs in different research programs.

As receptors and enzymes that play a key role in pathologies are progressively identified, they become the new targets for drug development. Methods range from biochemical techniques of enzyme inhibition, tissue and microorganism culture, peptide synthesis, and molecular biology techniques.

c) Natural Products

The use of natural products dates back centuries and still remains an important source of compounds for therapeutic use. Natural products include isolated compounds and plants, microorganisms, and marine organisms.

d) Pre-existing drugs

Currently used drugs in therapy can be modified for the development of new compounds, generally seeking to reduce possible adverse effects, resistance, and increase their effectiveness, tolerability, etc.

e) Pathology as a research model

Understanding the development of certain pathologies and obtaining susceptible animal models has allowed the discovery of several compounds with therapeutic potential. Once again, molecular biology has also contributed to this area, providing genetically modified animals to serve as research models.

f) Physiological mechanisms

The better understanding of physiological mechanisms and their endogenous agonists has allowed the use of a more rational and selective approach in the development of new drugs.

5.2 Macromolecules as a target for drug development

The emphasis on "rational drug design" widely used today does not mean that the other methods mentioned above are irrational and fruitless. While it is true that many great discoveries have been almost accidental, the vast majority of drugs originate from the best scientific procedures of the period in which they were developed. In the same way that, today, the development of new drugs is based on computer simulation studies as more and more new receptors have their structures resolved.

Many compounds interact with proteins, either via enzymes or via protein receptors; nucleic acids and carbohydrates are secondary targets, at least in number. The isolation and characterization of proteins, as mentioned earlier, had a greater impulse with molecular biology techniques, as they allowed the obtaining of large quantities of material originally scarce in the cell.

Although it is relatively easy nowadays to clone a specific gene and from this predict the primary polypeptide sequence, the prediction of the secondary and tertiary structures and, why not, quaternary, still remains an unsolved problem. What little can be achieved today is modeling from homologous proteins with already determined structures. The most accurate determination of the three-dimensional structure is obtained through X-ray crystallography and NMR techniques. Due to the difficulties of crystallization of the most different proteins and limitations of NMR techniques, the number of resolved structures is much lower than the number of primary sequences deposited in databases.

5.3 Modeling of drug-receptor interactions

Computational chemistry (modeling) techniques are extremely valuable. However, in drug-receptor interactions, solvent effects and additional interactive forces must be taken into account. This increases the complexity of the calculations by an order of magnitude.

A complex calculation of binding processes would produce the free energy of binding, reduced to terms of enthalpy and entropy, and would take into account the attraction and repulsion interaction that depends on the shape and electrostatic potential of the surface of the molecules. The forces considered include: ion-ion, ion-dipole interactions, hydrogen bonds, polarization, charge transfer, van der Waals interactions, loss of rotational and translational entropy, and hydrophobic interactions. The relative importance of each force depends on the nature of the species involved. Another technique takes into account the free energy perturbation (FEP) method and can be used to calculate some accurate values for differences in the binding energies of similar compounds on the same receptor.

5.4 Optimization of lead structures or prototypes

Several factors must be taken into account to optimize the activity of a potential drug, from physicochemical to quantum aspects. Changes in structure cause changes in several physical and physicochemical properties at once, making it difficult to identify which of these properties is most likely to control the activity of the compound. Some analysis processes are listed below.

a) Linear free energy relationships: partition coefficient, electronic effects, steric effects.

b) Hansch analysis.

c) Topliss analysis.

d) Batch replacement method.

e) Free-Wilson method.

f) Statistical methods: pattern recognition technique, correlation methods.

6. Bibliographic references

BRUNTON, L. L. et all. Goodman & Gilman: As Bases Farmacológicas da Terapêutica. 11ª Ed. Porto Alegre: Artmed, 2010.

KING, F.D. Medical chemistry: principles and pratice. Cambridge: The Royal Society of chemistry, 199. 313p.

TAYLOR, J. B.; KENNEWELL, P. º. Modern medicinal chemistry. New York : Ellis Horwood, 1993. 290p.