WO1998050537A1

WO1998050537A1 - Amino acid analogue maps and methods for using and designing the same

Info

Publication number: WO1998050537A1
Application number: PCT/US1998/009274
Authority: WO
Inventors: James A. Burton
Original assignee: Rsp Amino Acid Analogues, Inc.
Priority date: 1997-05-07
Filing date: 1998-05-06
Publication date: 1998-11-12
Also published as: AU7473298A

Abstract

The present invention involves maps of amino acid analogues each having a structure substantially isosteric with a corresponding parent amino acid selected from the group of the twenty genetically coded amino acids. Each of the analogues exhibits at least one common property. In addition or alternatively, each amino acid analogue has substantially the same conformation as a compound containing the corresponding parent amino acid without the amino acid analogue. Further, the present invention involves methods for designing and/or using the amino acid analogue maps in, for example, drug design.

Description

AMINO ACID ANALOGUE MAPS AND METHODS FOR USING AND DESIGNING THE SAME

The present invention relates to the development of pharmaceuticals from amino acid-containing lead compounds and, in particular, specific groups or maps of amino acid analogues aimed at facilitating this process and methods for designing the same.

BACKGROUND OF THE INVENTION

A major area of pharmaceutical research is the development of drugs from amino acid-containing lead compounds, such as, for example, peptides. Peptides are linear polymers of amino acids residues, with or without side chains, linked in a specific order through amide or "peptide" bonds. Historically, peptides are defined as such polymers containing up to 100 amino acid residues. If more than 100 amino acid residues are joined to form a single molecule, the polymer is defined as a protein. The vast majority of amino acid residues that occur in both peptides and proteins are the 20 amino acids specified by the genetic code.

As used herein, the term "amino acid residue", refers to an amino acid with at least one atom removed to permit linkage of the amino acid to other molecules.

To exert a biological or physical action, peptides (or proteins) must interact with their environment. As used herein, "environment" is a general term which includes, but is not limited to, the active site of an enzyme, the binding site of a receptor and a mucosal barrier. Biological actions can include, but again are not limited to, hydrolysis by a proteolytic enzyme, initiation of a physiologic response after binding to a receptor or passage across a mucosal barrier. The actual process of an interaction between an amino acid and its environment generally may be relatively highly correlated with a limited number of physical properties of the amino acid. Hansch, C. And Leo, A., Substitutent Constants for Correlation Analysis in Chemistry and Biology, John Wiley & Sons, New York (1979). Such properties include lipophilicity, H-bond acceptance ability, H-bond donation ability, charge-charge interactivity, and various types of electron polarizabilities. At present, most scientists working in this field might agree that about seven well defined types of properties are known.

Each amino acid can interact with its environment by one or more mechanisms. In addition, the mechanism by which an amino acid side chain interacts with its environment will vary from peptide to peptide or even at different locations within a peptide. There is no simple way to predict which type of interaction is operative.

In addition, an amino acid residue may not interact with its environment to a significant degree. A residue interaction may be "passive" when, for example, the residue functions merely as a spacer group so that other amino acid residues are correctly positioned for interaction with some key element in the environment.

Knowledge of the mechanism by which an amino acid analogue interacts with its environment would be useful. A drug designer could enhance a desirable biological activity of a compound, such as affinity, by replacing an amino acid of interest in the compound with a related analogue whose property or properties enhance the activity of the compound. Conversely, replacement of the amino acid of interest with a related analogue in which the property is absent could eliminate a troublesome interaction, such as rapid catabolism.

There are no systematic devices or methods for identifying, enhancing or suppressing the specific mechanisms by which an amino acid, or compounds containing amino acids, interact with the environment, however. Frequently, drug designers have used the "Aldrich Catalogue" approach, employing, for example low order combinatorial chemistry techniques. With this approach, commercially available amino acid analogues are substituted into a peptide and the resultant products are assayed for enhancement (or suppression) of a specific activity. In this way, the designer may identify the best commercially available option.

The "Aldrich Catalogue" approach relies on brute force and can be both time consuming and expensive, however. In addition, it is always possible for someone to identify a new analogue which better enhances or suppresses a particular activity of interest. Alternatively, drug designers have often used the "Model" approach (i.e., rational drug design), in which structural models of varying degrees of detail are constructed and used to propose various types of interaction between an amino acid-containing compound and its environment. Analogues which enhance or eliminate a proposed interaction are used to replace the amino acid and an enhancement, or diminution, of activity is then taken as proof of the validity of a predictive model. Usually, the Model approach requires that a molecular model of the peptide and its environment be constructed from physical data. The Model approach depends on a variety of intangibles that may be difficult to define and thus practice in a systematic way, however. Some parts of the environment, such as a mucosal surface or a peptide hormone receptor, are so complex that they may not readily allow models to be constructed. Further, the Model approach is inherently non- quantitative. It is extremely difficult to assign a quantitative value to a particular interaction. This makes it difficult both to rule out a particular interaction and to know the extent to which a particular analogue enhances or suppresses a particular interaction. Drug designers have also used mathematical equations to relate physical properties to biological activity. These studies, known as Quantitative Structure Activity Relationships (QSAR), may correlate, for example, a measured or calculated physical property of an amino acid to a biological activity such as affinity of a peptide containing the amino acid. QSAR studies may fail to take into account the effect of the size and shape of the molecule of interest on the biological activity of a compound containing that molecule.

Accordingly, there is a need for devices and methods for identifying the mechanism(s) by which an amino acid interacts with its environment.

Further, there is a need for devices and methods for identifying analogues of such amino acids which facilitate beneficial environmental interactions and, conversely, eliminate troublesome environmental interactions.

SUMMARY OF THE INVENTION

The present invention is directed to devices and methods for systematically determining how an amino acid and amino acid residues in amino acid-containing compounds are correlated with the environmental interactions of the compounds. In particular, the present invention is directed to maps, or kits, containing a selected number of amino acid analogues. Each amino acid analogue of a map has a structure which is substantially isosteric with a parent amino acid, that is, a corresponding genetically coded amino acid. The amino acid analogues of a map are characterized by at least one common property. In addition or alternatively, an amino acid-containing compound containing at least one of the amino acid analogues of the map has substantially the same conformation as the compound containing the corresponding parent amino acid in lieu of the amino acid analogue. The invention further provides methods of using the maps to assess activities of interest for compounds containing the amino acid analogues. In addition, the invention provides methods for designing the maps.

As used herein, the term "map" is used interchangeably with the word "kit" and is considered synonymous with the same.

As used herein, the term " isosteric", as in, for example, an amino acid analogue structure which is substantially isosteric with a corresponding parent amino acid, refers to an amino acid analogue which has a substantially similar size and shape as the corresponding parent amino acid.

As used herein, the term "parent amino acid" refers to genetically coded amino acid selected from the following group of amino acids:

Alanine, Glutamic Acid, Leucine, Serine, Arginine, Glutamine, Lysine, Threonine, Asparagine, Glycine, Methionine, Tryptophan, Aspartic Acid, Histidine, Phenylalanine, Tyrosine, and Cysteine, Isoleucine, Proline, Valine,

In one embodiment, the invention provides a map containing amino acid analogues each having a structure substantially isosteric with a corresponding parent amino acid. Each of the amino acid analogues is characterized by at least one common property.

In another embodiment, the invention provides a map containing amino acid analogues each having a structure substantially isosteric with a corresponding parent amino acid. A compound containing at least one of the amino acid analogues has substantially the same conformation as the compound containing the corresponding parent amino acid in lieu of the amino acid analogue.

In still another embodiment, the invention provides a method for assessing an activity of interest for variants of an amino acid-containing compound. This method includes the steps of providing a map containing amino acid analogues each having a structure substantially isosteric with a corresponding parent amino acid, wherein each of the amino acid analogues is characterized by at least one common property; substituting in succession one or more of the amino acid analogues of the map for an amino acid in the amino acid-containing compound to form a corresponding succession of substituted compounds; measuring the activity of interest of each of the substituted compounds; and determining a correlation of the measured activity of interest with each of the substituted compounds.

BRIEF DESCRIPTION OF THE DRAWING The foregoing and other objects of the invention, the various features thereof, as well as the invention itself, may be more fully understood from the following description, when read together with the accompanying drawing in which:

FIG. 1 is an illustration of a Difference Minimum Energy Ramachandran Plot according to an embodiment of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION In one embodiment, the invention provides a map containing amino acid analogues each having a structure substantially isosteric with a corresponding parent amino acid. For example, the amino acid analogue can have modified side chain(s), different side chain(s) or additional side chain(s) relative to the corresponding parent amino acid. Preferably, each of the amino acid analogues of the invention has a molecular volume which is about + 20% of the molecular volume of the corresponding coded parent amino acid.

In the above-identified embodiment, each of the analogues is characterized by at least one property common to the other amino acid analogues in the map. When an analogue of the map is substituted for the corresponding parent amino acid into a compound, the common property of the amino acid analogue determines, at least in part, how the compound containing the amino acid analogue (or its parent amino acid) interacts with its environment.

The common properties of the amino acid analogues of a map of the invention include one or more of size, length, breadth, thickness, molecular volume, surface area, molar refractive index, lipophilicity, parachor, electron polar izability, electron distribution, charge to charge interactivity, hydrogen bond donating ability, hydrogen bond accepting ability, and effect on conformation of an amino acid derivative into which the amino acid analogue component is incorporated. Preferably, the common properties include one or more of size, lipophilicity, electron polar izability, charge to charge interactivity, hydrogen bond donating ability, and hydrogen bond accepting ability.

As used herein, the term "size", refers to the root mean square fit of a molecule, such as, for example, an amino acid analogue of a map of the invention.

As used herein, the term "lipophilicity", refers to the ability of a molecule, such as, for example, an amino acid analogue (or its corresponding parent amino acid) to separate between immiscible phases. Typically, lipophilicity is measured as the ratio of the distribution of the molecule of interest in a phase such as octanol to the distribution of the molecule in an aqueous phase or a buffer such as phosphate.

As used herein, the term "parachor", refers to the ability of an amino acid analogue to bind with a water molecule, where the water molecule is held between two phases which are immiscible in water.

A map of the invention can present the properties associated with each of the amino acid analogues in, for example, a table.

At least some of the amino acid analogue properties identified in an amino acid map of the present invention can be based on physical measurement of the properties. Other amino acid analogue properties can be calculated using computer programs, such as Hyperchem, Autodesk, Inc., Sausalito, CA; and Alchemy 2000, Tripos, Inc., St. Louis, MO, known to those of ordinary skill in the art.

Each amino acid analogue of a map of the present invention can also include protecting groups which allow the incorporation of the analogue into a compound containing a corresponding parent amino acid. Such incorporation is accomplished using standard synthesis techniques where the analogue is substituted for the corresponding parent amino acid in the compound. Preferably, the protecting group is removed after an analogue is incorporated into the compound of interest.

A compound containing at least one of the amino acid analogues of a map of the present invention has substantially the same conformation as the compound containing the corresponding parent amino acid in lieu of the amino acid analogue.

Further, each of the present invention's amino acid analogues is substantially stable under predetermined conditions. In addition, the amino acid analogues can be easily synthesized by methods known to those of ordinary skill in the art. In another embodiment, the invention provides a map containing amino acid analogues each having a structure substantially isosteric with a corresponding parent amino acid, wherein a compound containing at least one of the amino acid analogues has substantially the same conformation as the compound containing the corresponding parent amino acid in lieu of the amino acid analogue. Like in the previously described embodiment, each of the amino acid analogues can also include one or more protecting groups. The protecting groups permit the analogue to replace a corresponding parent amino acid in a compound of interest. The protecting groups are possibly removable upon incorporation of the analogue into the compound. Each of the amino acid analogues is substantially stable under predetermined conditions; and/or easily synthesized.

In yet another embodiment, the invention provides a method for assessing an activity of interest for variants of an amino-acid containing compound. The method includes providing a map containing amino acid analogues each having a structure substantially isosteric with a corresponding parent amino acid, wherein each of the amino acids is characterized by a common property; substituting in succession one or more of the amino acid analogues of the map for an amino acid in the compound to form a corresponding succession of substituted compounds; measuring an activity of interest of each of the substituted compounds; and determining a correlation of the measured activity with each of the substituted compounds. When such correlations are above a desired value, the substituted compound can be selected as a compound of interest. The activities of interest can include one or more biological activities, such as rate of transfer across a biological barrier, ability to undergo hydrolysis by a proteolytic enzyme, ability to initiate a physiologic response after binding to a receptor, ability to inhibit an enzyme, and ability to inhibit a receptor. These activities of interest can also include one or more physical activities, such as affinity, half life in serum, binding energy, stability, and solubility.

The step of providing the map can also include providing the map wherein the compound containing at least one of the analogues has substantially the same conformation as the compound containing the corresponding parent amino acid in lieu of the analogue. Chemical synthesis of compounds can be facilitated by practicing this method of the invention. For example, in the pharmaceutical area, an amino acid or acids of interest in a compound such as a peptide or a protein can be replaced with an amino acid analogue or analogues of the map by various types of chemical synthesis known to those of ordinary skill in the art. The activities of interest of the substituted peptide or protein can then be measured or determined. These activities of interest can be correlated with the substituted compounds and their common properties in order to select compounds which enhance or suppress the specific activity of interest.

In still another embodiment, the present invention also involves a method for generating the amino acid maps described above. In a first identification step, amino acid analogues that have structures which are substantially isosteric with the corresponding parent amino acid are identified. This identification is based on standard principles known to those of ordinary skill in the art. For example, Rudinger's Principles of Isosteric Substitution, can be used to identify appropriate analogues. Other isosteric substitution principles known in the art can also be used to identify appropriate analogues. The analogues identified in this first identification step form Set A.

In a second selection step, model compounds containing the parent amino acid and/or each of the amino acid analogues in Set A are constructed using electronic techniques. For example, an electronic model of a blocked 5-residue peptide such as acetyl-alanyl-alanyl-XXX-alanyl-alanyl-acetyl-amide is constructed in which XXX is either the parent amino acid or one the analogues identified in Set A. Computer simulations of the three dimensional structures, that is, the conformations, of each of the model amino acid-containing compounds are made using any of a variety of computer programs known to those of ordinary skill in the art. Non-limiting examples of such computer programs include MM3, CHARM, AMBER, FRODO and Alchemy 2000. Generally, model compounds such as model peptides and proteins can assume a large number of conformations because of their flexible nature, although some of these conformations are preferred because of their lower energy.

The energies of the model amino acid-containing compounds are displayed as the compounds assume different conformations with Difference Minimum Energy Ramachandran (DMER) Plots. For example, the φ and ψ angles of the compound containing a parent amino acid are set at some value between 0° to 360°. The rotatable bonds (except those bonds involved in the set φ and ψ angles) are systematically incrementally rotated. For each rotation a new conformation of the compound results and its energy is calculated. Possibly as many as a billion conformations may result, and thus, preferably a fast force field such as the Sybil force field in the Alchemy 2000 program is used for such calculations. The conformation with the lowest energy is identified. The rotatable bonds of this low energy conformation are then further rotated in even smaller increments than those described above and the energies of the resulting conformations are calculated using a program such as MM3 described above to identify the conformation with the lowest possible energy.

The φ and ψ angles of the compound are then re-set and the systematic rotations and calculations repeated until all desired settings of the φ and ψ angles have been tested.

The above-described process is repeated for a compound containing a corresponding amino acid analogue. Subsequently, the minimum energy conformations of the model compound containing the parent amino acid are compared with those minimum energy conformations of the compound containing the corresponding amino acid analogue, yielding the Difference Minimum Energy Ramachandran Plot (DMER Plot). This DMER Plot demonstrates whether the substitution of the amino acid analogue for the corresponding parent amino acid produces a different effect on the conformation of the amino acid-containing compounds than that of the corresponding parent amino acid. FIG. 1 illustrates a DMER Plot. Based on the DMER Plot, amino acid analogues which affect the conformation of the amino acid-containing compound similarly to the way in which the parent amino acid affects the conformation of the compound are then selected for a Set B. Approximately 50% to 60% of the amino acid analogues identified in Set A will meet the restricted criteria for the Set B amino acid analogues.

In a third selection step, Set C is determined from Set B by excluding amino acid analogues from the amino acid analogue Set B according to different selection criteria. For example, amino acid analogues which are unstable under predetermined testing conditions or are difficult to chemically synthesize are excluded from Set B. Approximately 20% to 30% of the amino acid analogues in Set B will meet the restricted criteria and will be non- excluded to define the Set C amino acid analogues.

In a fourth selection step, a mathematical technique is applied to identify those members of set C whose physical properties most efficiently affect their interaction with the environment. Each parent amino acid or amino acid analogue has multiple properties expressed to varying degrees, and these properties are often inter-dependent. For example, if the size of a parent amino acid or an amino acid analogue is increased, the lipophilicity of the analogue is also usually increased. Thus, a matrix approach, such as, for example, a Leo-Hansch matrix, is used to systematically incrementally vary a single property, such as size, to identify the analogue whose respective single property independently of the analogue's other properties most efficiently affects the analogue's interaction with its environment.

The physical properties of the parent amino acids or the amino acid analogues are physically measured with techniques known to those of ordinary skill in the art. Alternatively, the properties of parent amino acids or their analogues are calculated using standard computer programs such as Hyperchem and Alchemy 2000, discussed above.

Preferably, the measured rather than computer program based properties serve as the basis for identifying analogues whose physical properties most efficiently affect their interaction with the environment.

Typically, approximately 50% of the amino acid analogues selected in Set C will meet the restricted criteria for the Set D amino acid analogues. Table I is an example of a series of about 20 analogues of the parent amino acid phenylalanine that meet the criteria for an amino acid analogue Set D.

Table I Amino Analogue Set For Parent Amino Acid Phenylalanine

This invention is further illustrated by the following Example which should not be construed as limiting.

EXAMPLE 1 : Amino Acid Analogue Phenylalanine Map

Table I illustrates the components of a non-limiting exemplary map, according to an embodiment of the invention. The parent genetically coded amino acid featured in this map is L form of phenylalanine (hereinafter L-Phe). Length, width, thickness, hydrophobicity and electron distribution are typical non-limiting examples of properties which are identified in the map as characteristic of the way L-Phe interacts with its environment. Accordingly, for a given analogue, at least one of these properties varies from that of the corresponding parent amino acid which in turn affects the way the analogue interacts with its environment.

Those of skill in the art will recognize that the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The presently described embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all variations of the invention which are encompassed within the meaning and range of equivalency of the claims are therefor intended to be embraced therein.

Claims

What is claimed is:

1. A map comprising, n amino acid analogues each having a structure substantially isosteric with a corresponding parent amino acid selected from the group of genetically coded amino acids consisting of:

wherein each of the amino acid analogues is characterized by at least one common property.

2. The map of claim 1, wherein the properties are selected from the group consisting of size, length, breadth, thickness, molecular volume, surface area, molar refractive index, lipophilicity, parachor, electron polarizability, electron distribution, charge to charge interactivity, hydrogen bond donating ability, hydrogen bond accepting ability, and effect on conformation of an amino acid-containing compound into which the amino acid analogue component is incorporated.

3. The map of claim 1, wherein the properties are selected from the group consisting of size, lipophilicity, electron polarizability, charge to charge interactivity, hydrogen bond donating ability, and hydrogen bond accepting ability.

4. The map of claim 1, wherein at least one of the amino acid analogues comprises a protecting group for allowing substitution of the corresponding parent amino acid in a compound with the amino acid analogue.

5. The map of claim 4, wherein the protecting group is removable from the amino acid analogue after substitution of the corresponding parent amino acid in the compound with the amino acid analogue.

6. The map of claim 1, wherein a compound containing at least one of the amino acid analogues has substantially the same conformation as the compound containing the parent amino acid in lieu of the amino acid analogue.

7. The map of claim 1, wherein each of the amino acid analogues is substantially stable under predetermined conditions.

8. The map of claim 1 , wherein each of the amino acid analogues can be easily synthesized.

9. A map comprising, n amino acid analogues each having a structure substantially isosteric with a corresponding parent amino acid selected from the group of genetically coded amino acids consisting of

wherein a compound containing at least one of the amino acid analogues has substantially the same conformation as the compound containing the corresponding parent amino acid in lieu of the amino acid analogue.

10. The map of claim 9, wherein at least one of the amino acid analogues comprises a protecting group for allowing substitution of the corresponding parent amino acid in the compound with the amino acid analogue.

11. The map of claim 10, wherein the protecting group is removable from the amino acid analogue after substitution of the corresponding parent amino acid in the compound with the amino acid analogue.

12. The map of claim 9, wherein each of the amino acid analogues is substantially stable under predetermined conditions.

13. The map of claim 9, wherein each of the amino acid analogues can be easily synthesized.

14. A method for assessing an activity of interest for variants of an amino acid- containing compound, comprising the steps of

a. providing a map containing n amino acid analogues each having a structure substantially isosteric with a corresponding parent amino acid selected from the group of genetically coded amino acids consisting of:

wherein each of the amino acid analogues is characterized by at least one common property; b. substituting in succession one or more of the amino acid analogues of the map for an amino acid in the amino acid-containing compound to form a corresponding succession of a plurality of substituted compounds;

c. measuring the activity of interest of each of the substituted compounds;

d. determining a correlation of the measured activity of interest with each of the substituted compounds.

15. The method of claim 14, wherein the activity of interest is selected from the group consisting of a physical activity of interest and a biological activity of interest.

16. The method of claim 14, wherein the activity of interest is selected from the group consisting of affinity, rate of transfer across a biological barrier, half life in serum, binding energy, stability, solubility, ability to undergo hydrolysis by a proteolytic enzyme, ability to initiate a physiologic response after binding to a receptor, ability to inhibit an enzyme, and ability to inhibit a receptor.

17. The method of claim 14, wherein the step of providing the map comprises providing the map wherein a compound containing at least one of the amino acid analogues has substantially the same conformation as the compound containing the parent amino acid in lieu of the amino acid analogue.