Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-12-10
2002-10-15
Venkat, Jyothsna (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C435S091500, C435S091500, C530S350000, C530S387300
Reexamination Certificate
active
06465192
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to compounds, compositions, and methods for blocking protein-protein interactions.
Many pharmaceutical drugs act by blocking the binding of an enzyme to its substrate. In order for these drugs to block this interaction effectively, they must bind to the target enzymes more tightly than the substrate binds. Binding strengths are determined in part by the number of favorable contacts between the compounds. Since most enzyme substrates are small molecules, small molecule drugs can be engineered to make as many (or, if desired, more) contacts with the enzyme than does the substrate, facilitating tight enzyme interactions. As an illustration,
FIG. 1A
shows an enzyme
1
bound to its substrate
2
.
FIG. 1B
shows the enzyme
1
bound to a small molecule drug
3
. In addition, because enzymes work by lowering the energy of the transition state between substrate and product, the enzyme binds to especially tightly to transition state analogues. Drugs that resemble the transition state can bind more tightly to an enzyme than does the normal substrate, again affording an opportunity for antagonist-type drug design.
In contrast, it is generally more difficult to engineer small molecule compounds that block the interaction of a target protein with another protein, because interacting proteins usually contact each other over large surface areas and make many favorable contacts. It is therefore difficult for a small molecule to have a greater number of favorable contacts with a protein than does another protein. As an illustration,
FIG. 1C
shows two such interacting proteins,
4
and
5
, and indicates the large number of favorable contacts between these proteins. In addition, interacting proteins do not possess transition states analogous to those exhibited by substrates and their products. Thus, there is no specialized conformation that a drug can mimic to bind more effectively to a target protein.
There are many instances in which it is therapeutically useful to block the interaction of a target protein with another protein. Examples of biological events that involve protein-protein interactions include signal transduction, transcription, protein ligand-receptor interactions, and protein assembly.
The ability to block these processes specifically facilitates the development of therapies for diseases that are currently difficult to treat. Accordingly, compounds that block interactions such as those described above represent potentially useful drugs for treating, preventing, or reducing the severity of certain diseases or their symptoms. For example, viral infections (such as herpes, hepatitis C, HIV, and influenza infections) could be treated with compounds that block the assembly of viral proteins, or with compounds that prevent the ligand-receptor interaction of a virus attaching to a host cell.
SUMMARY OF THE INVENTION
The invention features a compound having a molecular weight of less than 1500 daltons that non-covalently interacts with, and covalently bonds to, a target protein at an amino acid side chain that is not part of an enzyme active site; a covalently bound portion of the compound sterically blocks the binding of the target protein to a second protein. The compound bonds to the target protein with a forward reaction rate that is at least 100 times faster than the forward reaction rate at which the compound bonds to the side chain of the corresponding free amino acid under physiological conditions. The compound may bond in such a way that essentially all, most, or only a small portion, of the compound remains covalently attached to the target protein; another portion of the compound may serve as a leaving group. For example, in some instances, only an acyl group, preferably an acetyl group, remains attached to the target protein.
A preferred compound bonds to the target protein with a forward reaction rate that is at least 1000 times faster than the forward reaction rate at which the compound bonds to the side chain of the free amino acid under physiological conditions, and more preferably bonds with a rate 10,000 times, or 100,000 times faster. The compound is preferably a synthetic compound.
In addition, a preferred compound has a covalent bonding rate constant with the side chain of the free amino acid of less than 10
−5
/M/sec at room temperature under physiological conditions, and more preferably has a rate constant of less than 10
−6
/M/sec, or 10
−7
/M/sec at room temperature under physiological conditions.
The non-specific covalent bonding rate constant for penicillin to form stable bonds with amino acid side chains, such as those of serine and lysine, is about 8×10
−6
/M/sec. Because penicillin is a useful drug whose side effects, which are results of its reactivity, are considered to be acceptable, it is expected that the side effects resulting from the non-specific reactivity of drugs with similar or smaller covalent bonding rate constants will also be acceptable.
A preferred compound also includes a Specificity Group whose removal results in the bonding reaction rate with the side chain of the corresponding free amino acid being substantially unchanged, and the bonding reaction rate with the target protein being reduced to a rate that is substantially similar to the bonding reaction rate with the side chain of the free amino acid; the compound also includes a Bonding Group that forms the covalent bond with the target protein. In this compound, modification of the Specificity Group does not substantially alter the bonding reactivity of the Bonding Group. In such preferred compounds, the Bonding Group and Specificity Group are connected by appropriate linkers, so that, for example, electronic effects are not transmitted from the Specificity Group to the Bonding Group. An example of an appropriate linker is an alkyl chain having 2-12 carbon atoms.
Preferred target proteins include kinases, viral coat proteins, STAT proteins, oncogenes, transcription factors, and extracellular protein ligands, protein domains, and their receptors. More specifically, preferred target proteins include MCP-1, Fos, and IL-1 beta. In a preferred compound, the Bonding Group forms a covalent bond with the side chain of the amino acid of the target protein as a result of the intrinsic reactivity of the Bonding Group.
The purpose of the Specificity Group is to direct the compound to a particular protein target and to position the Bonding Group near an amino acid side chain on the target protein; the Bonding Group will therefore have a high effective concentration, relative to the amino acid side chain, and will react with the side chain. An initial Specificity Group may be obtained by screening conventional chemical compound libraries for compounds that non-covalently interact with the target protein. Alternatively, the initial Specificity Group could be obtained by rational drug design, or by using a peptide that is known to bind to the target protein.
For comparison purposes, the side chain of the free amino acid that corresponds to the amino acid that forms a covalent bond to the B group is used, rather than a side chain on a non-target protein. The side chain of the corresponding free amino acid can always be clearly defined; in addition, it will not be subject to environmental influences, such as steric factors, that may vary from one non-target protein to the next.
It is useful, during the improvement of Bifunctional Blockers that is described in detail below, for the Bonding Group and Specificity Group to be connected so that modification of one Group has little effect on the other. In brief, improvement of Bifunctional Blockers is accomplished by systematically improving the Specificity Group and weakening the reactivity of the Bonding Group. There is an extensive body of knowledge, well known to those skilled in the art of organic chemistry, that predicts the relative reactivity of possible Bonding Groups. This knowledge can be used to systematically alter and weaken a Bonding Group during the course
Clark & Elbing LLP
Venkat Jyothsna
Way Jeffrey C.
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