Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-06-02
2004-04-20
Celsa, Bennett (Department: 1639)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C436S518000, C530S300000
Reexamination Certificate
active
06723517
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the use of self-assembled monolayers attached to surfaces for the detection and probing of target molecule structure and function.
BACKGROUND OF THE INVENTION
Combinatorial chemistry techniques are used to synthesize diverse “libraries” of unique chemical compounds. These small molecule libraries often yield drug candidates that are capable of binding a specific biological target but because of their small size and relative simple chemical makeup, they characteristically interact with the target in a low affinity interaction. These low affinity interactions cannot adequately compete with larger more diverse natural ligands, like proteins and protein complexes, and thus provide little therapeutic value. Natural products, which are naturally occurring organisms isolated from soils, yeast, marine organisms, and the like are larger and chemically more interesting than small molecules from combinatorial libraries. Natural products are routinely screened for therapeutic activity against disease-related organisms. Many cancer drugs have been identified in this way. The problem with developing a natural product for the drug market is that they are large and chemically complicated, which means that elaborate and expensive schemes for their synthesis must be developed. Identifying a synthetic scheme that is commercially feasible is a technical challenge that at best takes years and millions of dollars to accomplish and at worst cannot be done. For this reason, there is interest in enhancing the affinity between small molecule drugs and their biologically relevant targets.
Knowles and colleagues, at Harvard, reported that they could enhance the binding affinity of a small molecule for a particular target by attaching a “greasy tail” to the small molecule. This hydrophobic tail was later shown to interact with a hydrophobic patch on the target molecule adjacent to the binding site.
Many biologically relevant target molecules present more than one binding site for a particular ligand. Some present pseudo identical binding sites with which they bind natural ligands that contain “repeats” of a binding motif. It is known that bivalent interactions (like antibody interactions) are higher affinity interactions than monovalent interactions, due to the cooperative binding effect. Therefore, one would like to link several small molecule drugs together to form a pseudo multivalent drug that would interact more strongly with a multi-binding-site target molecule. The problem with this logic is that the enthalpic advantage of the additional binding energy is offset by the large entropic energy cost of ordering the connected binding moieties. However, making the linker between the binding moieties a rigid linker would introduce order and thus minimize the entropic cost to yield a higher affinity interaction. In order to connect two binding moieties (the small molecule drugs) with a rigid linker, in a geometry that would encourage its binding to the target molecule, one would need to know apriori the distance between the binding sites on the target molecule. This inter-binding-site distance information is currently derived from X-ray or NMR structure determination of the target molecule. This process is time-consuming (years) and expensive.
The subject of this invention is how (self-assembled monolayers (SAMs) can be used to present discrete binding moieties, at varying densities, in a rigid 2-dimensional array, to multivalent target molecules in order to promote a higher affinity, cooperative interaction. Ligand densities within the SAM are varied to determine the critical distance between binding moieties that will promote simultaneous, cooperative binding of the target molecule. By monitoring the kinetics of binding events between the target molecule and the variable density ligand surfaces, one can empirically determine the lowest surface density that prompts a large shift in affinity for the multivalent target molecule. One can then use Poisson statistics to infer the distance between surface-immobilized ligands and thus also the distance between the binding sites on the target molecule. Once this distance information has been deduced, it can be used to rationally design bi- or multi-valent drugs or rigid-linkers to connect two binding moieties. Alternatively, the SAM itself can become a part of the “drug”; in this case, the SAM is used as the “rigid linker” between binding moieties to present multiple binding motifs, at the empirically determined critical density, to promote the higher affinity cooperative interaction. The SAM, presented ligands and underlying gold (may be gold colloids) are both the drug and the drug delivery system. Inert thiols of the SAMs can be terminated with lipid-like groups to facilitate drug delivery. Similarly, a biospecific ligand could be incorporated (at varying densities) into a liposome, at the critical presentation density determined, and used directly as a multivalent drug in its own delivery system.
SUMMARY OF THE INVENTION
Self-assembled monolayers are used as a rigid 2-dimensional matrix for presenting binding moieties, at varying distances from each other, to a target molecule. Two-component SAMs incorporate an inert spacer molecule and a biospecific molecule that can directly or indirectly present a binding moiety to a target molecule. The distance between the biospecific molecules in the array, the ligand density, is controlled by manipulating the concentrations of the two component thiols in solution before deposition onto gold. The affinity of the interaction between the surface immobilized ligands and the multivalent target molecule is monitored as a function of ligand density. The lowest ligand surface density that elicits a jump in affinity for the target molecule contains the critical information needed to extract the distance between binding sites on the target molecule. The dimensions of the hexagonal tiling pattern formed when the sulfurs from the thiols bind to gold solid are known. Therefore, Poisson statistics can be used to infer the distance between surface immobilized ligands, and thus the inter-binding-site distance on the target molecule, from the concentrations of the thiols in solution. Further, the gold surface itself and the attached SAM can be used as a scaffold to present binding moieties, in a controlled, higher affinity geometry, to a target molecule.
In a preferred embodiment, SAMs are generated that incorporate two thiol types: 1) an inert-tri-ethylene glycol-terminated thiol and 2) a nitrilo tri-acetic acid (NTA) terminated thiol that when complexed with Ni, captures histidine-tagged proteins or peptides. The density of NTA-thiol within the SAM is varied to present varying densities of a histidine-tagged binding moiety to a multi-valent target molecule. The affinity of the interaction is plotted as a function of ligand density within the SAM. A dramatic increase in the binding affinity occurs at a critical surface density when the presented ligands are close enough to each other to simultaneously bind to a common target molecule. The solution concentrations of the two thiol types and the dimensions of the tiling pattern that the thiols form on the gold substrate are input into Poisson distribution equations to extract the probable distance between binding sites on a target molecule.
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Ul
Celsa Bennett
Minerva Biotechnologies Corporation
Wolf Greenfield & Sacks PC
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