Reactive Immunization

110

159

Discovering small-molecule modulators of protein-protein interactions is a challenging task because of both the generally noncontiguous, large protein surfaces that form these interfaces and the shortage of high-throughput approaches capable of identifying such rare inhibitors. We describe here a robust and flexible methodology that couples disruption of protein-protein complexes to host cell survival. The feasibility of this approach was demonstrated through monitoring a small-molecule-mediated protein-protein association (FKBP12-rapamycin-FRAP) and two cases of dissociation (homodimeric HIV-1 protease and heterodimeric ribonucleotide reductase). For ribonucleotide reductase, we identified cyclic peptide inhibitors from genetically encoded libraries that dissociated the enzyme subunits. A solid-phase synthetic strategy and peptide ELISAs were developed to characterize these inhibitors, resulting in the discovery of cyclic peptides that operate in an unprecedented manner, thus highlighting the strengths of a functional approach. The ability of this method to process large libraries, coupled with the benefits of a genetic selection, allowed us to identify rare, uniquely active small-molecule modulators of protein-protein interactions at a frequency of less than one in 10 million.M any regulatory processes in living organisms are often a consequence of specific protein-protein contacts, and interference with such interactions provides a means to exert control over cellular events. The de novo discovery of small molecules capable of disrupting such protein-protein complexes has been fraught with challenges, yielding very few inhibitors at a low success rate (1-3). These difficulties suggest that large, functionally diverse libraries might be essential for finding unique molecules that are capable of perturbing the intracellular levels of specific protein-protein interactions. The major challenge in sifting through such vast compound pools is the shortage of functional high-throughput assays for detection of the protein complex dissociation (4).Genetic selection is uniquely capable of identifying individual molecules with desired properties from large libraries by using whole cells as reporters and correlating host growth to a desired functional property. Unlike recently popularized affinity-based selections (5), an intracellular genetic selection can directly assay for effects on enzymatic activity or the modulation of a proteinprotein complex, thus bypassing the inherent limitations of in vitro approaches. Additionally, library members must function within the context of the entire host proteome, requiring positive candidates to have an enhanced level of selectivity for their target. This feature represents an important advantage over traditional screen-based methods in drug discovery by allowing both target affinity and selectivity to be simultaneously optimized. The application of a genetic selection to the identification of small-molecule modulators may yield both potent and selective activities as well as uniq...

Section: Discussionmentioning

confidence: 99%

A systematic method for identifying small-molecule modulators of protein–protein interactions

Horswill

Savinov

Benkovic

2004

110

159

“…5B, Lower). Covalent modification by cocaine of IgG (22) did not contribute measurably to the immune response, because antibody binding to free cocaine was reversible (data not shown).…”

Section: Antibodies To Modified Proteins In Mouse Andmentioning

confidence: 99%

Covalent modification of proteins by cocaine

Deng

Bharat²,

Fischman³

et al. 2002

Cocaine covalently modifies proteins through a reaction in which the methyl ester of cocaine acylates the -amino group of lysine residues. This reaction is highly specific in vitro, because no other amino acid reacts with cocaine, and only cocaine's methyl ester reacts with the lysine side chain. Covalently modified proteins were present in the plasma of rats and human subjects chronically exposed to cocaine. Modified endogenous proteins are immunogenic, and specific antibodies were elicited in mouse and detected in the plasma of human subjects. Covalent modification of proteins could explain cocaine's autoimmune effects and provide a new biochemical approach to cocaine's long-term actions.

“…These antibody catalysts were found to be very useful for synthetic organic chemistry (8,9) in that they have all of the good properties of natural aldolases with the added advantage that they accept a much broader range of substrates (10). In reactive immunization (11), unlike normal immunization, highly reactive chemicals are used as immunogens so that a chemical reaction occurs in the binding site of an antibody during its induction. An analogue of this chemical reaction will later become part of a catalytic event.…”

mentioning

confidence: 99%

The antibody catalysis route to the total synthesis of epothilones

Sinha

Barbas

Lerner

1998

Self Cite

An efficient monoclonal aldolase antibody that proceeds by an enamine mechanism was generated by reactive immunization. Here, this catalyst has been used in the total synthesis of epothilones A (1) and C (3). The starting materials for the synthesis of these molecules have been obtained by using antibody-catalyzed aldol and retro-aldol reactions. These precursors were then converted to epothilones A (1) and C (3) to complete the total synthesis.