M. Muralidhar Reddy

Identification of Candidate IgG Biomarkers for Alzheimer's Disease via Combinatorial Library Screening

The adaptive immune system is thought to be a rich source of protein biomarkers, but diagnostically useful antibodies remain unknown for a large number of diseases. This is, in part, because the antigens that trigger an immune response in many diseases remain unknown. We present here a general and unbiased approach to the identification of diagnostically useful antibodies that avoids the requirement for antigen identification. This method involves the comparative screening of combinatorial libraries of unnatural, synthetic molecules against serum samples obtained from cases and controls. Molecules that retain far more IgG antibodies from the case samples than the controls are identified and subsequently tested as capture agents for diagnostically useful antibodies. The utility of this method is demonstrated using a mouse model for multiple sclerosis and via the identification of two candidate IgG biomarkers for Alzheimers disease.

Seamless Bead to Microarray Screening: Rapid Identification of the Highest Affinity Protein Ligands from Large Combinatorial Libraries

Several approaches have been developed for screening combinatorial libraries or collections of synthetic molecules for agonists or antagonists of protein function, each with its own advantages and limitations. In this report, we describe an experimental platform that seamlessly couples massively parallel bead-based screening of one-bead one-compound combinatorial libraries with microarray-based quantitative comparisons of the binding affinities of the many hits isolated from the bead library. Combined with other technical improvements, this technique allows the rapid identification of the best protein ligands in combinatorial libraries containing millions of compounds without the need for labor-intensive resynthesis of the hits.

Immobilized peptides as high-affinity capture agents for self-associating proteins

There is currently great interest in the fabrication of protein-detecting arrays comprised of large numbers of immobilized protein capture agents. While most efforts in this arena have focused on the use of biomolecules such as antibodies and nucleic acid aptamers as capture agents, synthetic species have many potential advantages. However, synthetic molecules isolated from combinatorial libraries generally do not bind target proteins with the high affinity necessary for array applications. Here, we demonstrate that simple linear peptides bind dimeric proteins tenaciously when immobilized, although they exhibit only modest affinity in solution. These data show that high-affinity bidentate capture agents for dimeric proteins can be created by simply immobilizing modest-affinity ligands on a surface at high density, bypassing the requirement for careful optimization of linker length and geometry that is normally required to create a high-affinity solution bidentate ligand.

Rapid identification of improved protein ligands using peptoid microarrays

A rapid array-based protocol is presented by which a modest affinity protein-binding small molecule can be appended to a library of peptoids via click chemistry. The array can then be screened for improved ligands that exhibit a higher affinity for the protein target.

Stereospecific synthesis of trans-arachidonic acids.

An effective synthesis is described for the preparation of all four mono trans isomers of arachidonic acid via deoxidation of epoxide precursors with lithium diphenylphosphide and quaternization with methyl iodide.

Discovery of biomarkers for systemic lupus erythematosus using a library of synthetic autoantigen surrogates.

Antibodies to a wide range of self-antigens, including those directed against nucleic acids or nucleic acid-binding proteins are the essential biomarkers for diseases such as systemic lupus erythematosus (SLE). Highly complex libraries of nonamers consisting of N-substituted glycines (peptoids) were screened for compounds that bound IgG from patients with SLE and earlier, incomplete autoimmune syndromes. Peptoids were identified that could identify subjects with SLE and related syndromes with a high sensitivity (70%) and specificity (97.5%). Immobilized peptoids were used to isolate IgG from both healthy subjects and SLE patients that reacted with known RNA-binding proteins. In the case of SLE patients, the peptoid-purified IgG reacted with several autoantigens, suggesting that the peptoids are capable of interacting with multiple, structurally similar molecules. These results show that the measurement of IgG binding to peptoids can identify subjects with high levels of pathogenic autoantibodies.

Serum Antibody Biomarkers for Alzheimer's Disease

Background: For disease states that are brought about or exacerbated by an immune response, if one could rapidly identify the offending antibodies and/ or T-cells, and identify neutralizingmolecules specific for them, a revolution in the treatment of these diseases would result. Then it would be possible to specifically interdict responses to specific antigens. Because patients with Alzheimer’s disease (AD) exhibit neurodegeneration in several brain regions, and immune system activation, there may well be numerous antibodies in the serum of AD patients that are specific to the disease that can serve as a biomarker for the disease. Methods: A novel unbiased approach to identifying disease-related antibodies, using combinatorial organic chemistry, involves the production of peptoid microarrays. Peptoids are oligo-Nsubstituted glycines which retain the binding characteristics of peptides, are easy to synthesize, and have been shown to be rich sources of protein ligands. Seven different amines (e.g., piperonylamine, methoxyethylamine, diaminobutane, etc.) were used to make the peptoids. Results: Using a microarray consisting of 15,000 unique 8-mer peptoids and serum from 6 AD, 6 Parkinson’s disease (PD) and 6 normal control subjects, we identified 3 peptoids that capture antibodies whose levels are 3-10-fold higher in each AD patient compared to each PD and each normal control subject. A different set of peptoids was found that is elevated in both AD and PD serum samples. We believe that the peptoids mimic some aspect of the shape of the (unknown) native antigen(s) recognized by the AD-related antibodies, and thus serve as relatively high affinity capture agents. To validate the 3 ADspecific peptoids, we used 49 AD, 20 PD and 25 normal control subjects, and there was >90% sensitivity and >90% specificity for the identification of AD for each of the peptoid biomarkers. Conclusions: These peptoids should represent useful serum biomarkers for AD. Furthermore, the identification of the IgG antibodies that are bound to the peptoids may provide insight for novel drug targets for the treatment of the disease.