The Zondlo Research Group
Laboratory of Organic Chemistry
and Molecular Design
Our research focuses on the design, synthesis and development of small molecules and minimalist polymers with biological activity, the elucidation of fundamental principles of and discovery of effectors of biological interactions, the development of novel, functional proteins, and the development of novel and practical methods of synthesis and enantioselective catalysis.
Our interests center on the general area of functional molecular recognition: the generation of novel molecular structures and architectures which interact specifically with target molecules. Biological targets represent a considerable test of our knowledge of the fundamental principles of molecular recognition. Effective modulation of biological events requires the generation of molecules with both high affinity and high specificity for the desired target. Researchers in my group utilize modern methods of organic synthesis and catalysis, combinatorial synthesis and high-throughput analysis, solid-phase and expression-based peptide and protein synthesis, advanced NMR and computational analysis, molecular biology, and biological assays.
PROTEIN PHOSPHORYLATION AND POST-TRANSLATIONAL MODIFICATIONS
The complexity of humans is dependent on post-translational modifications of proteins. The most common post-translational modification is the phosphorylation of serine, threonine, and tyrosine residues by protein kinases. Protein kinases are tightly regulated, and changes in kinase activity are associated with most human diseases, including cancer, heart disease, and Alzheimer's disease. We are developing approaches to understand how protein phosphorylation changes the structure of proteins toward understanding the mechanisms associating changes in kinase activity with human diseases. In addition, we are developing new tools to understand the changes in kinase activity associated with human disease. We have designed new protein structures, called protein kinase-inducible domains, whose structures are dependent on their phosphorylation state. These designed proteins are under the control of specific protein kinases and are non-fluorescent when not phosphorylated, but highly fluorescent when phosphorylated, and may act as genetically encoded sensors of protein kinase activity.
SMALL MOLECULE PROTEOMIMETICS
Work in genomics and proteomics is revealing vast numbers of interaction loci within the cellular milieu, and thus vast numbers of potential targets for agonists and antagonists of protein-protein, protein-DNA and protein-RNA interactions. Our focus is the use of molecular design and organic synthesis to develop small molecules which mimic larger biological structures. Our work involves the development of appropriate, readily accessible organic scaffolds, in solution and on solid phase, using modern methods of organic synthesis. To evaluate our scaffolds they are subjected to high-throughput testing for biological activity. Modularity in synthesis allows the combination of multiple structural elements to allow recognition of larger protein surfaces and the synthesis of multifunctional "proteins." The post-genomic era requires novel tools to elucidate the identity, classes and modes of protein-protein interactions in disparate cell types, developmental stages, and intracellular environments, in addition to changes due to age and disease states. The ability to generate small molecule mimics of protein recognition elements permits their use as chemical probes of protein-protein interactions.
PROTEIN MISFOLDING AND DISEASE
Numerous diseases, including Alzheimer's disease, Parkinson's disease and spongiform encephalopathies (i.e. mad cow disease and its human variants), are characterized by protein misfolding and precipitation which is central to the observed pathology. We are interested in the molecular mechanisms leading from the soluble, monomeric protein to the insoluble, polymeric protein forms. We are particularly interested in understanding the molecular mechanisms of Alzheimer's disease. Alzheimer's disease is characterized by two protein aggreagates in the brain: extracellular plaques and intracellular neurofibrillary tangles (NFTs). The major protein in neurofibrillary tangles is a hyperphosphorylated version of the protein tau. Tau normally stabilizes the elongated structure of neurons by binding to the microtubules. However, hyperphosphorylation of tau, phosphorylation on over 30 residues of tau, results in structural changes in tau that cause the precipitation of tau in neurofibrillary tangles. We are examining the molecular mechanisms by which hyperphosphorylation of tau leads to structural changes, protein aggregation, and neurofibrillary tangle formation. We have found that phosphothreonine induces a particularly ordered conformation.
SYNTHETIC METHODOLOGY: CROSS-COUPLING REACTIONS, STEREOSELECTIVE SYNTHESIS, AND SELECTIVE CATALYSIS
We are interested in developing enhanced proteins via the development of practical approaches to unnatural amino acids to complement the natural 20 amino acids. We have developed approaches to synthesize unnatural proline analogues in a manner to allow control of structure and the introduction of novel functional groups within proteins, including groups for spectroscopic probes and for bioorthogonal chemistry. We have become particularly interested in the amino acid 4-thiophenylalanine, a redox-active analogue of tyrosine with a reduced pKa (pKa 6) and enhanced reactivity. 4-Thiophenylalanine is a versatile probe of sulfur redox chemistry and is a functional hybrid of tyrosine and cysteine. In addition, effective, selective catalysis requires molecular functionality sufficient for catalytic activity, (regio- and stereo-) discrimination in substrate recognition enforced by reproducible transition state geometry, rapid association of substrate and dissociation of product to ensure turnover, and a partially open geometry to allow significant substrate scope. The incorporation of catalytic functionality within a designed structure is an important goal, and provides a critical test of our knowledge of the fundamental principles of molecular folding and catalysis. Due to their inherent chirality and structure, peptides and designed proteins are ideally situated to function as highly effective catalysts, despite limited success to date. Our approach is to design novel peptides cabable of functioning as effective catalysts. A second element to catalyst discovery is the ability to rapidly screen catalyst candidates. To address the scope of combinatorial space, both in terms of catalyst structure and substrate generality, we are developing new methods for high-throughput screening for catalysis. These methods are designed to be applicable not only to the discovery of protein- and peptide-based catalysts, but also toward the discovery of metal-complex-based catalysts and organocatalysts.
ELECTRONIC AND STEREOELECTRONIC EFFECTS IN PROTEINS AND THE DESIGN OF TUNABLE PROTEINS
One hallmark of native, functional proteins is the adoption of a stable, highly conformationally restricted ensemble of closely related structures. In peptides, small proteins, and natively disordered proteins, where the hydrophobic effect is reduced, it is difficult to overcome the entropic cost of conformational restriction. Strategies to enable conformational restriction and stabilization of peptides and proteins may be used to interrogate protein structure-function relationships and to develop novel mediators of biological activity. We are developing new approaches to control local conformation via small peptide motifs and practical and readily applied organic synthesis. We are examining two approaches to control protein conformation: controlling main chain conformation via controlling the ring pucker of proline residues, and controlling cis-trans isomerization via tuning of proline and/or aromatic ring electronics. We have demonstrated that the interactions between aromatic residues and proline residues are controlled by aromatic electronics. We are developing approaches to tunably control the structures of peptides and proteins using stereoelectronic effects to control proline conformation and the electronic effects of aromatic rings to control the interactions between aromatic rings and proline. We are applying these approaches to stabilize protein secondary structures in peptides, to stabilize protein structures, to develop novel inhibitors of protein-protein interactions, and to design tunable proteins which are able to act conditionally, producing one structure or response in one environment and producing a contrary structure or response in a different environment.
Costantini, N. V.; Ganguly, H. K.; Martin, M. I.; Wenzell, N. A.; Yap, G. P. A.; Zondlo, N. J. "T h e d i s t i n c t c o n f o r m a t i o n a l l a n d s c a p e s o f 4 S