Research

In the Raj lab, we develop novel probes, biosensors, molecular imaging agents, and new chemoselective reactions for global profiling of undruggable proteome, selective detection of lysine posttranslational modifications (PTMs), and the synthesis of mechanically interlocked structures, cyclic peptides, and bioconjugates. This research program leads to the discovery of novel protein biomarkers, enzyme inhibitors, affordable diagnostic tools for early detection of cancer, and endogenous protein partners thus facilitating the synthesis of biotherapeutics. We utilize organic chemistry tools, biochemical techniques, and structural characterization.

Project

Protein lysine methylation/acetylation is a reversible process controlled by lysine transferases and lysine erasers in human cells. Most studies on these lysine transferases and lysine erasers have focused mainly on their functions inside the nucleus. More than half of these lysine transferases and lysine erasers, which are present in the nucleus are also localized in the cytoplasm but very little is known about their functions in this subcellular compartment. The abnormalities in these enzymes are directly associated with cancers, inflammation and other diseases.Despite the critical importance of the lysine erasers in different subcellular compartments, there is a substantial gap between their global analysis and effective methods available to achieve it. The long-term goal of this research project is to develop a mechanistic understanding of how “lysine methylome” and “lysine acetylome” is maintained at a subcellular level and how these chemical markers modulate cell signaling. The current focus is to develop a new class of chemical tools that report on lysine erasers’ activities in living cells at a subcellular level.

Impact

These methods will have extensive applications in biomedical research by providing a better understanding of functions of lysine erasers in different subcellular compartments, the molecular mechanisms of diseases, thus assist in the discovery of novel protein biomarkers and combination-therapy for treatment of cancer.

Project

We are involved in the development of new chemical strategies for the synthesis of uniquely structured peptides and cyclic peptides. The unique structure of these peptides provides them extra-stability towards the degradation by enzymes. The design of these peptides is inspired by some classes of antimicrobial peptides produced by microbes in nature as a defense mechanism against other species. Also, these peptides mimic particular secondary structures. We are interested in utilizing these uniquely structured peptides and cyclic peptides for inhibiting Protein-Protein Interactions (PPIs), which are implicated in diseases and are mediated by secondary structures.

However, the laboratory synthesis of cyclic peptides has posed as a challenge because of the formation of undesirable dimers and cyclo-oligomers at high concentrations due to intermolecular reactions between linear peptides. We have recently developed a novel “CyClick” strategy for the cyclization of peptides that works in an exclusively intramolecular fashion thereby precluding the formation of dimers and oligomers via intermolecular reactions. The CyClick chemistry is highly chemoselective for the N-terminus of the peptide with a C-terminal aldehyde. In preliminary work, we have utilized this method for synthesis of various macrocycles of different ring sizes 12- to 24-membered rings with varying amino acid compositions, under mild reaction conditions. We seek to use the power of the “CyClick” strategy to synthesize libraries of cyclic peptides with photoaffinity tag in one pot followed by their incubation with cell lysate to discover new inhibitors of various protein targets.

Impact

This proposal would lead to the discovery of lead compounds for the synthesis of efficient therapeutics for various diseases. Our current focus is to develop antivirals for HIV, SARS-CoV-2 and anticancer agents.

Project

Lysine methylation is one of the most important Post-translational modifications(PTMs) of proteins because it regulates various biological processes including cell growth, division, gene expression, and DNA/RNA binding. Due to lysine’s unique structure, it undergoes mono, di, and tri-methylation; different levels of methylation give rise to different functions and localization within a cell. We focused our attention particularly on the mono- and di-methylation of lysine that occurs on both histones and non-histone proteins. Mono and di-methylated lysine PTMs have been implicated not only in transcriptional activation and silencing but the aberrant PTM levels have been linked to numerous diseases such as heart disease, cancer, and diabetes. Accordingly, there is great interest in mapping out the global role of mono and di-methylated lysine PTMs in development and diseases.

However, the identification of these methylation marks in the proteome is highly challenging because the addition of methyl groups leads to the negligible alteration in protein’s or peptide’s physicochemical properties. The first challenge is that methylation of lysine does not add a substantial steric bulk. Second, the lysine methylation does not neutralize their positive charge and does not significantly change their hydrophobicity. The main objectiveof this research work is to address these challenges by developingnew chemical tools with unique chemoselectivity towards mono- and di-methyl lysines. These chemical tools will form a strong covalent bond with methylated lysines in a selective manner, thus has the potential to detect methylated lysines on a global scale. Currently, there are no chemicals methods available in the literature to achieve this goal. We seek to achieve this by developing new chemical reactions selective for secondary and tertiary amines in a complex system under physiological conditions. The N-methylation of various nitrogen bases such as N6-methyladenine and N4-methylcytosine are also responsible for various pathogenesis and we will utilize these chemistries to detect N-methylations on DNA and RNA.

Impact

These methods will have extensive applications in biomedical research by providing a better understanding of how these modifications affect the cell state and the molecular mechanisms of diseases. This study would lead to the discovery of novel protein- and DNA biomarkers. It could also lead to the discovery of new therapies. These innovative tools for detecting PTMs would augment existing detection methods and expand the chemical tool kit available for epigenetics research.

Project

 Aldehydes are classified as cytotoxic because they damage DNA and proteins by forming interstrand cross-link in human cells. The human body tightly regulates the concentration of aldehydes by aldehyde dehydrogenases that convert toxic aldehydes to nontoxic acids. The mutation in the enzyme ALDH2*2 (present in approximately ~ 0.6 billion people worldwide) leads to accumulation of toxic aldehydes thus contribute to a variety of human diseases including cardiovascular diseases, diabetes, neurodegenerative diseases, stroke, and cancer. Despite the cytotoxicity associated with aldehydes, there are no pharmaceuticals available to eradicate these molecules. This is largely due to the lack of methods to measure the levels of multiple alkyl aldehydes in cells. The main goal of this research proposal is to fill the present gap in the range of available techniques to measure aldehyde levels in cells and the development of new therapeutics. We seek to develop a new family of chemical sensors to measure the total aldehyde levels inside the cell and the concentrations of individual aldehydes.

Impact

This proposal describes the development of new chemical sensors to monitor total aldehyde levels in cells. This research will enable a better understanding of how the excess of aldehydes and abnormalities in their metabolic pathways lead to various diseases including cancer, thus of extraordinary clinical interest as therapeutic targets. This study could lead to the development of a new and affordable point-of-care diagnostic tool and the discovery of new therapeutics for aldehyde-induced diseases.