Dr. Lindsay R. Comstock-Ferguson

Lindsay ComstockAssistant Professor

B.S., 2000, Northern Arizona University
Ph.D. (Pharmaceutical Sciences), University of Wisconsin-Madison
Postdoc (Biomolecular Chemistry, J.M. Denu), University of Wisconsin-Madison

Office: Salem 16A
Phone: (336) 758-5514
Email: comstolr@wfu.edu

Research Information

Traditionally, post-translational modifications have been difficult to study in native environments due to their inherent small size and recent efforts have focused on developing new strategies to identify both the substrate and specific location of these modifications on complex biological surfaces. While a variety of approaches are currently being pursued by others, we are developing a novel set of small molecules that exploit cellular machinery to transfer unique chemical functionalities to biological substrates. This approach utilizes both cellular enzymes and non-native cofactor mimics as biochemical tools to identify and isolate modified substrates. Instead of generating the native modification depicted here, a ligatable handle (R) is enzymatically transferred to the substrate. Once incorporated, this non-native, chemical functionality can be modified through various chemoselective ligations, such as the Staudinger ligation or Hüisgen [2+3] cycloaddition, to permit visualization of the modified substrate and hasten substrate isolation and identification from complex environments.

LRComstockResearch_clip_image002Utilizing this enzyme-based approach, two independent  projects unified by the common theme of exploring post-translational  modifications through cofactor mimics bearing ligatable handles, such as azides  and alkynes, is being pursued.  The first  project incorporates the synthesis of modified analogs of adenosine  triphosphate (ATP) and their subsequent biochemical evaluation as cofactors for  cellular kinases.  The second project entails  generating synthetic analogs of S-adenosyl-L-methionine   (SAM) bearing ligatable functionalities and evaluating their methyltransferase-dependent  modification of biological targets.

Ligatable Analogs of ATP as Probes  of Cellular Kinases and Their Substrates

Phosphorylation has been demonstrated to be the most  universal regulatory mechanism of protein function. Carried out by kinases,  phosphorylation occurs primarily on either a serine, threonine, or tyrosine  residue and results in a functional change of the target protein by altering  enzyme activity, cellular location, or its association with other proteins.   Aberrant expression of kinases has been linked to a variety of cancers,  leukemias, and neurodegenerative diseases and challenges still remain in identifying  the role of phosphorylation defects in these altered states.  With more than 500 known kinases in the human  genome, one of the largest tasks has been to identify the substrates of  specific kinases and the pathways in which they are involved.  While chemical genetics has emerged at the  forefront in developing new methodologies to investigate the role of cellular  kinases in physiological function, this methodology typically lacks generality  amongst a wide variety of kinases.

To circumvent this, an alternate approach is taken  here to develop a more universal analog of ATP that bears the ligatable handle  and is tolerated by several classes of kinases.   As depicted in the crystal structure of CK2, a tyrosine kinase, the g-phosphate of ATP is solvent  accessible and the incorporation of a small functionality at this position is  well-tolerated.  Thus, ATP analogs  bearing a ligatable handle on the g-phosphate would not only bind in the active site,  but also transfer the modified phosphate to substrate.  A small library of cofactor mimics is being synthesized bearing modified  linker structures and/or lengths between the g-phosphate and either alkynes or  azides, creating a diverse library of compounds to explore phosphorylation biology.


Development and Biochemical Evaluation of Ligatable Analogs of S-adenosyl-L-Methionine

Methylation of nucleic acids has been shown to play a  pivotal role in controlling cellular function through gene silencing, and  protein methylation, specifically on histones, is essential for transcriptional regulation via chromatin remodeling.  Aberrant  methylation of proteins/DNA is often responsible for the onset of disease  states, including carcinogenesis.  Catalyzing  the transfer of a methyl group from the naturally-occurring cofactor SAM, methyltransferases  function in either a site- or sequence-specific fashion.  Although this modification is highly efficient, the incorporation of a methyl group onto a  DNA base or amino acid can often traditionally been difficult to detect due to  its small size.  While strategies for detecting  methylated proteins and DNA have improved, the advent of cofactor mimics that utilize  naturally occurring enzymes may hold tremendous value in future research  efforts.

A novel approach in  exploring methyltransferases using synthetic cofactor mimics is illustrated in  the following reaction scheme.   Highlighting the utility of DNA methyltransferase M.TaqI, cofactor  mimics containing an aziridine ring in  lieu of the methyl-sulfonium (2)  undergo enzyme-dependent DNA alkylation.   Instead of generating the N6-methylated adenine  residue, substrate adenylation results from ring-opening of the aziridine to form  the modified-DNA complex.  While chemical  modifications (i.e. attachment of  biotin or fluorophore) of aziridine 2 have been carried out to improve its utility for biological imaging, such  agents are synthetically difficult to obtain.   Recent efforts introduce small ligatable functionalities to the aziridine  base of 2 and demonstrate their  versatility in undergoing chemoselective ligations on DNA.  Interestingly, synthetic cofactors of SAM are not restricted to 5′-aziridines,  as N-mustard containing analogs  generate the aziridinium in situ prior  to methyltransferase-dependent transfer.   The structural core of these N-mustards   serve as the basis for creating a diverse library of second generation cofactor  mimics bearing alkynes or azides to continue recent efforts in exploring  methylation biology.


Recent Publications

Mai, V. and Comstock, L.R. “Synthesis of an azide-bearing N-mustard analog of S-Adenosyl-L-methionine.” J. Org. Chem.,  76, 10319-10324.

Representative Publications

Comstock, L.R. and Rajski, S.R. “Expeditious synthesis of aziridine-based cofactor mimics.” Tetrahedron 2002, 58, 6019-6026.

Restituyo, J.A., Comstock, L.R., Petersen, S.G.,Stringfellow, T. Rajski, S.R. “Conversion of aryl azides to O-alkyl imidates via modified staudinger ligation.” Org. Lett. 2003, 5, 4357-4360.

Comstock, L.R.; Rajski, S.R. “Efficient synthesis of azido-based cofactor mimics.” J. Org. Chem. 2004, 69, 1425-1428.

Comstock, L.R., Rajski, S.R. “Conversion of DNA methyltransferases into azidonucleosidyl transferases via synthetic cofactors.” Nucleic Acids Res. 2005, 33, 1644-1652.

Comstock, L.R., Rajski, S.R. “Methyltransferase-directed DNA strand scission.” J. Am. Chem. Soc. 2005, 127, 14136-14137.

Weller, R.L. and Rajski, S.R. “DNA methyltransferase-moderated click chemistry.” Org. Lett. 2005, 7, 2141-2144.

Weller, R.L. and Rajski, S.R. “Design, synthesis, and preliminary biological evaluation of a DNA methyltransferase-directed alkylating agent.” ChemBioChem 2006, 7, 243-245.

Rajski, S.R, Comstock, L.R, Weller, R.L. “Synthetic cofactor analogs of S-adenosylmethionine as ligatable probes of biological methylation and methods for their use.”  US Patent 20071610072007.