Scott Singleton, PhD

RESEARCH

Summary:

The Singleton laboratory focuses on understanding the molecular basis of the evolution and transmission of drug resistance in pathogenic microorganisms.
 
In particular, our group studies DNA recombination and repair in relation to stress responses and antibiotic resistance in bacterial pathogens.  We focus on RecA, a bacterial protein that controls the SOS response system for DNA damage tolerance.  SOS is an important inducible gene in regards to antibiotic resistance because upregulation and mutation of SOS can promote survival.  RecA functions to upregulate SOS and restarts stalled replication forks via recombinational DNA repair.  We develop small molecule RecA inhibitors via high-throughput screening with novel assays and reagents.  We have previously identified more than 10 unique classes of RecA inhibitors and has demonstrated that these inhibitors can increase the efficacy of ciprofloxacin, ampicillin, kanamycin, chloramphenicol, and mitomycin C.
 
In addition to RecA, our group investigates the RecBCD enzyme, which recognizes damage on chromosomes and prepares them for SOS processing, and EndA, which is an extracellular nuclease that is an important factor in the virulence of Streptococcus pneumonia.  Inhibiting these protein targets has the potential to enhance commercial development of antibacterial drugs and satisfy currently unmet antibiotic needs.
 
The group also investigates how physical factors influence drug residence times on biomolecular targets in order to enhance the ability to manipulate biological interactions.  We test hypotheses between ligand-binding and intrinsic protein dynamics with small molecule inhibitors of dyhydrofolate reductase (DHFR), an enzyme essential in all organisms for nucleotide biosynthesis.

Tools

  • chemical synthesis
  • synthetic biology
  • combintorial biochemistry
  • molecular biophysics
  • high-throughput screening

To Study

  • synthetic nucleotide analogs, designed peptides, organometallic complexes
  • DNA repair and recombination related to drug resistance
  • genomic approaches for revealing antibacterial drug targets
  • designed genetic selections for directed evolution of unnatural enzymes

To Understand

  • molecular mechanisms related to the evolution of drug resistance
  • protease inhibitor resistance in human viruses

To Discover

  • new antimicrobial targets and strategies
  • new paradigms for antimicrobial drug development
  • new lead compounds via HTS
  • new macromolecular therapeutics
  • new enzymes

REPRESENTATIVE PUBLICATIONS

  • Peterson EJ, Kireev D, Moon AF, Midon M, Janzen WP, Pingoud A, Pedersen LC, Singleton SF. Inhibitors of Streptococcus pneumoniae surface endonuclease EndA discovered by high-throughput screening using a PicoGreen fluorescence assay. J Biomol Screen. 2013 Mar;18(3):247-57.
  • Carroll MJ, Mauldin RV, Gromova AV, Singleton SF, Collins EJ, Lee AL. Evidence for dynamics in proteins as a mechanism for ligand dissociation. Nat Chem Biol. 2012 Jan 15;8(3):246-52.
  • Peterson EJ, Janzen WP, Kireev D, Singleton SF. High-throughput screening for RecA inhibitors using a transcreener adenosine 5'-O-diphosphate assay. Assay Drug Dev Technol. 2012 Jun;10(3):260-8.
  • Carroll MJ, Gromova AV, Miller KR, Tang H, Wang XS, Tripathy A, Singleton SF, Collins EJ, Lee AL. Direct detection of structurally resolved dynamics in a multiconformation receptor-ligand complex. J Am Chem Soc. 2011 Apr 27;133(16):6422-8.
  • Sexton JZ, Wigle TJ, He Q, Hughes MA, Smith GR, Singleton SF, Williams AL, YehLA. Novel Inhibitors of E. coli RecA ATPase Activity. Curr Chem Genomics. 2010 May 26;4:34-42.
  • Wigle TJ, Sexton JZ, Gromova AV, Hadimani MB, Hughes MA, Smith GR, Yeh LA, Singleton SF. Inhibitors of RecA activity discovered by high-throughput screening: cell-permeable small molecules attenuate the SOS response in Escherichia coli. J Biomol Screen. 2009 Oct;14(9):1092-101. 
  • Lee, A.M., Wigle, T.J., and Singleton, S.F., “Two-Tiered High-Throughput Screening-Compatible Assay for Inhibitors of RecA Activities,” Analytical Biochemistry (in press).
  • Wigle, T.J., and Singleton, S.F., “Directed Molecular Screening for RecA ATPase Inhibitors,” Bioorganic & Medicinal Chemistry Letters, DOI 10.1016/j.bmcl.2007.04.013 (2007).
  • Cline, D.J., Holt, S.L., and Singleton, S.F., “Inhibition of Escherichia coli RecA by a Rationally Redesigned Helical Peptide,” Organic & Biomolecular Chemistry, DOI DOI: 10.1039/B703159A (2007).
  • Singleton, S.F., Roca, A.I., Lee, A.M., and Xiao, J., “Probing the Structures of RecA-DNA Filaments. Advantages of a Flurescent Guanine Analog,” Tetrahedron 63: 3553-3566 (2007).
  • Lee, A.M., Xiao, J., and Singleton, S.F., “Origins of Sequence Selectivity in Homologous Genetic Recombination: Insights from Rapid Kinetic Probing of RecA-mediated DNA Strand Exchange,” Journal of Molecular Biology 360: 343-359 (2006).
  • Xiao, J., Lee, A.M., and Singleton, S.F., “Direct evaluation of a kinetic model for RecA-mediated DNA strand exchange: Importance of nucleic acid dynamics and entropy during homologous genetic recombination,” ChemBioChem 7: 1265-1278 (2006).
  • Lee, A.M. and Singleton, S.F., “Intersubunit Electrostatic Complementarity in the RecA Nucleoprotein Filament Regulates Nucleotide Substrate Specificity and Conformational Activation,” Biochemistry 45: 4514-4529 (2006).
  • Wigle, T.J., Lee, A.M., and Singleton, S.F., “Conformationally Selective Binding of Nucleotide Analogs to Escherichia coli RecA: A Ligand-Based Analysis of the RecA ATP-Binding Site,” to Biochemistry 45: 4502-4513 (2006).
  • Xiao, J., Lee, A.M., and Singleton, S.F., “Construction and Evaluation of a Kinetic Scheme for RecA-mediated DNA Strand Exchange,” Biopolymers 81: 473-496 (2006).

CONTACT INFO

324 Beard Hall
Campus Box # 7568
301 Pharmacy Lane
Chapel Hill, NC 27599

Office: 919-966-7954

sfs@email.unc.edu

Singleton Lab Website