Research

Light-Driven Tools to Study Biological Systems

Photochemical methods in which light is used to trigger the release of a bioactive compound into a physiological environment are ideal for studying biological function and enable the conduct of experiments that would be difficult by other means.

Light-driven tools enable a high level of control of the timing, location, and amplitude of an event in a relatively non-invasive manner, especially when 2-photon excitation (2PE) is employed (Figure 1). 2PE enables activation of a bioactive messenger in a small volume relative to mammalian cells (1 fL, i.e., the volume of a single E. coli bacterium). Other advantages to 2PE include less photodamage to biological tissues and the ability to conduct experiments deeper inside tissues than can be achieved using conventional 1-photon excitation (1PE).

The Dore Laboratory designs, synthesizes, studies, and develops applications for photochemically active molecules, particularly those with sensitivity to 2PE. We were the first to recognize that 7-hydroxyquiolines could be used as photoremovable protecting groups (PPGs) to mediate the activation of biological effectors through 1PE or 2PE and conducted extensive work to understanding their complex photochemistry of releasing bioactive messengers. We also devise strategies to improve the sensitivity to 2PE of PPGs otherwise insensitive to this excitation mode.

We apply our photoactivation platforms to study neuron function, manipulate gene expression, and activate signal transduction pathways to address problems in neuroscience, developmental biology, and physiology and inform intervention in disease states, such as schizophrenia, addiction, epilepsy, birth defects, mood disorders, cancer, and others.

Figure 1. Comparison of the spatial selectivity of 1- and 2-photon excitation.

CaaX Proteases

The Ras subfamily of small GTP-binding proteins are CaaX proteins that play a prominent role in carcinogenesis; therefore, Ras proteins and Ras-regulatory proteins are considered targets for anticancer therapeutics.

The C-terminal end of the nascent protein contains a 4-residue motif where C is cysteine, a is typically an aliphatic amino acid, and X is one of several amino acids. Three post-translational modifications are critical for the proper localization and proper function of CaaX proteins (Figure 2): farnesylation by farnesyl transferase (FTase) or geranylgeranylation by geranylgeranyl transferase (GGTase); proteolysis by Ras converting enzyme endoprotease (Rce1); and carboxyl methyl esterification by isoprenylcysteine carboxyl methyltransferase (ICMT). Loss of Rce1 activity causes Ras mislocalization, diminished growth of Ras-transformed fibroblasts, and hypersensitivity to farnesyl transferase inhibitors.There is evidence that suggests Rce1 is essential for Plasmodium sp. and Trypanosoma brucei growth and it regulates the pathogenesis of Staphylococcus aureus and Streptococcus pneumonia.

Rce1 is membrane-bound and ER localized and crystal structure data from an Rce1 homolog in Methanococcus maripaludis point to a novel catalytic mechanism involving a histidine and glutamate-activated water molecule serving as the nucleophile. In collaboration with Walter Schmidt, we have discovered molecular scaffolds that provide the basis to design selective and potent inhibitors of Rce1. Small-molecule inhibitors of this protease would provide insight into the enzyme’s mechanism of action and could potentially have therapeutic value. Our approach involves computational modeling, virtual screening, and synthesis of hits for biochemical and cell-based assays, CaaX sequence mutations to probe the impact of the CaaX box on Ras localization and downstream signaling, and peptide mimetics to probe the proteolytic mechanism and develop a pharmacophoric model.

Figure 2. Canonical post-translational modifications to Ras and other CaaX proteins.

Target-Directed Discovery

We are leveraging our expertise in medicinal chemistry, structure-based drug design, and computational modeling and virtual screening to identify, design, and synthesize molecules to target proteins of therapeutic value. The goal is to deliver compounds with the desired pharmacodynamics and pharmacokinetic and toxicological profiles to potentially become leads for drug discovery. Our portfolio of targets includes proteins that are critical for the

  • survival of coronaviruses and related viruses, such as enteroviruses, coxsackieviruses, and rhinoviruses, whose members include the causative agents of influenza and the common cold; 
  • regulation of the life cycle of the malaria parasites Plasmodium falciparum and P. vivax; and 
  • process of tumorigenesis.