Lawrence J. Marnett

One of the most prevalent exocyclic DNA adducts is 3-(2-deoxy-β-D-erythro-pentofuranosyl) pyrimido[1,2-α]purin-10(3H)-one (M1dG), an oxidative DNA lesion that forms by the reaction of guanines in nucleic acids and pool nucleotides with oxidation-induced base propenals or with the lipid peroxidation product, malondialdehyde. Further oxidation converts M1dG to 6-oxo-M1dG, an even more deleterious lesion whose genotoxic and mutagenic properties have been characterized in vitro. The present work uses a site-specifically modified viral genome to evaluate the biochemical consequences of 6-oxo-M1dG in Escherichia coli cells and contrast them with the properties of the M1dG parent lesion. We found that 6-oxo-M1dG strongly inhibited replication, with a bypass efficiency of 1–2%, relative to an unmodified guanine. By contrast, under the same experimental conditions, the bypass efficiency of M1dG was 30–40%. Beyond its low bypass rate, 6-oxo-M1dG was 20 times more mutagenic than M1dG, with the majority of mutations being single base deletions. However, when the levels of bypass polymerases were increased by inducing the SOS response, the proportion of deletions decreased, at the expense of additional single base substitutions, primarily G → T and G → C mutations. Finally, two DNA repair pathways─the direct reversal dioxygenase AlkB and glycosylase MutY─were investigated for their putative activity on 6-oxo-M1dG. The results indicated that neither system was capable of repairing this highly mutagenic lesion.

Prostaglandin endoperoxide H synthase (PGHS) is a heme-containing enzyme that catalyzes the conversion of arachidonic acid (AA) into prostaglandin H2. The anti-inflammatory mediator nitro-arachidonic acid (NO2AA) has been identified as a potent inhibitor of both PGHS-1 and PGHS-2 isoforms. While this inhibition involves the displacement of the essential heme prosthetic group from the active site, the underlying molecular mechanism remains incompletely elucidated. Herein, we investigated the inhibitory action of NO2AA against murine PGHS-2 (mPGHS-2) using site-directed mutagenesis targeting the cyclooxygenase (COX) and peroxidase (POX) active sites, combined with kinetic analysis and mass spectrometry. NO2AA dose-dependently inactivates both COX and POX activities. Mutations of key histidine residues -H386A, H207A, and H388F- abrogated this inactivation in WT-mPGHS-2. The apoenzyme incubated with NO2AA and then reconstituted with heme, as well as the holoenzyme inhibited by the addition of NO2AA and then reconstituted with heme, did not recover their enzymatic activity. Despite the potential for NO2AA to modify residues that coordinate the heme group, we were unable to detect covalent nitroalkylation at these sites, suggesting that NO2AA induces heme release through a non-covalent, irreversible mechanism. Nano-scale differential scanning fluorimetry (nanoDSF) confirmed that NO2AA triggers a selective disruption of heme stabilization within the protein structure, a process hindered by mutations at His-207, His-386, and His-388. These results robustly support a model where NO2AA interacts with the POX active site, with residue H207, among others, emerging as a crucial participant in the interaction that triggers heme displacement. These insights enhance our understanding of the NO2AA mechanism of action and underscore its potential as a scaffold for targeted anti-inflammatory drug development.

Currently, on-demand treatment of pain (both chronic and acute) is primarily achieved using opioids that are delivered systemically. Unfortunately, these drugs are highly addictive; over 5 people per hour die from opioid abuse in the US alone. A safer, nonsystemic mechanism for pain relief is therefore needed. Nonsteroidal anti-inflammatory drugs (NSAIDs) have been explored for this purpose; they are nonaddictive, provide excellent pain relief, and can be delivered locally to minimize dosage and systemic side effects. However, an on-demand release method is needed to make local delivery of these drugs a viable, convenient replacement for opioids; external stimulus-triggered release from an implantable depot is one approach. Stimuli such as heat, light, ultrasound, and RF electromagnetic radiation have been used to trigger release of various drugs from implantable drug depots; however, these require energy input and complex apparatus and are thus not comparable to the ease of oral administration. We propose localized cooling as a safe, convenient stimulus. As icepacks are already widely applied to temporarily ease local pain, introducing a drug delivery mechanism switched "ON" by cooling could enable long duration, enhanced pain relief triggered by a method with which patients are already familiar. Herein, we demonstrate that cooling-triggered release of NSAIDs can be achieved by leveraging the gel-to-sol transition exhibited by physically cross-linked thermoresponsive polymer hydrogels upon cooling below their lower critical solution temperature (LCST). We demonstrate and characterize cooling-triggered release in simulated body fluid, in cell culture, in explanted tissue, and in a live animal wound model. We show that hydrogels loaded with an NSAID (Celecoxib) can be combined with a nonthermoresponsive membrane material to create implantable devices that demonstrate up to a ∼40× increase in drug release rate upon mild cooling (29 °C) and that support multiple cycles of triggered release. These results demonstrate that cooling-triggered release of therapeutics is a promising concept that could allow patients to use a familiar method (applying an icepack to pain points) to achieve enhanced pain relief.

We report the coencapsulation of fluorocoxib Q (FQ) and chemocoxib A (CA) in micellar nanoparticles (FQ-CA-NPs) of a new PPS₁₃₅-b-POEGA₁₇ diblock polymer, which exhibited a hydrodynamic diameter of 109.2 ± 4.1 nm and a zeta potential (ζ) of -1.59 ± 0.3 mV. The uptake of FQ-CA-NPs by 4T1 mouse mammary cancer cells and intracellular cargo release were assessed by fluorescence microscopy that resulted in increased fluorescence in 4T1 cells compared to cells pretreated with celecoxib. The viability of primary human mammary epithelial cells (HMECs) or 4T1 mouse mammary carcinoma cells treated with FQ-CA-NPs were assessed, which showed decreased growth of 4T1 breast cancer cells but showed no effect on the growth of primary human mammary epithelial cells (HMECs). Intravenous dosing of FQ-CA-NPs in mice enabled ROS-induced cargo (FQ and CA) release and fluorescence activation of FQ and resulted in increased fluorescence in breast tumors compared to the tumors of animals pretreated with tempol or celecoxib, and minimum fluorescence was detected in the tumors of animals treated with nothing or empty-NPs. In addition, tumor tissues from treated animals were analyzed ex vivo by liquid chromatography-mass spectrometry (LC-MS)/MS, and identified increased levels of cargo delivery and retention in the tumor compared to tempol- or celecoxib-pretreated animal tumors. These in vivo and ex vivo results confirmed the targeted delivery of loaded NPs followed by ROS-mediated cargo release and fluorescence activation for targeted visualization of drug delivery in breast tumors and CA-induced therapeutic effect in an in vivo tumor growth inhibition assay and an ex vivo hematoxylin and eosin (H&E) staining of tumor tissues. Thus, coencapsulation of FQ and CA into polymeric micellar nanoparticles (FQ-CA-NPs) enabled their ROS-sensitive release followed by fluorescence activation and COX-2-dependent tumor targeting and retention in the visualization of CA delivery in solid breast tumors.

2-Arachidonoyl glycerol (2-AG) is the principal endogenously produced ligand for the cannabinoid CB1 and CB2 receptors (CBRs). The lack of potent and efficacious 2-AG ligands with resistance against metabolizing enzymes represents a significant void in the armamentarium of research tools available for studying eCB system molecular constituents and their function. Herein we report the first endocannabinoid glyceride templates with remarkably high potency and efficacy at CBRs. Two of our lead chiral 2-AG analogs, namely, (13S)- and (13R)-Me-2-AGs, potently inhibit excitatory neurotransmission via CB1 while they are endowed with excellent resistance to the oxidizing enzyme COX-2. Our SAR results are supported by docking studies of the key analog and 2-AG on the crystal structures of CB1.