Nicholas W. Griggs

Glucose and SIRT2 reciprocally mediate the regulation of keratin 8 by lysine acetylation.

Keratin 8 lysine acetylation, which is enhanced by hyperglycemia and reduced by SIRT2, alters filament organization and reduces solubility.

Dynamic coupling and allosteric networks in the α subunit of heterotrimeric G proteins

G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gαi. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function.

Further Optimization and Evaluation of Bioavailable, Mixed-Efficacy μ-Opioid Receptor (MOR) Agonists/δ-Opioid Receptor (DOR) Antagonists: Balancing MOR and DOR Affinities.

In a previously described peptidomimetic series, we reported the development of bifunctional μ-opioid receptor (MOR) agonist and δ-opioid receptor (DOR) antagonist ligands with a lead compound that produced antinociception for 1 h after intraperitoneal administration in mice. In this paper, we expand on our original series by presenting two modifications, both of which were designed with the following objectives: (1) probing bioavailability and improving metabolic stability, (2) balancing affinities between MOR and DOR while reducing affinity and efficacy at the κ-opioid receptor (KOR), and (3) improving in vivo efficacy. Here, we establish that, through N-acetylation of our original peptidomimetic series, we are able to improve DOR affinity and increase selectivity relative to KOR while maintaining the desired MOR agonist/DOR antagonist profile. From initial in vivo studies, one compound (14a) was found to produce dose-dependent antinociception after peripheral administration with an improved duration of action of longer than 3 h.

Asymmetric Synthesis and in Vitro and in Vivo Activity of Tetrahydroquinolines Featuring a Diverse Set of Polar Substitutions at the 6 Position as Mixed-Efficacy μ Opioid Receptor/δ Opioid Receptor Ligands

We previously reported a small series of mixed-efficacy μ opioid receptor (MOR) agonist/δ opioid receptor (DOR) antagonist peptidomimetics featuring a tetrahydroquinoline scaffold and showed the promise of this series as effective analgesics after intraperitoneal administration in mice. We report here an expanded structure-activity relationship study of the pendant region of these compounds and focus in particular on the incorporation of heteroatoms into this side chain. These analogues provide new insight into the binding requirements for this scaffold at MOR, DOR, and the κ opioid receptor (KOR), and several of them (10j, 10k, 10m, and 10n) significantly improve upon the overall MOR agonist/DOR antagonist profile of our previous compounds. In vivo data for 10j, 10k, 10m, and 10n are also reported and show the antinociceptive potency and duration of action of compounds 10j and 10m to be comparable to those of morphine.

Lamin aggregation is an early sensor of porphyria-induced liver injury

Summary Oxidative liver injury during steatohepatitis results in aggregation and transglutaminase-2 (TG2)-mediated crosslinking of the keratin cytoplasmic intermediate filament proteins (IFs) to form Mallory-Denk body (MDB) inclusions. The effect of liver injury on lamin nuclear IFs is unknown, though lamin mutations in several human diseases result in lamin disorganization and nuclear shape changes. We tested the hypothesis that lamins undergo aggregation during oxidative liver injury using two MDB mouse models: (i) mice fed the porphyrinogenic drug 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and (ii) mice that harbor a mutation in ferrochelatase (fch), which converts protoporphyrin IX to heme. Dramatic aggregation of lamin A/C and B1 was noted in the livers of both models in association with changes in lamin organization and nuclear shape, as determined by immunostaining and electron microscopy. The lamin aggregates sequester other nuclear proteins including transcription factors and ribosomal and nuclear pore components into high molecular weight complexes, as determined by mass-spectrometry and confirmed biochemically. Lamin aggregate formation is rapid and precedes keratin aggregation in fch livers, and is seen in liver explants of patients with alcoholic cirrhosis. Exposure of cultured cells to DDC, protoporphyrin IX or N-methyl-protoporphyrin, or incubation of purified lamins with protoporphyrin IX, also results in lamin aggregation. In contrast, lamin aggregation is ameliorated by TG2 inhibition. Therefore, lamin aggregation is an early sensor of porphyria-associated liver injury and might serve to buffer oxidative stress. The nuclear shape and lamin defects associated with porphyria phenocopy the changes seen in laminopathies and could result in transcriptional alterations due to sequestration of nuclear proteins.

CD73 (ecto‐5′‐nucleotidase) hepatocyte levels differ across mouse strains and contribute to mallory‐denk body formation

Formation of hepatocyte Mallory‐Denk bodies (MDBs), which are aggregates of keratins 8 and 18 (K8/K18), ubiquitin, and the ubiquitin‐binding protein, p62, has a genetic predisposition component in humans and mice. We tested the hypothesis that metabolomic profiling of MDB‐susceptible C57BL and MDB‐resistant C3H mouse strains can illuminate MDB‐associated pathways. Using both targeted and unbiased metabolomic analyses, we demonstrated significant differences in intermediates of purine metabolism. Further analysis revealed that C3H and C57BL livers differ significantly in messenger RNA (mRNA) level, protein expression, and enzymatic activity of the adenosine‐generating enzyme, ecto‐5′‐nucleotidase (CD73), which was significantly lower in C57BL livers. CD73 mRNA levels were also dramatically decreased in human liver biopsies from hepatitis C and nonalcoholic fatty liver disease patients. Feeding mice with a diet containing the MDB‐inducing agent, 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC), significantly decreased CD73 protein and activity in C57BL livers and resulted in loss of plasma membrane CD73 expression and activity in isolated mouse hepatocytes. To further examine the role of CD73 in MDB formation in vivo, we fed wild‐type (WT) and CD73−/− mice a DDC‐containing diet. Liver enlargement, p62 induction, and disappearance of the K8/K18 cytoskeleton were attenuated in CD73−/−, compared to WT livers. MDB formation, as assessed by biochemical and immunofluorescence detection of keratin and ubiquitin complexes, was nearly absent in CD73−/− mice. Conclusion: Purine metabolism and CD73 expression are linked to susceptibility to MDB formation in livers of different mouse strains. Expression of the adenosine‐generating enzyme, CD73, contributes to experimental MDB induction and is highly regulated in MDB‐associated liver injury in mice and in chronic human liver disease. (Hepatology 2013;58:1790–1800)

Effects of N-Substitutions on the Tetrahydroquinoline (THQ) Core of Mixed-Efficacy μ-Opioid Receptor (MOR)/δ-Opioid Receptor (DOR) Ligands.

N-Acetylation of the tetrahydroquinoline (THQ) core of a series of μ-opioid receptor (MOR) agonist/δ-opioid receptor (DOR) antagonist ligands increases DOR affinity, resulting in ligands with balanced MOR and DOR affinities. We report a series of N-substituted THQ analogues that incorporate various carbonyl-containing moieties to maintain DOR affinity and define the steric and electronic requirements of the binding pocket across the opioid receptors. 4h produced in vivo antinociception (ip) for 1 h at 10 mg/kg.

Benzylideneoxymorphone: A new lead for development of bifunctional mu/delta opioid receptor ligands

Opioid analgesic tolerance remains a considerable drawback to chronic pain management. The finding that concomitant administration of delta opioid receptor (DOR) antagonists attenuates the development of tolerance to mu opioid receptor (MOR) agonists has led to interest in producing bifunctional MOR agonist/DOR antagonist ligands. Herein, we present 7-benzylideneoxymorphone (6, UMB 246) displaying MOR partial agonist/DOR antagonist activity, representing a new lead for designing bifunctional MOR/DOR ligands.

In vivo effects of μ‐opioid receptor agonist/δ‐opioid receptor antagonist peptidomimetics following acute and repeated administration

Agonists at μ‐opioid receptors (μ‐receptors) are used for pain management but produce adverse effects including tolerance, dependence and euphoria. The co‐administration of a μ‐receptor agonist with a δ‐opioid receptor (δ‐receptor) antagonist has been shown to produce antinociception with reduced development of some side effects. We characterized the effects of three μ‐receptor agonist/δ‐receptor antagonist peptidomimetics in vivo after acute and repeated administration to determine if this profile provides a viable alternative to traditional opioid analgesics.

Synthesis and Pharmacological Evaluation of Novel C-8 Substituted Tetrahydroquinolines as Balanced-Affinity Mu/Delta Opioid Ligands for the Treatment of Pain

The use of opioids for the treatment of pain, while largely effective, is limited by detrimental side effects including analgesic tolerance, physical dependence, and euphoria, which may lead to opioid abuse. Studies have shown that compounds with a μ-opioid receptor (MOR) agonist/δ-opioid receptor (DOR) antagonist profile reduce or eliminate some of these side effects including the development of tolerance and dependence. Herein we report the synthesis and pharmacological evaluation of a series of tetrahydroquinoline-based peptidomimetics with substitutions at the C-8 position. Relative to our lead peptidomimetic with no C-8 substitution, this series affords an increase in DOR affinity and provides greater balance in MOR and DOR binding affinities. Moreover, compounds with carbonyl moieties at C-8 display the desired MOR agonist/DOR antagonist profile whereas alkyl substitutions elicit modest DOR agonism. Several compounds in this series produce a robust antinociceptive effect in vivo and show antinociceptive activity for greater than 2 h after intraperitoneal administration in mice.