Robert Dullea

The differential effects of IL-1 and TNF-α on proinflammatory cytokine and matrix metalloproteinase expression in human chondrosarcoma cells

AbstractObjective and design:Interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and matrix metalloproteinases (MMPs) play important roles in the pathogenesis of osteoarthritis (OA). In the present study, using Affymetrix oligonucleotide array technology and real-time quantitative RT-PCR we have investigated the molecular mechanisms underlying the differential effect of IL-1 and TNF-α on gene expression in the human chondrosarcoma cell line, SW1353. Materials and methods:SW1353 cells were stimulated singularly with IL-1α, TNF-α, Phorbol 12-myristate 13-acetate (PMA), or treated with the combination of cytokine and PMA. Total RNA was collected at multiple time points over a 24-h period followed by biotinylated cRNA target preparation and hybridization onto the Affymetrix HG-U95Av2 array. The differential expression patterns of several cytokine and MMP genes were further confirmed by real time quantitative RT-PCR, Western blot, and ELISA. Results:Our microarray experiments have broadly confirmed previously published data on chondrocyte gene expression regulated by IL-1 and TNF-α. The expression pattern of proIL-1β, MMP-1, and MMP-13 in chondrocytes is differentially regulated when stimulated with proinflammatory cytokines. IL-1, but not TNF-α, can induce IL-6, bone morphogenic protein 2 (BMP-2), and cyclooxygenase (COX-2) expression in SW1353 cells. Additionally, our Western blot results provide the first evidence that IL-1β is produced in the proform in IL-1α-activated chondrosarcoma cells and that additional signals are required for its posttranslational processing/activation. Conclusions:IL-1 and TNF-α each activate a distinct set of genes in chondrosarcoma cells, and gene expression in these cells is regulated by groups of genes related in part by their function. Chondrocyte IL-1α appears to serve an important role in the pathogenesis OA contributing to joint inflammation and cartilage destruction.

UTILITY OF A NOVEL OATP1B2 KNOCKOUT MOUSE MODEL FOR EVALUATING THE ROLE OF OATP1B2 IN THE HEPATIC UPTAKE OF MODEL COMPOUNDS

We generated the organic anion transporting polypeptide (Oatp) 1b2 knockout (KO) mouse model and assessed its utility to study hepatic uptake using model compounds: cerivastatin, lovastatin acid, pravastatin, simvastatin acid, rifampicin, and rifamycin SV. A selective panel of liver cytochromes P450 (P450s) (Cyp3a11, Cyp3a13, Cyp3a16, Cyp2c29, and Cyp2c39) and transporters [Oatp1b2, Oatp1a1, Oatp1a4, Oatp1a5; organic anion transporter (Oat) 1, Oat2, Oat3; multidrug resistance gene 1 (Mdr1) a, Mdr1b; bile salt export pump, multidrug resistance associated protein (Mrp) 2, Mrp3; breast cancer resistance protein] were measured by reverse transcription-polymerase chain reaction in both KO and wild-type (WT) male mice. Male KO and WT mice received each model compound s.c. at 3 mg/kg. Blood and liver samples were obtained at 0, 0.5, and 2 h postdose and analyzed using liquid chromatography/tandem mass spectrometry. Liver/plasma concentration ratio (Kp,liver) was calculated. Students t test was used to compare the mRNA and Kp,liver between the KO and WT mice. A similar mRNA expression was observed between the KO and WT for the selected P450s and transporters except for Oatp1b2, for which the level was negligible in the KO but prominent in the WT mice with P < 0.0001. The in vivo results showed a differential effect of Oatp1b2 on hepatic uptake of the model compounds, indicating that Oatp1b2 plays a more significant role in the hepatobiliary disposition of rifampicin and lovastatin than the other compounds tested. This study suggests the Oatp1b2 mouse as a useful in vivo tool to understand drug targeting and disposition in the liver.

Design and Synthesis of Truncated EGF-A Peptides that Restore LDL-R Recycling in the Presence of PCSK9 In Vitro

Disrupting the binding interaction between proprotein convertase (PCSK9) and the epidermal growth factor-like domain A (EGF-A domain) in the low-density lipoprotein receptor (LDL-R) is a promising strategy to promote LDL-R recycling and thereby lower circulating cholesterol levels. In this study, truncated 26 amino acid EGF-A analogs were designed and synthesized, and their structures were analyzed in solution and in complex with PCSK9. The most potent peptide had an increased binding affinity for PCSK9 (KD = 0.6 μM) compared with wild-type EGF-A (KD = 1.2 μM), and the ability to increase LDL-R recycling in the presence of PCSK9 in a cell-based assay.

Molecular Characterization of Novel and Selective Peroxisome Proliferator-Activated Receptor α Agonists with Robust Hypolipidemic Activity in Vivo

The nuclear receptor peroxisome proliferator-activated receptor α (PPARα) is recognized as the primary target of the fibrate class of hypolipidemic drugs and mediates lipid lowering in part by activating a transcriptional cascade that induces genes involved in the catabolism of lipids. We report here the characterization of three novel PPARα agonists with therapeutic potential for treating dyslipidemia. These structurally related compounds display potent and selective binding to human PPARα and support robust recruitment of coactivator peptides in vitro. These compounds markedly potentiate chimeric transcription systems in cell-based assays and strikingly lower serum triglycerides in vivo. The transcription networks induced by these selective PPARα agonists were assessed by transcriptional profiling of mouse liver after short- and long-term treatment. The induction of several known PPARα target genes involved with fatty acid metabolism were observed, reflecting the expected pharmacology associated with PPARα activation. We also noted the down-regulation of a number of genes related to immune cell function, the acute phase response, and glucose metabolism, suggesting that these compounds may have anti-inflammatory action in the mammalian liver. Whereas these compounds are efficacious in acute preclinical models, extended safety studies and further clinical testing will be required before the full therapeutic promise of a selective PPARα agonist is realized.

Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome that allows small molecules to specifically block translation of individual transcripts.

Efficient Liver Targeting by Polyvalent Display of a Compact Ligand for the Asialoglycoprotein Receptor

A compact and stable bicyclic bridged ketal was developed as a ligand for the asialoglycoprotein receptor (ASGPR). This compound showed excellent ligand efficiency, and the molecular details of binding were revealed by the first X-ray crystal structures of ligand-bound ASGPR. This analogue was used to make potent di- and trivalent binders of ASGPR. Extensive characterization of the function of these compounds showed rapid ASGPR-dependent cellular uptake in vitro and high levels of liver/plasma selectivity in vivo. Assessment of the biodistribution in rodents of a prototypical Alexa647-labeled trivalent conjugate showed selective hepatocyte targeting with no detectable distribution in nonparenchymal cells. This molecule also exhibited increased ASGPR-directed hepatocellular uptake and prolonged retention compared to a similar GalNAc derived trimer conjugate. Selective release in the liver of a passively permeable small-molecule cargo was achieved by retro-Diels-Alder cleavage of an oxanorbornadiene linkage, presumably upon encountering intracellular thiol. Therefore, the multicomponent construct described here represents a highly efficient delivery vehicle to hepatocytes.

Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing

CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.

Structural basis for selective stalling of human ribosome nascent chain complexes by a drug-like molecule

Small molecules that target the ribosome generally have a global impact on protein synthesis. However, the drug-like molecule PF-06446846 (PF846) binds the human ribosome and selectively blocks the translation of a small subset of proteins by an unknown mechanism. In high-resolution cryo-electron microscopy (cryo-EM) structures of human ribosome nascent chain complexes stalled by PF846, PF846 binds in the ribosome exit tunnel in a eukaryotic-specific pocket formed by the 28S ribosomal RNA (rRNA), and redirects the path of the nascent polypeptide chain. PF846 arrests the translating ribosome in the rotated state, with peptidyl-tRNA occupying a mixture of A/A and hybrid A/P sites. These results provide a structural foundation for a new strategy for developing small molecules that selectively inhibit the production of proteins of therapeutic interest. One sentence summary A drug-like small molecule binds in a newly-identified site in the exit tunnel of the human ribosome and selectively stalls translation by trapping the rotated state of the ribosome, likely preventing efficient translocation of the mRNA and tRNAs.

Liver-Targeted Small-Molecule Inhibitors of Proprotein Convertase Subtilisin/Kexin Type 9 Synthesis

Targeting of the human ribosome is an unprecedented therapeutic modality with a genome-wide selectivity challenge. A liver-targeted drug candidate is described that inhibits ribosomal synthesis of PCSK9, a lipid regulator considered undruggable by small molecules. Key to the concept was the identification of pharmacologically active zwitterions designed to be retained in the liver. Oral delivery of the poorly permeable zwitterions was achieved by prodrugs susceptible to cleavage by carboxylesterase 1. The synthesis of select tetrazole prodrugs was crucial. A cell-free in vitro translation assay containing human cell lysate and purified target mRNA fused to a reporter was used to identify active zwitterions. In vivo PCSK9 lowering by oral dosing of the candidate prodrug and quantification of the drug fraction delivered to the liver utilizing an oral positron emission tomography 18 F-isotopologue validated our liver-targeting approach.

Small Molecule Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibitors: Hit to Lead Optimization of Systemic Agents

The optimization of a new class of small molecule PCSK9 mRNA translation inhibitors is described. The potency, physicochemical properties, and off-target pharmacology associated with the hit compound (1) were improved by changes to two regions of the molecule. The last step in the synthesis of the congested amide center was enabled by three different routes. Subtle structural changes yielded significant changes in pharmacology and off-target margins. These efforts led to the identification of 7l and 7n with overall profiles suitable for in vivo evaluation. In a 14-day toxicology study, 7l demonstrated an improved safety profile vs lead 7f. We hypothesize that the improved safety profile is related to diminished binding of 7l to nontranslating ribosomes and an apparent improvement in transcript selectivity due to the lower strength of 7l stalling of off-target proteins.