Aberrant trafficking of a Leu89Pro connexin32 mutant associated with X-linked dominant Charcot–Marie–Tooth disease

<p><b>Objective:</b> To determine the functional abnormalities of the Leu89Pro mutation in connexin32 (CX32), which we have previously reported is present within an X-linked dominant Charcot–Marie–Tooth disease family. In this family, male patients were moderately to severely affected.</p> <p><b>Methods:</b> We performed immunofluorescence to investigate whether the Leu89Pro CX32 protein was transported to the cell membrane in HeLa and Schwann cells. First, we constructed the eukaryotic express plasmids expressing CX32 (wild-type or Leu89Pro) and enhanced green fluorescent protein by the gene recombination technology. Then the recombinant plasmids were transiently transfected into communication-incompetent HeLa cells and human Schwann cells by the lipofectamine method. Later, we double-labeled cells for both CX32 and markers of the ER (calnexin) or the Golgi (58-kDa protein) at 24 h or 48 h. The images were collected using a Leica TCS SP5 II confocal microscope.</p> <p><b>Results:</b> The mutant CX32 protein was localized in the endoplasmic reticulum and failed to reach the cell membrane to form gap junctions.</p> <p><b>Conclusion:</b> Our results indicated that the Leu89Pro substitution in the second transmembrane domain of CX32 disrupts the trafficking of the protein, inhibiting the assembly of CX32 gap junctions, which in turn may result in peripheral neuropathy. This functional abnormality may explain the moderate to severe phenotype seen in Leu89Pro patients, and as such represents a promising therapeutic target in the treatment of this subset of CMTX patients.</p

An exposure–response analysis based on rifampin suggests CYP3A4 induction is driven by AUC: an <i>in vitro</i> investigation

<p>1. Induction is an important mechanism contributing to drug–drug interactions. It is most commonly evaluated in the human hepatocyte assay over 48-h or 72-h incubation period. However, whether the overall exposure (i.e. Area Under the Curve (AUC) or C_ave) or maximum exposure (i.e. C_max) of the inducer is responsible for the magnitude of subsequent induction has not been thoroughly investigated. Additionally, <i>in vitro</i> induction assays are typically treated as static systems, which could lead to inaccurate induction potency estimation. Hence, European Medicines Agency (EMA) guidance now specifies quantitation of drug levels in the incubation.</p> <p>2. This work treated the typical <i>in vitro</i> evaluation of rifampin induction as an <i>in vivo</i> system by generating various target engagement profiles, measuring free rifampin concentration over 3 d of incubation and evaluating the impact of these factors on final induction response.</p> <p>3. This rifampin-based analysis demonstrates that the induction process is driven by time-averaged target engagement (i.e. AUC-driven). Additionally, depletion of rifampin in the incubation medium over 3 d as well as non-specific/specific binding were observed.</p> <p>4. These findings should help aid the discovery of clinical candidates with minimal induction liability and further expand our knowledge in the quantitative translatability of <i>in vitro</i> induction assays.</p

Table_1_Identification of the CFTR c.1666A>G Mutation in Hereditary Inclusion Body Myopathy Using Next-Generation Sequencing Analysis.XLS

<p>Hereditary inclusion body myopathy (HIBM) is a rare autosomal recessive adult onset muscle disease which affects one to three individuals per million worldwide. This disease is autosomal dominant and occurs in adulthood. Our previous study reported a new subtype of HIBM linked to the susceptibility locus at 7q22.1-31.1. The present study is aimed to identify the candidate gene responsible for the phenotype in HIBM pedigree. After multipoint linkage analysis, we performed targeted capture sequencing on 16 members and whole-exome sequencing (WES) on 5 members. Bioinformatics filtering was performed to prioritize the candidate pathogenic gene variants, which were further genotyped by Sanger sequencing. Our results showed that the highest peak of LOD score (4.70) was on chromosome 7q22.1-31.1.We identified 2 and 22 candidates using targeted capture sequencing and WES respectively, only one of which as CFTRc.1666A>G mutation was well cosegregated with the HIBM phenotype. Using transcriptome analysis, we did not detect the differences of CFTR's mRNA expression in the proband compared with healthy members. Due to low incidence of HIBM and there is no other pedigree to assess, mutation was detected in three patients with duchenne muscular dystrophyn (DMD) and five patients with limb-girdle muscular dystrophy (LGMD). And we found that the frequency of mutation detected in DMD and LGMD patients was higher than that of being expected in normal population. We suggested that the CFTRc.1666A>G may be a candidate marker which has strong genetic linkage with the causative gene in the HIBM family.</p

Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging

Bioorthogonal reactions, including the strain-promoted azide–alkyne cycloaddition (SPAAC) and inverse electron demand Diels–Alder (iEDDA) reactions, have become increasingly popular for live-cell imaging applications. However, the stability and reactivity of reagents has never been systematically explored in the context of a living cell. Here we report a universal, organelle-targetable system based on HaloTag protein technology for directly comparing bioorthogonal reagent reactivity, specificity, and stability using clickable HaloTag ligands in various subcellular compartments. This system enabled a detailed comparison of the bioorthogonal reactions in live cells and informed the selection of optimal reagents and conditions for live-cell imaging studies. We found that the reaction of sTCO with monosubstituted tetrazines is the fastest reaction in cells; however, both reagents have stability issues. To address this, we introduced a new variant of sTCO, Ag-sTCO, which has much improved stability and can be used directly in cells for rapid bioorthogonal reactions with tetrazines. Utilization of Ag complexes of conformationally strained <i>trans</i>-cyclooctenes should greatly expand their usefulness especially when paired with less reactive, more stable tetrazines

2-(Pyrrolidin-1-yl)ethyl-3,4-dihydroisoquinolin-1(<i>2H</i>)-one Derivatives as Potent and Selective Histamine-3 Receptor Antagonists

On the basis of the previously reported benzimidazole 1,3′-bipyrrolidine benzamides (<b>1</b>), a new class of 2-(pyrrolidin-1-yl)­ethyl-3,4-dihydroisoquinolin-1­(2<i>H</i>)-one derivatives (<b>3</b>–<b>50</b>) were synthesized and evaluated as potent H₃ receptor antagonists. In particular, compound <b>39</b> exhibited potent in vitro binding and functional activities at the H₃ receptor, good selectivities against other neurotransmitter receptors and ion channels, acceptable pharmacokinetic properties, and a favorable in vivo profile

A Second-Generation Oral SARS-CoV‑2 Main Protease Inhibitor Clinical Candidate for the Treatment of COVID-19

Despite the record-breaking discovery, development and approval of vaccines and antiviral therapeutics such as Paxlovid, coronavirus disease 2019 (COVID-19) remained the fourth leading cause of death in the world and third highest in the United States in 2022. Here, we report the discovery and characterization of PF-07817883, a second-generation, orally bioavailable, SARS-CoV-2 main protease inhibitor with improved metabolic stability versus nirmatrelvir, the antiviral component of the ritonavir-boosted therapy Paxlovid. We demonstrate the in vitro pan-human coronavirus antiviral activity and off-target selectivity profile of PF-07817883. PF-07817883 also demonstrated oral efficacy in a mouse-adapted SARS-CoV-2 model at plasma concentrations equivalent to nirmatrelvir. The preclinical in vivo pharmacokinetics and metabolism studies in human matrices are suggestive of improved oral pharmacokinetics for PF-07817883 in humans, relative to nirmatrelvir. In vitro inhibition/induction studies against major human drug metabolizing enzymes/transporters suggest a low potential for perpetrator drug–drug interactions upon single-agent use of PF-07817883

A Second-Generation Oral SARS-CoV‑2 Main Protease Inhibitor Clinical Candidate for the Treatment of COVID-19

Despite the record-breaking discovery, development and approval of vaccines and antiviral therapeutics such as Paxlovid, coronavirus disease 2019 (COVID-19) remained the fourth leading cause of death in the world and third highest in the United States in 2022. Here, we report the discovery and characterization of PF-07817883, a second-generation, orally bioavailable, SARS-CoV-2 main protease inhibitor with improved metabolic stability versus nirmatrelvir, the antiviral component of the ritonavir-boosted therapy Paxlovid. We demonstrate the in vitro pan-human coronavirus antiviral activity and off-target selectivity profile of PF-07817883. PF-07817883 also demonstrated oral efficacy in a mouse-adapted SARS-CoV-2 model at plasma concentrations equivalent to nirmatrelvir. The preclinical in vivo pharmacokinetics and metabolism studies in human matrices are suggestive of improved oral pharmacokinetics for PF-07817883 in humans, relative to nirmatrelvir. In vitro inhibition/induction studies against major human drug metabolizing enzymes/transporters suggest a low potential for perpetrator drug–drug interactions upon single-agent use of PF-07817883