A novel small molecule inhibitor of human Drp1

Mitochondrial dynamin-related protein 1 (Drp1) is a large GTPase regulator of mitochondrial dynamics and is known to play an important role in numerous pathophysiological processes. Despite being the most widely used Drp1 inhibitor, the specificity of Mdivi-1 towards human Drp1 has not been definitively proven and there have been numerous issues reported with its use including off-target effects. In our hands Mdivi-1 showed varying binding affinities toward human Drp1, potentially impacted by compound aggregation. Herein, we sought to identify a novel small molecule inhibitor of Drp1. From an initial virtual screening, we identified DRP1i27 as a compound which directly bound to the human isoform 3 of Drp1 via surface plasmon resonance and microscale thermophoresis. Importantly, DRP1i27 was found to have a dose-dependent increase in the cellular networks of fused mitochondria but had no effect in Drp1 knock-out cells. Further analogues of this compound were identified and screened, though none displayed greater affinity to human Drp1 isoform 3 than DRP1i27. To date, this is the first small molecule inhibitor shown to directly bind to human Drp1

Gene Transfer of Pro-opiomelanocortin Prohormone Suppressed the Growth and Metastasis of Melanoma: Involvement of ␣-Melanocyte-Stimulating Hormone-Mediated Inhibition of the Nuclear Factor B/Cyclooxygenase-2 Pathway

ABSTRACT Pro-opiomelanocortin (POMC) is a prohormone of various neuropeptides, including corticotropin, ␣-melanocyte-stimulating hormone (␣-MSH), and ␤-endorphin (␤-EP) . POMC neuropeptides are potent inflammation inhibitors and immunosuppressants and may exert opposite influences during tumorigenesis. However, the role of POMC expression in carcinogenesis remains elusive. We evaluated the antineoplastic potential of POMC gene delivery in a syngenic B16-F10 melanoma model. Adenovirus-mediated POMC gene delivery in B16-F10 cells increased the release of POMC neuropeptides in cultured media, which differentially regulated the secretion of pro-and anti-inflammatory cytokines in lymphocytes. POMC gene transfer significantly reduced the anchorage-independent growth of melanoma cells. Moreover, pre-or post-treatment with POMC gene delivery effectively retarded the melanoma growth in mice. Intravenous injection of POMC-transduced B16-F10 cells resulted in reduced foci formation in lung by 60 to 70% of control. The reduced metastasis of POMC-transduced B16-F10 cells could be attributed to their attenuated migratory and adhesive capabilities. POMC gene delivery reduced the cyclooxygenase-2 (COX-2) expression and prostaglandin (PG) E 2 synthesis in melanoma cells and tumor tissues. In addition, application of NS-398, a selective COX-2 inhibitor, mimicked the antineoplastic functions of POMC gene transfer in melanoma. The POMC-mediated COX-2 down-regulation was correlated with its inhibition of nuclear factor B (NFB) activities. Exogenous supply of ␣-MSH inhibited NFB activities, whereas application of the ␣-MSH antagonist growth hormone-releasing peptide-6 (GHRP-6) abolished the POMC-induced inhibition of NFB activities and melanoma growth in mice. In summary, POMC gene delivery suppresses melanoma via ␣-MSH-induced inhibition of NFB/COX-2 pathway, thereby constituting a novel therapy for melanoma. POMC is a multifunctional polycistronic gene located on human chromosome 2p23.3. POMC is a 31 kDa prohormone that is processed into various neuropeptides, including corticotropin, melanotropins (␣-, ␤-, and ␥-MSH), lipotropins, and ␤-endorphin (␤-EP

sFlt Multivalent Conjugates Inhibit Angiogenesis and Improve Half-Life In Vivo

We would like to thank Jonathan Winger and Xiao Zhu for guidance with the insect cell protein expression system and providing reagents. We would like to acknowledge Ann Fischer for help with expressing the sFlt protein in the Tissue Culture Facility at UC Berkeley and Dawn Spelke and Anusuya Ramasubramanian for help optimizing protein purification from insect cells. We are also grateful for the help from Leah Byrne and John Flannery at in the Helen Wills Neuroscience Institute at UC Berkeley for aiding us in the development of the rat intravitreal residence time model and for allowing us to use their facilities.Current anti-VEGF drugs for patients with diabetic retinopathy suffer from short residence time in the vitreous of the eye. In order to maintain biologically effective doses of drug for inhibiting retinal neovascularization, patients are required to receive regular monthly injections of drug, which often results in low patient compliance and progression of the disease. To improve the intravitreal residence time of anti-VEGF drugs, we have synthesized multivalent bioconjugates of an anti-VEGF protein, soluble fms-like tyrosine kinase-1 (sFlt) that is covalently grafted to chains of hyaluronic acid (HyA), conjugates that are termed mvsFlt. Using a mouse corneal angiogenesis assay, we demonstrate that covalent conjugation to HyA chains does not decrease the bioactivity of sFlt and that mvsFlt is equivalent to sFlt at inhibiting corneal angiogenesis. In a rat vitreous model, we observed that mvsFlt had significantly increased intravitreal residence time compared to the unconjugated sFlt after 2 days. The calculated intravitreal half-lives for sFlt and mvsFlt were 3.3 and 35 hours, respectively. Furthermore, we show that mvsFlt is more effective than the unconjugated form at inhibiting retinal neovascularization in an oxygen-induced retinopathy model, an effect that is most likely due to the longer half-life of mvsFlt in the vitreous. Taken together, our results indicate that conjugation of sFlt to HyA does not affect its affinity for VEGF and this conjugation significantly improves drug half-life. These in vivo results suggest that our strategy of multivalent conjugation could substantially improve upon drug half-life, and thus the efficacy of currently available drugs that are used in diseases such as diabetic retinopathy, thereby improving patient quality of life.Yeshttp://www.plosone.org/static/editorial#pee

Screening of CRISPR/Cas base editors to target the AMD high-risk Y402H complement factor H variant

Purpose: To evaluate the efficacy of using a CRISPR/Cas-mediated strategy to correct a common high-risk allele that is associated with age-related macular degeneration (AMD; rs1061170; NM_000186.3:c.1204T>C; NP_000177.2:p.His402Tyr) in the complement factor H (CFH) gene. Methods: A human embryonic kidney cell line (HEK293A) was engineered to contain the pathogenic risk variant for AMD (HEK293A-CFH). Several different base editor constructs (BE3, SaBE3, SaKKH-BE3, VQR-BE3, and Target-AID) and their respective single-guide RNA (sgRNA) expression cassettes targeting either the pathogenic risk variant allele in the CFH locus or the LacZ gene, as a negative control, were evaluated head-to-head for the incidence of a cytosine-to-thymine nucleotide correction. The base editor construct that showed appreciable editing activity was selected for further assessment in which the base-edited region was subjected to next-generation deep sequencing to quantify on-target and off-target editing efficacy. Results: The tandem use of the Target-AID base editor and its respective sgRNA demonstrated a base editing efficiency of facilitating a cytosine-to-thymine nucleotide correction in 21.5% of the total sequencing reads. Additionally, the incidence of insertions and deletions (indels) was detected in only 0.15% of the sequencing reads with virtually no off-target effects evident across the top 11 predicted off-target sites containing at least one cytosine in the activity window (n = 3, pooled amplicons). Conclusions: CRISPR-mediated base editing can be used to facilitate a permanent and stably inherited cytosine-to-thymine nucleotide correction of the rs1061170 SNP in the CFH gene with minimal off-target effects

AAV-mediated CRISPR/Cas gene editing of retinal cells in vivo

PURPOSE. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPRassociated protein (Cas) has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt yellow fluorescent protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo. METHODS. Single guide RNA (sgRNA) plasmids were designed to target YFP, and after in vitro validation, selected guides were cloned into a dual AAV system. One AAV2 construct was used to deliver Streptococcus pyogenes Cas9 (SpCas9), and the other delivered sgRNA against YFP or LacZ (control) in the presence of mCherry. Five weeks after intravitreal injection, retinal function was determined using electroretinography, and CRISPR/Cas-mediated gene modifications were quantified in retinal flat mounts. RESULTS. Adeno-associated virus 2-mediated in vivo delivery of SpCas9 with sgRNA targeting YFP significantly reduced the number of YFP fluorescent cells of the inner retina of our transgenic mouse model. Overall, we found an 84.0% (95% confidence interval [CI]: 81.8-86.9) reduction of YFP-positive cells in YFP-sgRNA-infected retinal cells compared to eyes treated with LacZ-sgRNA. Electroretinography profiling found no significant alteration in retinal function following AAV2-mediated delivery of CRISPR/Cas components compared to contralateral untreated eyes. CONCLUSIONS. Thy1-YFP transgenic mice were used as a rapid quantifiable means to assess the efficacy of CRISPR/Cas-based retinal gene modification in vivo. We demonstrate that genomic modification of cells in the adult retina can be readily achieved by viral-mediated delivery of CRISPR/Cas