Staphylococcal nuclease domain-containing protein 1 (SND1) is a multifunctional oncoprotein overexpressed in breast cancer. Binding of metadherin (MTDH) to SND1 results in the stabilization of SND1 and is important in the initiation and progression of breast cancer. Disruption of such interaction is a potential therapeutic for breast cancer. SN1/2 domain of SND1 was used as bait in a phage display screening to identify a 12-amino acid peptide 4-2. The activity of peptide 4-2 was evaluated by ELISA, coimmunoprecipitation, MTS, Western blot analysis, and xenograft mouse model. Peptide 4-2 could disrupt SND1–MTDH interaction. Cell penetrating derivative of peptide 4–2 (CPP-4–2) could penetrate and kill breast cancer cells by disrupting SND1–MTDH interaction and degrading SND1. Tryptophan 10 (W10) of peptide 4-2 was essential in mediating cytotoxicity, SND1 interaction, SND1–MTDH disruption, and SND1 degradation. CPP-4-2 could inhibit the growth of breast cancer in a xenograft mouse model. The SND1-interacting peptide 4-2 could kill breast cancer cells both in vitro and in vivo by interacting with SND1, disrupting SND1–MTDH interaction, and inducing SND1 degradation. W10 was an essential amino acid in the activity of peptide 4-2.

Age-related macular degeneration (AMD) is a degenerative eye disease leading to central vision loss and is characterized by dysregulated autophagy of the retinal pigment epithelium (RPE) layer. Recent studies have suggested that rho-associated protein kinase (ROCK) inhibitors may enhance autophagy in neurodegenerative diseases and promote the survival of RPE cells. This study investigated the effect of ROCK inhibitors on autophagy gene expression and autophagic vacuole formation in a human RPE (ARPE-19) cell line. The highly selective and potent ROCK inhibitor Y-39983 enhanced the expression of autophagy genes in ARPE-19 cells and increased autophagic vacuole formation. A proteomic analysis using mass spectrometry was performed to further characterize the effects of ROCK inhibition at the protein level. Y-39983 downregulated thrombospondin-1 (THBS1), and suppression of THBS1 in ARPE-19 cells resulted in an increase in autophagic vacuole formation. Our data showed that ROCK inhibitor-induced autophagy was mediated by THBS1 downregulation. We identified ROCK and THBS1 as potential novel therapeutic targets in AMD.

Chloroquine was a cheap, extremely effective drug against Plasmodium falciparum until resistance arose. One approach to reversing resistance is the inhibition of chloroquine efflux from its site of action, the parasite digestive vacuole. Chloroquine accumulation studies have traditionally relied on radiolabelled chloroquine, which poses several challenges. There is a need for development of a safe and biologically relevant substitute. We report here a commercially-available green fluorescent chloroquine-BODIPY conjugate, LynxTag-CQ_GREEN, as a proxy for chloroquine accumulation. This compound localized to the digestive vacuole of the parasite as observed under confocal microscopy, and inhibited growth of chloroquine-sensitive strain 3D7 more extensively than in the resistant strains 7G8 and K1. Microplate reader measurements indicated suppression of LynxTag-CQ_GREEN efflux after pretreatment of parasites with known reversal agents. Microsomes carrying either sensitive- or resistant-type PfCRT were assayed for uptake; resistant-type PfCRT exhibited increased accumulation of LynxTag-CQ_GREEN, which was suppressed by pretreatment with known chemosensitizers. Eight laboratory strains and twelve clinical isolates were sequenced for PfCRT and Pgh1 haplotypes previously reported to contribute to drug resistance, and pfmdr1 copy number and chloroquine IC₅₀s were determined. These data were compared with LynxTag-CQ_GREEN uptake/fluorescence by multiple linear regression to identify genetic correlates of uptake. Uptake of the compound correlated with the logIC₅₀ of chloroquine and, more weakly, a mutation in Pgh1, F1226Y.

Rho-associated protein kinase (ROCK) inhibitors reduce cytoskeletal tension in trabecular meshwork (TM) cells, but the mechanisms linking this acute response to sustained extracellular matrix remodelling remain incompletely understood. In this study, we investigated whether the ROCK inhibitor Y39983 regulates thrombospondin-1 (THBS1) and DNA methylation programs, elements known to modulate extracellular matrix (ECM) remodelling, in primary human trabecular meshwork (hTM) cells. Primary hTM cells were treated with Y39983, 5-azacytidine (5-Aza, a DNA methyltransferase inhibitor), or both in combination. Gene expression of THBS1, DNA methyltransferase, ECM, and cytoskeletal molecules was assessed using RT-qPCR. THBS1 promoter methylation was examined through methylation-specific PCR, while genome-wide methylation remodelling was profiled using whole-genome bisulphite sequencing (WGBS) followed by pathway enrichment analysis. 5-Azacytidine abolished Y39983-dependent effects on DNMT1 and DNMT3A and attenuated THBS1 regulation. Methylation-specific PCR suggested altered THBS1 promoter methylation after Y39983 treatment, while whole-genome bisulphite sequencing identified 8845 differentially methylated genes, including 5608 hypomethylated and 3237 hypermethylated genes, enriched in ErbB signalling, focal adhesion, and regulation of the actin cytoskeleton. Independently, Y39983 reduced the basal and stimulus-induced expression of THBS1, as well as ECM- and cytoskeleton-associated genes, including COL4A1, COL6A1, and ACTG1. These findings demonstrate that ROCK inhibition is associated with changes in DNMT-related gene expression and DNA methylation patterns in hTM cells. However, because 5-azacytidine has effects beyond selective DNMT inhibition, further studies using orthogonal approaches are required to establish whether DNA methylation directly mediates transcriptional responses to Y-39983.