Biology subjects

Scott, D. A.

Publications and source records attributed to Scott, D. A..

26 records · Page 2Linked to original sources

bioRxiv2021

MPI8 is Potent Against SARS-CoV-2 by Inhibiting Dually and Selectively the SARS-CoV-2 Main Protease and the Host Cathepsin L

A number of inhibitors have been developed for the SARS-CoV-2 main protease (MPro) as potential COVID-19 medications but little is known about their selectivity. Using enzymatic assays, we characterized inhibition of TMPRSS2, furin, and cathepsins B/K/L by more than a dozen of previously developed MPro inhibitors including MPI1-9, GC376, 11a, 10-1, 10-2, and 10- 3. MPI1-9, GC376 and 11a all contain an aldehyde for the formation of a reversible covalent hemiacetal adduct with the MPro active site cysteine and 10-1, 10-2 and 10-3 contain a labile ester to exchange with the MPro active site cysteine for the formation of a thioester. Our data revealed that all these inhibitors are inert toward TMPRSS2 and furin. Diaryl esters also showed low inhibition of cathepsins. However, all aldehyde inhibitors displayed high potency in inhibiting three cathepsins. Their determined IC50 values vary from 4.1 to 380 nM for cathepsin B, 0.079 to 2.3 nM for cathepsin L, and 0.35 to 180 nM for cathepsin K. All aldehyde inhibitors showed similar inhibition levels toward cathepsin L. A cellular analysis indicated high potency of MPI5 and MPI8 in inhibiting lysosomal activity, which is probably attributed to their inhibition of cathepsins. Among all aldehyde inhibitors, MPI8 shows the best selectivity toward cathepsin L. With respect to cathepsins B and K, the selective indices are 192 and 150, respectively. MPI8 is the most potent compound among all aldehyde inhibitors in cellular MPro inhibition potency and anti-SARS-CoV-2 activity in Vero E6 cells. Cathepsin L has been demonstrated to play a critical role in the SARS-CoV-2 cell entry. By selectively inhibiting both SARS-CoV-2 MPro and the host cathepsin L, MPI8 potentiates dual inhibition effects to synergize its overall antiviral potency and efficacy. Due to its high selectivity toward cathepsin L that reduces potential toxicity toward host cells and high cellular and antiviral potency, we urge serious consideration of MPI8 for preclinical and clinical investigations for treating COVID-19.

pharmacology and toxicology↗

bioRxiv2020

SOX7 deficiency causes ventricular sepal defects through its effects on endocardial-to-mesenchymal transition and the expression of Wnt4 and Bmp2

SOX7 is located in a region on chromosome 8p23.1 that is recurrently deleted in individuals with septal defects. Sox7-/- embryos die of heart failure around E11.5 due to defects in vascular remodeling. These embryos have hypocellular endocardial cushions with severely reduced numbers of mesenchymal cells. We also observed a ventricular septal defect in a rare Sox7flox/-;Tie2-Cre embryo that escaped early lethality. This led us to hypothesize that SOX7 plays a critical developmental role in the endocardium of the atrioventricular (AV) canal. We subsequently used AV explant studies to show that SOX7 deficiency leads to a severe reduction in endocardial-to-mesenchymal transition (EndMT). Since SOX7 is a transcription factor, we hypothesized that it functions in the endocardium by regulating the expression of EndMT-related genes. To identify these genes in an unbiased manner, we performed RNA-seq on pooled E9.5 hearts tubes harvested from Sox7-/- embryos and their wild-type littermates. We found that Wnt4 transcript levels were severely reduced, which we confirmed by RNA in situ hybridization. Previous studies have shown that WNT4 is expressed in the endocardium and promotes EndMT by acting in a paracrine manner to increase the expression of BMP2 in the myocardium. Consistent with these findings, we found that Bmp2 transcript levels were diminished in Sox7-/- embryonic hearts. We conclude that SOX7 promotes EndMT in the developing AV canal by modulating the expression of Wnt4 and Bmp2. These data also provide additional evidence that haploinsufficiency of SOX7 contributes to the congenital heart defects seen in individuals with recurrent 8p23.1 microdeletions. SUMMARY STATEMENTIn the developing atrioventricular canal, SOX7 promotes endocardial-to-mesenchymal transition (EndMT) by positively regulating Wnt4 and Bmp2 expression. SOX7 deficiency leads to the development of hypocellular endothelial cushions and ventricular septal defects.

developmental biology↗

bioRxiv2020

The deacylase SIRT5 supports melanoma viability by regulating chromatin dynamics

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieves a durable remission. SIRT5 is a member of the sirtuin family of protein deacylases that regulate metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we show that SIRT5 is required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 is required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf;Pten-driven melanoma model. Via metabolite and transcriptomic analyses, we find that SIRT5 is required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably include MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a novel, druggable genotype-independent addiction in melanoma.

cancer biology↗

bioRxiv2020

A Speedy Route to Multiple Highly Potent SARS-CoV-2 Main Protease Inhibitors

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro) to digest two of its translated polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replication in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro), we have designed and synthesized a series of SC2MPro inhibitors that contain {beta}-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active site cysteine C145. All inhibitors display high potency with IC50 values at or below 100 nM. The most potent compound MPI3 has as an IC50 value as 8.5 nM. Crystallographic analyses of SC2MPro bound to 7 inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549 cells. Two inhibitors MP5 and MPI8 completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 M and A549 cells at 0.16-0.31 M. Their virus inhibition potency is much higher than some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with extreme potency. Due to the urgent matter of the COVID-19 pandemic, MPI5 and MPI8 may be quickly advanced to preclinical and clinical tests for COVID-19.

biochemistry↗

bioRxiv2020

An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity

Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to -ketoglutarate (-KG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasing kcat values for -KG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKa values. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKa value upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue [~]12 [A] from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.

biochemistry↗

bioRxiv2019

Defining and targeting adaptations to oncogenic KRASG12C inhibition using quantitative temporal proteomics

Covalent inhibitors of the KRASG12C oncoprotein have recently been developed and are being evaluated in clinical trials. Resistance to targeted therapies is common and likely to limit long-term efficacy of KRAS inhibitors (KRASi). To identify pathways of adaptation to KRASi and to predict drug combinations that circumvent resistance, we used a mass spectrometry-based quantitative temporal proteomics and bioinformatics workflow to profile the temporal proteomic response to KRASG12C inhibition in pancreatic and lung cancer 2D and 3D cellular models. We quantified 10,805 proteins across our datasets, representing the most comprehensive KRASi proteomics effort to date. Our data reveal common mechanisms of acute and long-term response between KRASG12C-driven tumors. To facilitate discovery in the cancer biology community, we generated an interactive KRASi proteome website (https://manciaslab.shinyapps.io/KRASi/). Based on these proteomic data, we identified potent combinations of KRASi with PI3K, HSP90, CDK4/6, and SHP2 inhibitors, in some instances converting a cytostatic response to KRASi monotherapy to a cytotoxic response to combination treatment. Overall, using our quantitative temporal proteomics-bioinformatics platform, we have comprehensively characterized the proteomic adaptations to KRASi and identified combinatorial regimens to induce cytotoxicity with potential therapeutic utility.

cancer biology↗

bioRxiv2017

A Survey of Genome Editing Activity for 16 Cpf1 orthologs

The recently discovered class 2 CRISPR-Cas endonuclease Cpf1 offers several advantages over Cas9, including the ability to process its own array and requirement for just a single RNA guide. These attributes make Cpf1 promising for many genome engineering applications. To further expand the suite of Cpf1 tools available, we tested 16 Cpf1 orthologs for activity in eukaryotic cells. Four of these new enzymes demonstrated targeted activity, one of which, from Moraxella bovoculi AAX11_00205 (Mb3Cpf1), exhibited robust indel formation. We also show that Mb3Cpf1 displays some tolerance for a shortened PAM (TTN versus the canonical Cpf1 PAM TTTV). The addition of these enzymes to the genome editing toolbox will further expand the utility of this powerful technology.

molecular biology↗

bioRxiv2016

BLISS: quantitative and versatile genome-wide profiling of DNA breaks in situ

We present a method for genome-wide DNA double-strand Breaks (DSBs) Labeling In Situ and Sequencing (BLISS) which, compared to existing methods, introduces several key features: 1) high efficiency and low input requirement by in situ DSB labeling in cells or tissue sections directly on a solid surface; 2) easy scalability by performing in situ reactions in multi-well plates; 3) high sensitivity by linearly amplifying tagged DSBs using in vitro transcription; and 4) accurate DSB quantification and control of PCR biases by using unique molecular identifiers. We demonstrate the ability to use BLISS to quantify natural and drug-induced DSBs in low-input samples of cancer cells, primary mouse embryonic stem cells, and mouse liver tissue sections. Finally, we applied BLISS to compare the specificity of CRISPR-associated RNA-guided endonucleases Cas9 and Cpf1, and found that Cpf1 has higher specificity than Cas9. These results establish BLISS as a versatile, sensitive, and efficient method for genome-wide DSB mapping in many applications.

molecular biology↗