Biology subjects

Itkin, Z.

Publications and source records attributed to Itkin, Z..

14 recordsLinked to original sources

bioRxiv2024

Identification of potent HSV antivirals using 3D bioprinted human skin equivalents

Herpes simplex virus (HSV) infection has worldwide public health concerns and lifelong medical impacts. The standard therapy, acyclovir, has limited efficacy in preventing HSV subclinical virus shedding, and drug resistance occurs in immunocompromised patients, highlighting the need for novel therapeutics. HSV manifests in the skin and mucosal epithelium. Here, we found acyclovir significantly less effective in skin-derived keratinocytes than donor-matched fibroblasts. To recapitulate in vivo tissue architecture, we 3D bioprinted human skin equivalents (HSE) in a 96-well plate format amenable for antiviral screening and preclinical testing. We screened a library of 738 compounds with broad targets and mechanisms of action and identified potent antivirals, including 23 known or experimental HSV treatments, validating the translational relevance of our assay. Unlike acyclovir, antivirals against HSV helicase/primase or host replication pathways displayed similar potency across cell types and donor sources in 2D and 3D models. Our 3D bioprinted platform allowed for integrating patient-derived cells and incorporating genetic variability early in drug development. The reduced potency in keratinocytes helps explain the limited benefit acyclovir and its congeners play in reducing sexual transmission. These data indicate that the 3D bioprinted HSE assay platform provides a more physiologically relevant approach to identifying potential antivirals for HSV. One Sentence SummaryHigh-throughput screen using 3D bioprinted human skin equivalents to identify antivirals against HSV and evaluate cell-type specific effects.

bioengineering↗

bioRxiv2024

A comprehensive collection of pain and addiction targets and compounds library for high throughput screening and AI-driven drug discovery

As part of the NIH Helping to End Addiction Long-term (HEAL) Initiative, the National Center for Advancing Translational Sciences (NCATS) is dedicated to the development of new pharmacological tools and investigational drugs for managing and treating pain, as well as the prevention and treatment of opioid misuse and addiction. In line with these objectives, we created a comprehensive, annotated small molecule library including drugs, probes, and tool compounds that act on published pain and addiction-relevant targets. Nearly 3,000 small molecules associated with approximately 200 known and hypothesized HEAL targets have been assembled, curated, and annotated in one collection. Physical samples of the library compounds have been acquired and plated in 1536-well format, enabling rapid and efficient high throughput screen (HTS) against a wide range of assays. The creation of the HEAL Targets and Compounds Library, coupled with an integrated computational platform for AI-driven machine learning (ML), structural modeling, and virtual screening (VS), provides a valuable source for strategic drug repurposing, innovative profiling, and hypothesis testing of novel targets related to pain and opioid use disorder (OUD). The library is available to investigators for screening pain and OUD-relevant phenotypes.

bioinformatics↗

bioRxiv2022

High-throughput Approaches to Uncover Synergistic Drug Combinations in Leukemia

We report a comprehensive drug synergy study in acute myeloid leukemia (AML). In this work, we investigate 11 cell lines spanning both MLL-rearranged and non-rearranged subtypes. The work comprises a resource for the community, with many synergistic drug combinations that could not have been predicted a priori, and open source code for automation and analyses. We base our definitions of drug synergy on the Chou-Talalay method, which is useful for visualizations of synergy experiments in isobolograms, and median-effects plots, among other representations. Our key findings include drug synergies affecting the chromatin state, specifically in the context of regulation of the modification state of histone H3 lysine-27. We report open source high throughput methodology such that multidimensional drug screening can be accomplished with equipment that is accessible to most laboratories. This study will enable preclinical investigation of new drug combinations in a lethal blood cancer, with data analysis and automation workflows freely available to the community. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/518409v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1e85ef9org.highwire.dtl.DTLVardef@b3369eorg.highwire.dtl.DTLVardef@1c6d705org.highwire.dtl.DTLVardef@14ec6b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

Application of Temperature-Responsive HIS-Tag Fluorophores to Differential Scanning Fluorimetry Screening of Small Molecule Libraries

Differential scanning fluorimetry is a rapid and economical biophysical technique used to monitor perturbations to protein structure during a thermal gradient most often by detecting protein unfolding events through an environment-sensitive fluorophore. By employing an NTA-complexed fluorophore that is sensitive to nearby structural changes in 6xHIS-tagged protein, a robust and sensitive differential scanning fluorimetry (DSF) assay is established with the specificity of an affinity tag-based system. We developed, optimized, and miniaturized this histidine-tag DSF assay (HIS-DSF) into a 1536-well high-throughput biophysical platform using the Borrelial high temperature requirement A protease (BbHtrA) as a proof of concept for the workflow. A production run of the BbHtrA HIS-DSF assay showed a tight negative control group distribution of Tm values with an average coefficient of variation of 0.51% and median coefficient of variation of compound Tm of 0.26%. The HIS-DSF platform will provide an additional assay platform for future drug discovery campaigns with applications in buffer screening and optimization, target engagement screening, and other biophysical assay efforts.

biochemistry↗

bioRxiv2022

A Suite of TMPRSS2 Assays for Screening Drug Repurposing Candidates as Potential Treatments of COVID-19

SARS-CoV-2 is the causative viral pathogen driving the COVID-19 pandemic that prompted an immediate global response to the development of vaccines and antiviral therapeutics. For antiviral therapeutics, drug repurposing allowed for rapid movement of existing clinical candidates and therapies into human clinical trials to be tested as COVID-19 therapies. One effective antiviral treatment strategy used early in symptom onset is to prevent viral entry. SARS-CoV-2 enters ACE2-expressing cells when the receptor-binding domain of the spike protein on the surface of SARS-CoV-2 binds to ACE2 followed by cleavage at two cut sites on the spike protein. TMPRSS2 has a protease domain capable of cleaving the two cut sites; therefore, a molecule capable of inhibiting the protease activity of TMPRSS2 could be a valuable antiviral therapy. Initially, we used a fluorogenic high-throughput screening assay for the biochemical screening of 6030 compounds in NCATS annotated libraries. Then, we developed an orthogonal biochemical assay that uses mass spectrometry detection of product formation to ensure that hits from the primary screen are not assay artifacts from the fluorescent detection of product formation. Finally, we assessed the hits from the biochemical screening in a cell-based SARS-CoV-2 pseudotyped particle entry assay. Of the six molecules advanced for further studies, two are approved drugs in Japan (camostat and nafamostat), two have entered clinical trials (PCI-27483 and otamixaban), while the other two molecules are peptidomimetic inhibitors of TMPRSS2 taken from the literature that have not advanced into clinical trials (compounds 92 and 114). This work demonstrates a suite of assays for the discovery and development of new inhibitors of TMPRSS2.

biochemistry↗

bioRxiv2021

mTOR Inhibition Overcomes RSK3-mediated Resistance to BET Inhibitors in Small Cell Lung Cancer

PurposeSCLC is a recalcitrant malignancy with limited treatment options. BET inhibitors have shown promising preclinical activity in SCLC, but their broad sensitivity spectrum limits their clinical prospects in this malignancy. Drug combination could be a solution. Experimental designWe performed high-throughput drug combination screens in SCLC cell lines to identify potential therapeutics synergizing with BET inhibitors. Validation was performed in SCLC cell lines and patient-derived xenograft models. Genome-wide RNA sequencing of xenograft tumors was performed to determine the mechanism underlying the synergy of the drug combination. ResultsInhibitors of the PI-3K-AKT-mTOR pathway were the top candidates from the screens. Among the therapeutics targeting this pathway, mTOR inhibitors showed the highest degree of synergy with BET inhibitors in vitro. Furthermore, the combination of these two classes of drugs showed superior antitumor efficacy and tolerability in vivo. Using both in vitro and in vivo SCLC models, we demonstrate that BET inhibitors activate the intrinsic apoptotic cascade, and mTOR inhibitors further enhance these apoptotic effects. Mechanistically, BET inhibitors activate the TSC2-mTOR-p70S6K1 signaling cascade by upregulating RSK3, an upstream kinase of TSC2. Activation of p70S6K1 leads to BAD phosphorylation and cell survival. mTOR inhibition blocks this survival signaling cascade and potentiates the antitumor effects of BET inhibitors. ConclusionsOur results demonstrate that RSK3 upregulation is a novel resistance mechanism of BET inhibitors in SCLC, and mTOR inhibition can overcome this resistance and enhance apoptosis. These findings provide a rationale to evaluate the combination of mTOR and BET inhibitors in patients with SCLC.

cancer biology↗

bioRxiv2021

The humanized nanobody RBD-1-2G tolerates the spike N501Y mutation to neutralize SARS-CoV-2

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

synthetic biology↗

bioRxiv2021

A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.

pharmacology and toxicology↗

bioRxiv2020

Drug Repurposing Screen for Compounds Inhibiting the Cytopathic Effect of SARS-CoV-2

Drug repurposing is a rapid approach to identifying therapeutics for the treatment of emerging infectious diseases such as COVID-19. To address the urgent need for treatment options, we carried out a quantitative high-throughput screen using a SARS-CoV-2 cytopathic assay with a compound collection of 8,810 approved and investigational drugs, mechanism-based bioactive compounds, and natural products. Three hundred and nineteen compounds with anti-SARS-CoV-2 activities were identified and confirmed, including 91 approved drug and 49 investigational drugs. Among these confirmed compounds, the anti-SARS-CoV-2 activities of 230 compounds, including 38 approved drugs, have not been previously reported. Chlorprothixene, methotrimeprazine, and piperacetazine were the three most potent FDA approved drugs with anti-SARS-CoV-2 activities. These three compounds have not been previously reported to have anti-SARS-CoV-2 activities, although their antiviral activities against SARS-CoV and Ebola virus have been reported. These results demonstrate that this comprehensive data set of drug repurposing screen for SARS-CoV-2 is useful for drug repurposing efforts including design of new drug combinations for clinical trials.

microbiology↗

bioRxiv2020

Modulation of triple artemisinin-based combination therapy pharmacodynamics by Plasmodium falciparum genotype

The first-line treatments for uncomplicated Plasmodium falciparum malaria are artemisinin-based combination therapies (ACTs), consisting of an artemisinin derivative combined with a longer acting partner drug. However, the spread of P. falciparum with decreased susceptibility to artemisinin and partner drugs presents a significant challenge to malaria control efforts. To stem the spread of drug resistant parasites, novel chemotherapeutic strategies are being evaluated, including the implementation of triple artemisinin-based combination therapies (TACTs). Currently, there is limited knowledge on the pharmacodynamics and pharmacogenetic interactions of proposed TACT drug combinations. To evaluate these interactions, we established an in vitro high-throughput process for measuring the drug dose-response to three distinct antimalarial drugs present in a TACT. Sixteen different TACT combinations were screened against fifteen parasite lines from Cambodia, with a focus on parasites with differential susceptibilities to piperaquine and artemisinins. Analysis revealed drug-drug interactions unique to specific genetic backgrounds, including antagonism between piperaquine and pyronaridine associated with gene amplification of plasmepsin II/III, two aspartic proteases that localize to the parasite digestive vacuole. From this initial study, we identified parasite genotypes with decreased susceptibility to specific TACTs, as well as potential TACTs that display antagonism in a genotype-dependent manner. Our assay and analysis platform can be further leveraged to inform drug implementation decisions and evaluate next-generation TACTs.One Sentence Summary In vitro process to evaluate triple-drug combinations for prioritizing antimalarial combinations for in vivo evaluation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗

bioRxiv2020

Discovery of Synergistic and Antagonistic Drug Combinations against SARS-CoV-2 In Vitro

COVID-19 is undoubtedly the most impactful viral disease of the current century, afflicting millions worldwide. As yet, there is not an approved vaccine, as well as limited options from existing drugs for treating this disease. We hypothesized that combining drugs with independent mechanisms of action could result in synergy against SARS-CoV-2. Using in silico approaches, we prioritized 73 combinations of 32 drugs with potential activity against SARS-CoV-2 and then tested them in vitro. Overall, we identified 16 synergistic and 8 antagonistic combinations, 4 of which were both synergistic and antagonistic in a dose-dependent manner. Among the 16 synergistic cases, combinations of nitazoxanide with three other compounds (remdesivir, amodiaquine and umifenovir) were the most notable, all exhibiting significant synergy against SARS-CoV-2. The combination of nitazoxanide, an FDA-approved drug, and remdesivir, FDA emergency use authorization for the treatment of COVID-19, demonstrate a strong synergistic interaction. Notably, the combination of remdesivir and hydroxychloroquine demonstrated strong antagonism. Overall, our results emphasize the importance of both drug repurposing and preclinical testing of drug combinations for potential therapeutic use against SARS-CoV-2 infections.

microbiology↗

bioRxiv2020

Targeting ACE2-RBD interaction as a platform for COVID19 therapeutics: Development and drug repurposing screen of an AlphaLISA proximity assay

The COVID-19 pandemic, caused by SARS-CoV-2, is a pressing public health emergency garnering rapid response from scientists across the globe. Host cell invasion is initiated through direct binding of the viral spike protein to the host receptor angiotensin-converting enzyme 2 (ACE2). Disrupting the spike-ACE2 interaction is a potential therapeutic target for treating COVID-19. We have developed a proximity-based AlphaLISA assay to measure binding of SARS-CoV-2 spike protein Receptor Binding Domain (RBD) to ACE2. Utilizing this assay platform, a drug-repurposing screen against 3,384 small molecule drugs and pre-clinical compounds was performed, yielding 25 high-quality, small-molecule hits that can be evaluated in cell-based models. This established AlphaLISA RBD-ACE2 platform can facilitate evaluation of biologics or small molecules that can perturb this essential viral-host interaction to further the development of interventions to address the global health pandemic.

biochemistry↗

bioRxiv2020

An OpenData portal to share COVID-19 drug repurposing data in real time

The National Center for Advancing Translational Sciences (NCATS) has developed an online open science platform - named the NCATS OpenData Portal (ODP) - for quickly and freely sharing complete NCATS translational datasets via an open-access, user-friendly interface. This paper describes the establishment of the ODP, initially deployed during the COVID-19 crisis, and provides a detailed analysis of COVID-19 drug repurposing screening datasets that served as the first large-scale use case for the platform. Over 10,000 compounds were tested across 17 quantitative high-throughput assays, covering a wide spectrum of the SARS-CoV-2 life cycle. In total, over 87,000 concentration-response curves and 426,000 data points were made publicly available on ODP in near real-time, enabling immediate access to complete datasets. The resource is flexible in accommodating various types and structures of data, and it has already expanded since its launch to host additional datasets for COVID-19, other viruses of pandemic potential, and beyond. The OpenData Portal has been designed as a scalable platform for real-time data sharing across drug discovery campaigns, regardless of disease area, with the overarching goal of accelerating discovery at NCATS, the NIH, and the greater scientific community.

microbiology↗

bioRxiv2020

The SARS-CoV-2 cytopathic effect is blocked with autophagy modulators

SARS-CoV-2 is a new type of coronavirus capable of rapid transmission and causing severe clinical symptoms; much of which has unknown biological etiology. It has prompted researchers to rapidly mobilize their efforts towards identifying and developing anti-viral therapeutics and vaccines. Discovering and understanding the virus’ pathways of infection, host-protein interactions, and cytopathic effects will greatly aid in the design of new therapeutics to treat COVID-19. While it is known that chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including inhibiting autophagy, there are also dose-limiting toxicities in patients that make clearly establishing their potential mechanisms-of-action problematic. Therefore, we evaluated a range of other autophagy modulators to identify an alternative autophagy-based drug repurposing opportunity. In this work, we found that 6 of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero-E6 cells with EC50 values ranging from 2.0 to 13 µM and selectivity indices ranging from 1.5 to >10-fold. Immunofluorescence staining for LC3B and LysoTracker dye staining assays in several cell lines indicated their potency and efficacy for inhibiting autophagy correlated with the measurements in the SARS-CoV-2 cytopathic effect assay. Our data suggest that autophagy pathways could be targeted to combat SARS-CoV-2 infections and become an important component of drug combination therapies to improve the treatment outcomes for COVID-19.One Sentence Summary Blocking SARS-CoV-2 cytopathic effects with selective autophagy inhibitors underlying the clinical benefits of chloroquine and hydroxychloroquine.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗