Novel quinoline substituted autophagy inhibitors attenuate Zika virus replication in ocular cells.

Zika virus (ZIKV) is a re-emerging RNA virus that is known to cause ocular and neurological abnormalities in infants. ZIKV exploits autophagic processes in infected cells to enhance its replication and spread. Thus, autophagy inhibitors have emerged as a potent therapeutic target to combat RNA viruses, with Hydroxychloroquine (HCQ) being one of the most promising candidates. In this study, we synthesized several novel small-molecule quinoline derivatives, assessed their antiviral activity, and determined the underlying molecular mechanisms. Among the nine synthesized analogs, two lead candidates, labeled GL-287 and GL-382, significantly attenuated ZIKV replication in human ocular cells, primarily by inhibiting autophagy. These two compounds surpassed the antiviral efficacy of HCQ and other existing autophagy inhibitors, such as ROC-325, DC661, and GNS561. Moreover, unlike HCQ, these novel analogs did not exhibit cytotoxicity in the ocular cells. Treatment with compounds GL-287 and GL-382 in ZIKV-infected cells increased the abundance of LC3 puncta, indicating the disruption of the autophagic process. Furthermore, compounds GL-287 and GL-382 effectively inhibited the ZIKV-induced innate inflammatory response in ocular cells. Collectively, our study demonstrates the safe and potent antiviral activity of novel autophagy inhibitors against ZIKV.

Leveraging hypoxia in triple-negative breast cancer as a promising treatment strategy.

Current treatment strategies for triple-negative breast cancer (TNBC) are based upon conventional chemotherapy, immunotherapy, or a combination of both. The treatment regimen for chemotherapy is often a combination of two or more drugs, either dose dense or low dose for synergy. Anthracyclines, alkylating agents, antimicrotubule agents, and antimetabolites for early-stage TNBC; and antimetabolites, non-taxane microtubule inhibitors, and cross-linker platinums for late-stage TNBC are usually administered in the clinical setting. Newer options for patients with advanced TNBC, such as poly (ADP-ribose) polymerase (PARP) inhibitors and immune checkpoint inhibitors, have recently emerged for cases where surgery is not a viable option and the disease has metastasized. This review outlines the current trends in hypoxia-inspired treatment strategies for TNBC with a focus on clinical trials.

PROTAC'ing oncoproteins: targeted protein degradation for cancer therapy.

Molecularly targeted cancer therapies substantially improve patient outcomes, although the durability of their effectiveness can be limited. Resistance to these therapies is often related to adaptive changes in the target oncoprotein which reduce binding affinity. The arsenal of targeted cancer therapies, moreover, lacks coverage of several notorious oncoproteins with challenging features for inhibitor development. Degraders are a relatively new therapeutic modality which deplete the target protein by hijacking the cellular protein destruction machinery. Degraders offer several advantages for cancer therapy including resiliency to acquired mutations in the target protein, enhanced selectivity, lower dosing requirements, and the potential to abrogate oncogenic transcription factors and scaffolding proteins. Herein, we review the development of proteolysis targeting chimeras (PROTACs) for selected cancer therapy targets and their reported biological activities. The medicinal chemistry of PROTAC design has been a challenging area of active research, but the recent advances in the field will usher in an era of rational degrader design.

Targeting the Epidermal Growth Factor Receptor with Molecular Degraders: State-of-the-Art and Future Opportunities.

Epidermal growth factor receptor (EGFR) is an oncogenic drug target and plays a critical role in several cellular functions including cancer cell growth, survival, proliferation, differentiation, and motility. Several small-molecule tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs) have been approved for targeting intracellular and extracellular domains of EGFR, respectively. However, cancer heterogeneity, mutations in the catalytic domain of EGFR, and persistent drug resistance limited their use. Different novel modalities are gaining a position in the limelight of anti-EGFR therapeutics to overcome such limitations. The current perspective reflects upon newer modalities, importantly the molecular degraders such as PROTACs, LYTACs, AUTECs, and ATTECs, etc., beginning with a snapshot of traditional and existing anti-EGFR therapies including small molecule inhibitors, mAbs, and antibody drug conjugates (ADCs). Further, a special emphasis has been made on the design, synthesis, successful applications, state-of-the-art, and emerging future opportunities of each discussed modality.

Ku-DNA binding inhibitors modulate the DNA damage response in response to DNA double-strand breaks.

The DNA-dependent protein kinase (DNA-PK) plays a critical role in the DNA damage response (DDR) and non-homologous end joining (NHEJ) double-strand break (DSB) repair pathways. Consequently, DNA-PK is a validated therapeutic target for cancer treatment in certain DNA repair-deficient cancers and in combination with ionizing radiation (IR). We have previously reported the discovery and development of a novel class of DNA-PK inhibitors with a unique mechanism of action, blocking the Ku 70/80 heterodimer interaction with DNA. These Ku-DNA binding inhibitors (Ku-DBi's) display nanomolar activity in vitro, inhibit cellular DNA-PK, NHEJ-catalyzed DSB repair and sensitize non-small cell lung cancer (NSCLC) cells to DSB-inducing agents. In this study, we demonstrate that chemical inhibition of the Ku-DNA interaction potentiates the cellular effects of bleomycin and IR via p53 phosphorylation through the activation of the ATM pathway. This response is concomitant with a reduction of DNA-PK catalytic subunit (DNA-PKcs) autophosphorylation at S2056 and a time-dependent increase in H2AX phosphorylation at S139. These results are consistent with Ku-DBi's abrogating DNA-PKcs autophosphorylation to impact DSB repair and DDR signaling through a novel mechanism of action, and thus represent a promising anticancer therapeutic strategy in combination with DNA DSB-inducing agents.

Senescence-associated secretory proteins induced in lung adenocarcinoma by extended treatment with dexamethasone enhance migration and activation of lymphocytes.

There is a need to improve response rates of immunotherapies in lung adenocarcinoma (AC). Extended (7-14 days) treatment of high glucocorticoid receptor (GR) expressing lung AC cells with dexamethasone (Dex) induces an irreversible senescence phenotype through chronic induction of p27. As the senescence-associated secretory phenotype (SASP) may have either tumor supporting or antitumor immunomodulatory effects, it was interest to examine the effects of Dex-induced senescence of lung AC cells on immune cells. Dex-induced senescence resulted in sustained production of CCL2, CCL4, CXCL1 and CXCL2, both in vitro and in vivo. After Dex withdrawal, secretion of these chemokines by the senescent cells attracted peripheral blood monocytes, T-cells, and NK cells. Following treatment with Dex-induced SASP protein(s), the peripheral blood lymphocytes exhibited higher cell count and tumor cytolytic activity along with enhanced Ki67 and perforin expression in T and NK cells. This cytolytic activity was partially attributed to NKG2D, which was upregulated in NK cells by SASP while its ligand MICA/B was upregulated in the senescent cells. Enhanced infiltrations of T and NK cells were observed in human lung AC xenografts in humanized NSG mice, following treatment with Dex. The findings substantiate the idea that induction of irreversible senescence in high-GR expressing subpopulations of lung AC tumors using Dex pretreatment enhances tumor immune infiltration and may subsequently improve the clinical outcome of current immunotherapies.

Nanomedicine approaches to reduce cytokine storms in severe infections.

During a cytokine storm, dysregulated proinflammatory cytokines are produced in excess. Cytokine storms occur in multiple infectious diseases, including Coronavirus 2019 (COVID-19). Thus, eliminating cytokine storms to enhance patient outcomes is crucial. Given the numerous cytokines involved, individual therapies might have little effect. Traditional cytokines might be less effective than medicines that target malfunctioning macrophages. Nanomedicine-based therapeutics reduce cytokine production in animal models of proinflammatory illnesses. The unique physicochemical features and controlled nano-bio interactions of nanotechnology show promise in healthcare and could be used to treat several stages of this virus-induced sickness, including cytokine storm mortality. Macrophage-oriented nanomedicines can minimize cytokine storms and associated harmful effects, enhancing patient outcomes. Here, we also discuss engineering possibilities for enhancing macrophage efficacy with nanodrug carriers.

Synthesis and Biological Evaluation of Cardiac Glycosides for Cancer Therapy by Targeting the DNA Damage Response.

Cardiac glycosides (CGs) are bioactive compounds originally used to treat heart diseases, but recent studies have demonstrated their anticancer activity. We previously demonstrated that Antiaris toxicaria 2 (AT2) possesses anticancer activity in KRAS mutated lung cancers via impinging on the DNA damage response (DDR) pathway. Toward developing this class of molecules for cancer therapy, herein we report a multistep synthetic route utilizing k-strophanthidin as the initial building block for determination of structure-activity relationships (SARs). A systematic structural design approach was applied that included modifications of the sugar moiety, the glycoside linker, stereochemistry, and lactone ring substitutions to generate a library of O-glycosides and MeON-neoglycosides derivatives. These molecules were screened for their anticancer activities and their impact on DDR signaling in KRAS mutant lung cancer cells. These results demonstrate the ability to chemically synthesize CG derivatives and define the SARs to optimize AT2 as a cancer therapeutic.

Recent Advances in the Development of Non-PIKKs Targeting Small Molecule Inhibitors of DNA Double-Strand Break Repair.

The vast majority of cancer patients receive DNA-damaging drugs or ionizing radiation (IR) during their course of treatment, yet the efficacy of these therapies is tempered by DNA repair and DNA damage response (DDR) pathways. Aberrations in DNA repair and the DDR are observed in many cancer subtypes and can promote de novo carcinogenesis, genomic instability, and ensuing resistance to current cancer therapy. Additionally, stalled or collapsed DNA replication forks present a unique challenge to the double-strand DNA break (DSB) repair system. Of the various inducible DNA lesions, DSBs are the most lethal and thus desirable in the setting of cancer treatment. In mammalian cells, DSBs are typically repaired by the error prone non-homologous end joining pathway (NHEJ) or the high-fidelity homology directed repair (HDR) pathway. Targeting DSB repair pathways using small molecular inhibitors offers a promising mechanism to synergize DNA-damaging drugs and IR while selective inhibition of the NHEJ pathway can induce synthetic lethality in HDR-deficient cancer subtypes. Selective inhibitors of the NHEJ pathway and alternative DSB-repair pathways may also see future use in precision genome editing to direct repair of resulting DSBs created by the HDR pathway. In this review, we highlight the recent advances in the development of inhibitors of the non-phosphatidylinositol 3-kinase-related kinases (non-PIKKs) members of the NHEJ, HDR and minor backup SSA and alt-NHEJ DSB-repair pathways. The inhibitors described within this review target the non-PIKKs mediators of DSB repair including Ku70/80, Artemis, DNA Ligase IV, XRCC4, MRN complex, RPA, RAD51, RAD52, ERCC1-XPF, helicases, and DNA polymerase θ. While the DDR PIKKs remain intensely pursued as therapeutic targets, small molecule inhibition of non-PIKKs represents an emerging opportunity in drug discovery that offers considerable potential to impact cancer treatment.

Nanomedicine for overcoming therapeutic and diagnostic challenges associated with pancreatic cancer.

Pancreatic cancer is the second leading cause of cancer-related death in the USA. The 5-year survival rate for pancreatic cancer is as low as 10%, making it one of the most deadly cancers. This dismal prognosis is caused, in part, by the lack of early detection and screening options, leading to late-stage detection of the disease, at a point at which chemotherapy is no longer effective. However, nanoparticle (NP) drug delivery systems have increased the efficacy of chemotherapeutics by improving the targeting ability of drugs to the tumor site, while also decreasing the risk of local and systemic toxicity. Such efforts can contribute to the development of early diagnosis and routine screening tests, which will drastically improve the survival rates and prognosis of patients with pancreatic cancer.

In Vivo Targeting Replication Protein A for Cancer Therapy.

Replication protein A (RPA) plays essential roles in DNA replication, repair, recombination, and the DNA damage response (DDR). Retrospective analysis of lung cancer patient data demonstrates high RPA expression as a negative prognostic biomarker for overall survival in smoking-related lung cancers. Similarly, relative expression of RPA is a predictive marker for response to chemotherapy. These observations are consistent with the increase in RPA expression serving as an adaptive mechanism that allows tolerance of the genotoxic stress resulting from carcinogen exposure. We have developed second-generation RPA inhibitors (RPAis) that block the RPA-DNA interaction and optimized formulation for in vivo analyses. Data demonstrate that unlike first-generation RPAis, second-generation molecules show increased cellular permeability and induce cell death via apoptosis. Second-generation RPAis elicit single-agent in vitro anticancer activity across a broad spectrum of cancers, and the cellular response suggests existence of a threshold before chemical RPA exhaustion induces cell death. Chemical RPA inhibition potentiates the anticancer activity of a series of DDR inhibitors and traditional DNA-damaging cancer therapeutics. Consistent with chemical RPA exhaustion, we demonstrate that the effects of RPAi on replication fork dynamics are similar to other known DDR inhibitors. An optimized formulation of RPAi NERx 329 was developed that resulted in single-agent anticancer activity in two non-small cell lung cancer models. These data demonstrate a unique mechanism of action of RPAis eliciting a state of chemical RPA exhaustion and suggest they will provide an effective therapeutic option for difficult-to-treat lung cancers.

Implications of the USP10-HDAC6 axis in lung cancer - A path to precision medicine.

Lung cancer is the leading cause of cancer death among both men and women in the United States. Because lung cancer is genetically heterogeneous, tailored therapy alone or in combination with chemotherapy would increase patient overall survival as compared with the one-size-fits-all chemotherapy. TP53-mutant lung cancer accounts for more than half of all lung cancer cases and is oftentimes more aggressive and resistant to chemotherapy. Directly targeting mutant p53 has not yet been successful, so identification of novel therapy targets and biomarkers in the TP53-mutant lung cancer is urgently needed to increase the overall survival in this subgroup. Deubiquitinating enzymes (DUBs) regulate a vast majority of proteins (DUBs' substrates) via removal of ubiquitin moieties or ubiquitin chains from these proteins, thereby altering the stability and/or functions of these substrates. In this review, we will focus on a DUB, referred to as ubiquitin-specific peptidase 10 (USP10) whose substrates include both oncogenic proteins and tumor suppressors. Therefore, targeting USP10 in cancer is highly context-dependent. Here, we will discuss USP10's functions in cancer by examining its various known substrates. In particular, we will elaborate our recent findings in the oncogenic role of USP10 in the TP53-mutant subgroup of lung cancer, focusing on USP10's function in the DNA damage response (DDR) via histone deacetylase 6 (HDAC6). Overall, these findings support the notion that targeting USP10 in the TP53-mutant subgroup of NSCLC would sensitize patients to cisplatin-based chemotherapy. Generating potent and specific clinically relevant USP10 inhibitors would benefit the TP53-mutant subgroup of NSCLC patients.

Design, synthesis, biological evaluation of 3,5-diaryl-4,5-dihydro-1H-pyrazole carbaldehydes as non-purine xanthine oxidase inhibitors: Tracing the anticancer mechanism via xanthine oxidase inhibition.

Xanthine oxidase (XO) has been primarily targeted for the development of anti-hyperuriciemic /anti-gout agents as it catalyzes the conversion of xanthine and hypoxanthine into uric acid. XO overexpression in various cancer is very well correlated due to reactive oxygen species (ROS) production and metabolic activation of carcinogenic substances during the catalysis. Herein, we report the design and synthesis of a series of 3,5-diaryl-4,5-dihydro-1H-pyrazole carbaldehyde derivatives (2a-2x) as xanthine oxidase inhibitors (XOIs). A docking model was developed for the prediction of XO inhibitory activity of our novel compounds. Furthermore, our compounds anticancer activity results in low XO expression and XO-harboring cancer cells both in 2D and 3D-culture models are presented and discussed. Among the array of synthesized compounds, 2b and 2m emerged as potent XO inhibitors having IC50 values of 9.32 ± 0.45 µM and 10.03 ± 0.43 µM, respectively. Both compounds induced apoptosis, halted the cell cycle progression at the G1 phase, elevated ROS levels, altered mitochondrial membrane potential, and inhibited antioxidant enzymes. The levels of miRNA and expression of redox sensors in cells were also altered due to increase oxidative stress induced by our compounds. Compounds 2b and 2m hold a great promise for further development of XOIs for the treatment of XO-harboring tumors.

Pro-inflammatory cytokines and chemokines initiate multiple prostate cancer biologic pathways of cellular proliferation, heterogeneity and metastasis in a racially diverse population and underlie the genetic/biologic mechanism of racial disparity: Update.

Pro-inflammatory cytokine and chemokines genes drive prostate cancer progression and metastasis: molecular mechanism update and the science that underlies racial disparity. comprehensive review article. Isaac J. Powell, S. Chinni, S.S. Reddy, Alexander Zaslavsky, Navnath Gavande Introduction: In 2013 we reported that with the use of bioinformatics and ingenuity pathway network analysis we were able to identify functional driver genes that were differentially expressed among a large population of African American men (AAM) and European American men (EAM). Pro-inflammatory cytokine genes were found to be more interactive and more expressed among AAM and have been found to be functional drivers of aggressive prostate cancer (CaP) and aggressiveness in other solid tumors. We examined these genes and biological pathways initiated by these cytokines in primary CaP tissue. Method We unravel the gene network and identified biologic pathways that impacted activation of the androgen receptor, mesenchymal epithelial transition (invasion) and chemokines associated with metastasis in the CaP tissue from 639 radical prostatectomy specimens. Results Biologic pathways identified by unraveling pro-inflammatory genes from our network, more expressed among AAM compared to EAM, were tumor necrosis factor (TNF), IL1b, IL6, and IL8. IL6 and IL8 are downstream of TNF activity and are known activators of androgen receptor and through mediators promote CaP cell proliferation. TNF and IL1b mediate tumor cell invasiveness through the activation of MMP (matrix metalloproteinase) which down regulates E-Cadherin to initiate epithelial mesenchymal transition which allows cells to become invasive in the microenvironment. Ultimately our network analysis indicates that TNF and IL1b activate CXCR4 receptor on CaP cells, which facilitates metastatic progression reportedly by binding to CXCL12 on lipid rafts and tumor implantation in the bone marrow. Conclusion Our retrospective biologic mechanistic model reveals a set of pro-inflammatory cytokines and chemokines that drive CaP aggressiveness, tumor heterogeneity, progression and metastasis. A prospective multi-institutional study needs to be conducted for clinical validation as well consideration of targeted therapy.

Discovery and development of novel DNA-PK inhibitors by targeting the unique Ku-DNA interaction.

DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) repair pathway and the DNA damage response (DDR). DNA-PK has therefore been pursued for the development of anti-cancer therapeutics in combination with ionizing radiation (IR). We report the discovery of a new class of DNA-PK inhibitors that act via a novel mechanism of action, inhibition of the Ku-DNA interaction. We have developed a series of highly potent and specific Ku-DNA binding inhibitors (Ku-DBi's) that block the Ku-DNA interaction and inhibit DNA-PK kinase activity. Ku-DBi's directly interact with the Ku and inhibit in vitro NHEJ, cellular NHEJ, and potentiate the cellular activity of radiomimetic agents and IR. Analysis of Ku-null cells demonstrates that Ku-DBi's cellular activity is a direct result of Ku inhibition, as Ku-null cells are insensitive to Ku-DBi's. The utility of Ku-DBi's was also revealed in a CRISPR gene-editing model where we demonstrate that the efficiency of gene insertion events was increased in cells pre-treated with Ku-DBi's, consistent with inhibition of NHEJ and activation of homologous recombination to facilitate gene insertion. These data demonstrate the discovery and application of new series of compounds that modulate DNA repair pathways via a unique mechanism of action.

Structure-Guided Optimization of Replication Protein A (RPA)-DNA Interaction Inhibitors.

Replication protein A (RPA) is the major human single stranded DNA (ssDNA)-binding protein, playing essential roles in DNA replication, repair, recombination, and DNA-damage response (DDR). Inhibition of RPA-DNA interactions represents a therapeutic strategy for cancer drug discovery and has great potential to provide single agent anticancer activity and to synergize with both common DNA damaging chemotherapeutics and newer targeted anticancer agents. In this letter, a new series of analogues based on our previously reported TDRL-551 (4) compound were designed to improve potency and physicochemical properties. Molecular docking studies guided molecular insights, and further SAR exploration led to the identification of a series of novel compounds with low micromolar RPA inhibitory activity, increased solubility, and excellent cellular up-take. Among a series of analogues, compounds 43, 44, 45, and 46 hold promise for further development of novel anticancer agents.

The flavonoid, 2'-methoxy-6-methylflavone, affords neuroprotection following focal cerebral ischaemia.

Tonic inhibitory currents, mediated by extrasynaptic GABAA receptors, are elevated at a delay following stroke. Flavonoids minimise the extent of cellular damage following stroke, but little is known about their mode of action. We demonstrate that the flavonoid, 2'-methoxy-6-methylflavone (0.1-10 µM; 2'MeO6MF), increases GABAA receptor tonic currents presumably via δ-containing GABAA receptors. Treatment with 2'MeO6MF 1-6 h post focal ischaemia dose dependently decreases infarct volume and improves functional recovery. The effect of 2'MeO6MF was attenuated in δ-/- mice, indicating that the effects of the flavonoid were mediated via δ-containing GABAA receptors. Further, as flavonoids have been shown to have multiple modes of action, we investigated the anti-inflammatory effects of 2'MeO6MF. Using a macrophage cell line, we show that 2'MeO6MF can dampen an LPS-induced elevation in NFkB activity. Assessment of vehicle-treated stroke animals revealed a significant increase in circulating IL1ß, TNFα and IFγ levels. Treatment with 2'MeO6MF dampened the stroke-induced increase in circulating cytokines, which was blocked in the presence of the pan-AKT inhibitor, GSK690693. These studies support the hypothesis that compounds that potentiate tonic inhibition via δ-containing GABAA receptors soon after stroke can afford neuroprotection.

Modulating DNA Repair Pathways to Improve Precision Genome Engineering.

Programmable nucleases like the popular CRISPR/Cas9 system allow for precision genome engineering by inducing a site-specific DNA double strand break (DSB) within a genome. The DSB is repaired by endogenous DNA repair pathways, either nonhomologous end joining (NHEJ) or homology directed repair (HDR). The predominant and error-prone NHEJ pathway often results in small nucleotide insertions or deletions that can be used to construct knockout alleles. Alternatively, HDR activity can result in precise modification incorporating exogenous DNA fragments into the cut site. However, genetic recombination in mammalian systems through the HDR pathway is an inefficient process and requires cumbersome laboratory methods to identify the desired accurate insertion events. This is further compromised by the activity of the competing DNA repair pathway, NHEJ, which repairs the majority of nuclease induced DNA DSBs and also is responsible for mutagenic insertion and deletion events at off-target locations throughout the genome. Various methodologies have been developed to increase the efficiency of designer nuclease-based HDR mediated gene editing. Here, we review these advances toward modulating the activities of the two critical DNA repair pathways, HDR and NHEJ, to enhance precision genome engineering.

Design and Structure-Guided Development of Novel Inhibitors of the Xeroderma Pigmentosum Group A (XPA) Protein-DNA Interaction.

XPA is a unique and essential protein required for the nucleotide excision DNA repair pathway and represents a therapeutic target in oncology. Herein, we are the first to develop novel inhibitors of the XPA-DNA interaction through structure-guided drug design efforts. Ester derivatives of the compounds 1 (X80), 22, and 24 displayed excellent inhibitory activity (IC50 of 0.82 ± 0.18 µM and 1.3 ± 0.22 µM, respectively) but poor solubility. We have synthesized novel amide derivatives that retain potency and have much improved solubility. Furthermore, compound 1 analogs exhibited good specificity for XPA over RPA (replication protein A), another DNA-binding protein that participates in the nucleotide excision repair (NER) pathway. Importantly, there were no significant interactions observed by the X80 class of compounds directly with DNA. Molecular docking studies revealed a mechanistic model for the interaction, and these studies could serve as the basis for continued analysis of structure-activity relationships and drug development efforts of this novel target.