Suppression of HIV-TAT and cocaine-induced neurotoxicity and inflammation by cell penetrable itaconate esters.

HIV-associated neurological disorder (HAND) is a serious complication of HIV infection marked by neurotoxicity induced by viral proteins like Tat. Substance abuse exacerbates neurocognitive impairment in people living with HIV. There is an urgent need for therapeutic strategies to combat HAND comorbid with Cocaine Use Disorder (CUD). Our analysis of HIV and cocaine-induced transcriptomes in primary cortical cultures revealed significant overexpression of the macrophage-specific gene aconitate decarboxylase 1 (Acod1). The ACOD1 protein converts the tricarboxylic acid intermediate cis-aconitate into itaconate during the activation of inflammation. Itaconate then facilitates cytokine production and activates anti-inflammatory transcription factors, shielding macrophages from infection-induced cell death. However, the immunometabolic function of itaconate was unexplored in HIV and cocaine-exposed microglia. We assessed the potential of 4-octyl-itaconate (4OI), a cell-penetrable ester form of itaconate known for its anti-inflammatory properties. When primary cortical cultures exposed to Tat and cocaine were treated with 4OI, microglial cell number increased and the morphological altercations induced by Tat and cocaine were reversed. Microglial cells also appeared more ramified, resembling the quiescent microglia. 4OI treatment inhibited secretion of the proinflammatory cytokines IL-1α, IL-1ß, IL-6, and MIP1-α induced by Tat and cocaine. Transcriptome profiling determined that Nrf2 target genes were significantly activated in Tat and 4OI treated cultures relative to Tat alone. Further, genes associated with cytoskeleton dynamics in inflammatory microglia were downregulated by 4OI treatment. Together, the results strongly suggest 4-octyl-itaconate holds promise as a potential candidate for therapeutic development to treat HAND coupled with CUD comorbidities.

Improving oncology biosimilar launches in the EU, the USA, and Japan: an updated Policy Review from the Southern Network on Adverse Reactions.

The EU, the USA, and Japan account for the majority of biological pharmacotherapy use worldwide. Biosimilar regulatory approval pathways were authorised in the EU (2006), in Japan (2009), and in the USA (2015), to facilitate approval of biological drugs that are highly similar to reference products and to encourage market competition. Between 2007 and 2020, 33 biosimilars for oncology were approved by the European Medicines Agency (EMA), 16 by the US Food and Drug Administration (FDA), and ten by the Japan Pharmaceuticals and Medical Devices Agency (PMDA). Some of these approved applications were initially rejected because of manufacturing concerns (four of 36 [11%] with the EMA, seven of 16 [44%] with the FDA, none of ten for the PMDA). Median times from initial regulatory submission before approval of oncology biosimilars were 1·5 years (EMA), 1·3 years (FDA), and 0·9 years (PMDA). Pharmacists can substitute biosimilars for reference biologics in some EU countries, but not in the USA or Japan. US regulation prohibits substitution, unless the biosimilar has been approved as interchangeable, a designation not yet achieved for any biosimilar in the USA. Japan does not permit biosimilar substitution, as prescribers must include the product name on each prescription and that specific product must be given to the patient. Policy Reviews published in 2014 and 2016 in The Lancet Oncology focused on premarket and postmarket policies for oncology biosimilars before most of these drugs received regulatory approval. In this Policy Review from the Southern Network on Adverse Reactions, we identify factors preventing the effective launch of oncology biosimilars. Introduction to the market has been more challenging with therapeutic than for supportive care oncology biosimilars. Addressing region-specific competition barriers and educational needs would improve the regulatory approval process and market launches for these biologics, therefore expanding patient access to these products in the EU, the USA, and Japan.

Human Papillomavirus Type 16 L2 DNA Methylation in Exfoliated Cervical Cells From College-Age Women.

OBJECTIVES: The Carolina Women's Care Study (CWCS) at the University of South Carolina followed 467 young women with the goal of identifying biomarkers of human papillomavirus (HPV) persistence. In this study, we analyzed the methylation of HPV16 DNA. METHODS: The aims of this study were to determine the methylation status of the HPV16 L2 gene in DNA isolated from exfoliated cervical cells collected longitudinally as part of the CWCS and to determine the prevalence of polymorphisms (single nucleotide polymorphisms [SNPs]) in folate metabolizing enzymes and DNA repair enzymes known to affect DNA methylation in blood-derived genomic DNA from CWCS participants. For methylation studies, DNA samples were bisulfite converted and amplified with the EpiTect Whole Bisulfitome kit. Polymerase chain reaction was performed for amplicons containing 5 CpG sites in L2. Pyrosequencing was carried out using EpigenDx and analyzed with PyroMark Software. Taqman genotyping assays were performed to determine selected SNP alleles in the CWCS cohort. RESULTS AND CONCLUSIONS: Methylation data were obtained for 82 samples from 27 participants. Of these, 22 participants were positive for HPV16 for 3 or more visits (≥12 months). Methylation in L2 was detectable, but methylation levels varied and were not associated with HPV16 persistence. No linearity of methylation levels over time was observed in participants for whom longitudinal data could be analyzed. Analysis of 9 selected SNPs did not reveal an association with persistence. We conclude that at early stages of infection methylation of HPV16 L2 DNA in Pap test samples is not a predictive biomarker of HPV persistence.

Regulatory and clinical considerations for biosimilar oncology drugs.

Biological oncology products are integral to cancer treatment, but their high costs pose challenges to patients, families, providers, and insurers. The introduction of biosimilar agents-molecules that are similar in structure, function, activity, immunogenicity, and safety to the original biological drugs-provide opportunities both to improve health-care access and outcomes, and to reduce costs. Several international regulatory pathways have been developed to expedite entry of biosimilars into global marketplaces. The first wave of oncology biosimilar use was in Europe and India in 2007. Oncology biosimilars are now widely marketed in several countries in Europe, and in Australia, Japan, China, Russia, India, and South Korea. Their use is emerging worldwide, with the notable exception of the USA, where several regulatory and cost barriers to biosimilar approval exist. In this Review, we discuss oncology biosimilars and summarise their regulatory frameworks, clinical experiences, and safety concerns.

The contribution of heavy metals in cigarette smoke condensate to malignant transformation of breast epithelial cells and in vivo initiation of neoplasia through induction of a PI3K-AKT-NFκB cascade.

Cigarette smoking is a crucial factor in the development and progression of multiple cancers including breast. Here, we report that repeated exposure to a fixed, low dose of cigarette smoke condensate (CSC) prepared from Indian cigarettes is capable of transforming normal breast epithelial cells, MCF-10A, and delineate the biochemical basis for cellular transformation. CSC transformed cells (MCF-10A-Tr) were capable of anchorage-independent growth, and their anchorage dependent growth and colony forming ability were higher compared to the non-transformed MCF-10A cells. Increased expression of biomarkers representative of oncogenic transformation (NRP-1, Nectin-4), and anti-apoptotic markers (PI3K, AKT, NFκB) were also noted in the MCF-10A-Tr cells. Short tandem repeat (STR) profiling of MCF-10A and MCF-10A-Tr cells revealed that transformed cells acquired allelic variation during transformation, and had become genetically distinct. MCF-10A-Tr cells formed solid tumors when implanted into the mammary fat pads of Balb/c mice. Data revealed that CSC contained approximately 1.011µg Cd per cigarette equivalent, and Cd (0.0003µg Cd/1×10(7) cells) was also detected in the lysates from MCF-10A cells treated with 25µg/mL CSC. In similar manner to CSC, CdCl2 treatment in MCF-10A cells caused anchorage independent colony growth, higher expression of oncogenic proteins and increased PI3K-AKT-NFκB protein expression. An increase in the expression of PI3K-AKT-NFκB was also noted in the mice xenografts. Interestingly, it was noted that CSC and CdCl2 treatment in MCF-10A cells increased ROS. Collectively, results suggest that heavy metals present in cigarettes of Indian origin may substantially contribute to tumorigenesis by inducing intercellular ROS accumulation and increased expression of PI3K, AKT and NFκB proteins.

Synthesis and Monoamine Oxidase Inhibitory Activity of Halogenated Flavones.

Small molecule neurotransmitters containing amines are metabolized by monoamine oxidase (MAO) in the nervous system. Monoamine oxidase inhibitors are a valuable class of drugs prescribed for the management of neurological disorders, including depression. A series of halogenated flavonoids similar to the dietary flavonoid acacetin were designed as selective MAO-B inhibitors. MAO-A and -B inhibition of 36 halogenated flavones were tested. The halogens (fluorine and chlorine) were placed at positions 5 and 7 on ring A of the flavone scaffold. All compounds were selective MAO-B inhibitors with micro- and nanomolar IC50 values. Compounds 9f, 10a-c, 11a-c, 11g,h, and 11l displayed inhibitory activity toward MAO-B with IC50 values between 16 to 74 nM. We conclude that halogenated flavonoids are promising molecules in pursuit of developing new agents for neurological disorders.

AI-based mining of biomedical literature: Applications for drug repurposing for the treatment of dementia.

Neurodegenerative pathologies such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis, Multiple sclerosis, HIV-associated neurocognitive disorder, and others significantly affect individuals, their families, caregivers, and healthcare systems. While there are no cures yet, researchers worldwide are actively working on the development of novel treatments that have the potential to slow disease progression, alleviate symptoms, and ultimately improve the overall health of patients. Huge volumes of new scientific information necessitate new analytical approaches for meaningful hypothesis generation. To enable the automatic analysis of biomedical data we introduced AGATHA, an effective AI-based literature mining tool that can navigate massive scientific literature databases, such as PubMed. The overarching goal of this effort is to adapt AGATHA for drug repurposing by revealing hidden connections between FDA-approved medications and a health condition of interest. Our tool converts the abstracts of peer-reviewed papers from PubMed into multidimensional space where each gene and health condition are represented by specific metrics. We implemented advanced statistical analysis to reveal distinct clusters of scientific terms within the virtual space created using AGATHA-calculated parameters for selected health conditions and genes. Partial Least Squares Discriminant Analysis was employed for categorizing and predicting samples (122 diseases and 20889 genes) fitted to specific classes. Advanced statistics were employed to build a discrimination model and extract lists of genes specific to each disease class. Here we focus on drugs that can be repurposed for dementia treatment as an outcome of neurodegenerative diseases. Therefore, we determined dementia-associated genes statistically highly ranked in other disease classes. Additionally, we report a mechanism for detecting genes common to multiple health conditions. These sets of genes were classified based on their presence in biological pathways, aiding in selecting candidates and biological processes that are exploitable with drug repurposing. Author Summary: This manuscript outlines our project involving the application of AGATHA, an AI-based literature mining tool, to discover drugs with the potential for repurposing in the context of neurocognitive disorders. The primary objective is to identify connections between approved medications and specific health conditions through advanced statistical analysis, including techniques like Partial Least Squares Discriminant Analysis (PLSDA) and unsupervised clustering. The methodology involves grouping scientific terms related to different health conditions and genes, followed by building discrimination models to extract lists of disease-specific genes. These genes are then analyzed through pathway analysis to select candidates for drug repurposing.

Al-based mining of biomedical literature: Applications for drug repurposing for the treatment of dementia.

Neurodegenerative pathologies such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis, Multiple sclerosis, HIV-associated neurocognitive disorder, and others significantly affect individuals, their families, caregivers, and healthcare systems. While there are no cures yet, researchers worldwide are actively working on the development of novel treatments that have the potential to slow disease progression, alleviate symptoms, and ultimately improve the overall health of patients. Huge volumes of new scientific information necessitate new analytical approaches for meaningful hypothesis generation. To enable the automatic analysis of biomedical data we introduced AGATHA, an effective AI-based literature mining tool that can navigate massive scientific literature databases, such as PubMed. The overarching goal of this effort is to adapt AGATHA for drug repurposing by revealing hidden connections between FDA-approved medications and a health condition of interest. Our tool converts the abstracts of peer-reviewed papers from PubMed into multidimensional space where each gene and health condition are represented by specific metrics. We implemented advanced statistical analysis to reveal distinct clusters of scientific terms within the virtual space created using AGATHA-calculated parameters for selected health conditions and genes. Partial Least Squares Discriminant Analysis was employed for categorizing and predicting samples (122 diseases and 20889 genes) fitted to specific classes. Advanced statistics were employed to build a discrimination model and extract lists of genes specific to each disease class. Here we focus on drugs that can be repurposed for dementia treatment as an outcome of neurodegenerative diseases. Therefore, we determined dementia-associated genes statistically highly ranked in other disease classes. Additionally, we report a mechanism for detecting genes common to multiple health conditions. These sets of genes were classified based on their presence in biological pathways, aiding in selecting candidates and biological processes that are exploitable with drug repurposing.

Collateral damage of NUDT15 deficiency in cancer provides a cancer pharmacogenetic therapeutic window with thiopurines.

Genome instability is a hallmark of cancer and are driven by mutations in oncogenes and tumor suppressor genes. Despite successes seen with select targeted therapeutics, this type of personalized medicine is only beneficial for a small subpopulation of cancer patients who have one of a few actionable genetic changes. Most tumors also contain hundreds of passenger mutations that offered no fitness advantage or disadvantage during tumor evolution. Mutations in known pharmacogenetic (PGx) loci for which germline variants encode variability in drug response can cause somatically acquired drug sensitivity. The NUDT15 gene is a known PGx locus that participates in the rate-limiting metabolism of thiopurines. People with two defective germline alleles of NUDT15 are hypersensitive to the toxic effects of thiopurines. NUDT15 is located adjacent to the Retinoblastoma ( RB1 ) tumor suppressor gene, which often undergoes homozygous deletion in retinoblastomas and other epithelial cancers. We observed that RB1 undergoes homozygous deletions in 9.4% of prostate adenocarcinomas and 2.5% of ovarian cancers, and in nearly all of these cases NUDT15 is also lost. Moreover, 44% of prostate adenocarcinomas and over 60% of ovarian cancers have lost one allele of NUDT15, which predicts that a majority of all prostate and ovarian cancers have somatically acquired hypersensitivity to thiopurine treatment. We performed a retrospective analysis of >16,000 patients in the US Veterans Administration health care system and found concurrent xanthine oxidase inhibition (XOi) and thiopurine usage for non-cancer indications is significantly associated with reduced incidence of prostate cancer. The hazard ratio for the development of prostate cancer in patients treated with thiopurines and XOi was 0.562 (0.301-1.051) for the unmatched cohort and 0.389 (0.185-0.819) for the propensity score matched cohort. We experimentally depleted NUDT15 from ovarian and prostate cancer cell lines and observed a dramatic sensitization to thiopurine-induced and DNA damage-dependent toxicity. These results indicate that somatic loss of NUDT15 predicts therapeutic sensitivity to a low cost and well tolerated drug with a broad therapeutic window.

Exposure to angiotensin-converting enzyme inhibitors that cross the blood-brain barrier and the risk of dementia among patients with human immunodeficiency virus.

More than one million people in the United States and over 38 million people worldwide are living with human immunodeficiency virus (HIV) infection. Antiretroviral therapy (ART) greatly improves the health of people living with HIV (PLWH); however, the increased life longevity of PLWH has revealed consequences of HIV-associated comorbidities. HIV can enter the brain and cause inflammation even in individuals with well-controlled HIV infection. The quality of life for PLWH can be compromised by cognitive deficits and memory loss, termed HIV-associated neurological disorders (HAND). HIV-associated dementia is a related but distinct diagnosis. Common causes of dementia in PLWH are similar to the general population and can affect cognition. There is an urgent need to identify treatments for the aging PWLH population. We previously developed AI-based biomedical literature mining systems to uncover a potential novel connection between HAND the renin-angiotensin system (RAAS), which is a pharmacological target for hypertension. RAAS-targeting anti-hypertensives are gaining attention for their protective benefits in several neurocognitive disorders. To our knowledge, the effect of RAAS-targeting drugs on the cognition of PLWH development of dementia has not previously been analyzed. We hypothesized that exposure to angiotensin-converting enzyme inhibitors (ACEi) that cross the blood brain barrier (BBB) reduces the risk/occurrence of dementia in PLWH. We report a retrospective cohort study of electronic health records (EHRs) to examine the proposed hypothesis using data from the United States Department of Veterans Affairs, in which a primary outcome of dementia was measured in controlled cohorts of patients exposed to BBB-penetrant ACEi versus those unexposed to BBB-penetrant ACEi. The results reveal a statistically significant reduction in dementia diagnosis for PLWH exposed to BBB-penetrant ACEi. These results suggest there is a potential protective effect of BBB ACE inhibitor exposure against dementia in PLWH that warrants further investigation.

Lycopene synergistically enhances quinacrine action to inhibit Wnt-TCF signaling in breast cancer cells through APC.

We previously reported that quinacrine (QC) has anticancer activity against breast cancer cells. Here, we examine the mechanism of action of QC and its ability to inhibit Wnt-TCF signaling in two independent breast cancer cell lines. QC altered Wnt-TCF signaling components by increasing the levels of adenomatous polyposis coli (APC), DAB2, GSK-3ß and axin and decreasing the levels of ß-catenin, p-GSK3ß (ser 9) and CK1. QC also reduced the activity of the Wnt transcription factor TCF/LEF and its downstream targets cyclin D1 and c-MYC. Using a luciferase-based Wnt-TCF transcription factor assay, it was shown that APC levels were inversely associated with TCF/LEF activity. Induction of apoptosis and DNA damage was observed after treatment with QC, which was associated with increased expression of APC. The effects induced by QC depend on APC because the inhibition of Wnt-TCF signaling by QC is lost in APC-knockdown cells, and consequently, the extent of apoptosis and DNA damage caused by QC is reduced compared with parental cells. Because we previously showed that QC inhibits topoisomerase, we examined the effect of another topoisomerase inhibitor, etoposide, on Wnt signaling. Interestingly, etoposide treatment also reduced TCF/LEF activity, ß-catenin and cyclin D1 levels commensurate with induction of DNA damage and apoptosis. Lycopene, a plant-derived antioxidant, synergistically increased QC activity and inhibited Wnt-TCF signaling in cancer cells without affecting the MCF-10A normal breast cell line. Collectively, the data suggest that QC-mediated Wnt-TCF signal inhibition depends on APC and that the addition of lycopene synergistically increases QC anticancer activity.

SMUG1 but not UNG DNA glycosylase contributes to the cellular response to recovery from 5-fluorouracil induced replication stress.

5-Fluorouracil (5-FU) is a widely utilized cancer chemotherapeutic that causes DNA damage via two mechanisms. Its active metabolite inhibits thymidylate synthase, which deprives cells of TTP and causes the introduction of uracil in DNA. Also, 5-FU is directly incorporated into DNA. Both uracil and 5-FU in DNA are recognized by uracil-DNA glycosylases (UDGs), which initiate base excision repair. UNG and SMUG1 are the two human UDGs most likely to combat the genomic incorporation of uracil and 5-FU during replication. In this study, we examined the roles of UNG and SMUG1 in the initial cellular response to 5-FU and compared continuous exposure to a 24h exposure followed by incubation in drug-free media, which mimics what occurs clinically. Loss of UNG did not alter cellular sensitivity to 5-FU in two human cell lines, despite its predominant biochemical activity for uracil and 5-FU in DNA. Loss of SMUG1 corresponded with >2-fold increase in sensitivity to 5-FU, but only with a 24h treatment followed by recovery. There was no difference between SMUG1 proficient and depleted cells following continuous exposure. We observed that 5-FU treatment induced an enhanced S-phase arrest and CHK1 activation plus an increase in the formation of strand breaks and alkali-labile sites in all sublines. However, SMUG1-depleted cells showed a prolonged S-phase arrest, a transient increase in DNA double-strand breaks following 5-FU treatment and an altered phosphorylation of CHK1 following removal of drug. Collectively, the results suggest that SMUG1 has a role in the resumption of replication following 5-FU treatment.

Structural Basis for Variations in Polo-like Kinase 1 Conformation and Intracellular Stability Induced by ATP-Competitive and Novel Noncompetitive Abbapolin Inhibitors.

Polo-like kinase 1 (PLK1) is an essential protein kinase with multiple roles in mitotic progression. PLK1 consists of a kinase domain (KD) and a phosphopeptide-binding polobox domain (PBD), which is responsible for substrate recognition and subcellular localization. The regulation of PLK1 involves an autoinhibitory conformation in which KD and PBD interact. Our previous work identified PBD-binding molecules termed abbapolins that inhibit the cellular phosphorylation of a PLK1 substrate and induce the loss of intracellular PLK1. Here, we describe a comparison of the abbapolin activity with that of KD inhibitors to gain insight into conformational features of PLK1. As measured by a cellular thermal shift assay, abbapolins produce ligand-induced thermal stabilization of PLK1. In contrast, KD inhibitors decreased the soluble PLK1, suggesting that catalytic-site binding causes a less thermally stable PLK1 conformation. Binding measurements with full-length PLK1 and a KD inhibitor also demonstrated a conformational change. Interestingly, the cellular consequences of KD versus PBD engagement contrast as KD binding causes the accumulation of intracellular PLK1, whereas PBD binding produces a striking loss of nuclear PLK1. These data are consistent with the relief of autoinhibited PLK1 by KD binders; an explanation for these observations is presented using structures for the catalytic domain and full-length PLK1 predicted by AlphaFold. Collectively, the results highlight an underappreciated aspect of targeting PLK1, namely, conformational perturbations induced by KD versus PBD binding. In addition to their significance for PBD-binding ligands, these observations have implications for the development of ATP-competitive PLK1 inhibitors because catalytic inhibitors may conversely promote PLK1 noncatalytic functions, which may explain their lack of clinical efficacy to date.

Inhibitors of the PLK1 polo-box domain: drug design strategies and therapeutic opportunities in cancer.

INTRODUCTION: Polo Like Kinase 1 (PLK1) is a key regulator of mitosis and its overexpression is frequently observed in a wide variety of human cancers, while often being associated with poor survival rates. Therefore, it is considered a potential and attractive target for cancer therapeutic development. The Polo like kinase family is characterized by the presence of a unique C terminal polobox domain (PBD) involved in regulating kinase activity and subcellular localization. Among the two functionally essential, druggable sites with distinct properties that PLK1 offers, targeting the PBD presents an alternative approach for therapeutic development. AREAS COVERED: Significant progress has been made in progressing from the peptidic PBD inhibitors first identified, to peptidomimetic and recently drug-like small molecules. In this review, the rationale for targeting the PBD over the ATP binding site is discussed, along with recent progress, challenges, and outlook. EXPERT OPINION: The PBD has emerged as a viable alternative target for the inhibition of PLK1, and progress has been made in using compounds to elucidate mechanistic aspects of activity regulation and in determining roles of the PBD. Studies have resulted in proof of concept of in vivo efficacy suggesting promise for PBD binders in clinical development.

Suppression of HIV and cocaine-induced neurotoxicity and inflammation by cell penetrable itaconate esters.

HIV-associated neurological disorder (HAND) is a serious complication of HIV infection, marked by neurotoxicity induced by viral proteins like Tat. Substance abuse exacerbates neurocognitive impairment in people living with HIV. There is an urgent need for effective therapeutic strategies to combat HAND comorbid with Cocaine Use Disorder (CUD). Our analysis of the HIV and cocaine-induced transcriptomes in primary cortical cultures revealed a significant overexpression of the macrophage-specific gene, aconitate decarboxylase 1 (Acod1), caused by the combined insults of HIV and cocaine. ACOD1 protein converts the tricarboxylic acid intermediate cis-aconitate into itaconate during the activation of inflammation. The itaconate produced facilitates cytokine production and subsequently activates anti-inflammatory transcription factors, shielding macrophages from infection-induced cell death. While the role of itaconate' in limiting inflammation has been studied in peripheral macrophages, its immunometabolic function remains unexplored in HIV and cocaine-exposed microglia. We assessed in this model system the potential of 4-octyl-itaconate (4OI), a cell-penetrable esterified form of itaconate known for its potent anti-inflammatory properties and potential therapeutic applications. We administered 4OI to primary cortical cultures exposed to Tat and cocaine. 4OI treatment increased the number of microglial cells in both untreated and Tat±Cocaine-treated cultures and also reversed the morphological altercations induced by Tat and cocaine. In the presence of 4OI, microglial cells also appeared more ramified, resembling the quiescent microglia. Consistent with these results, 4OI treatment inhibited the secretion of the proinflammatory cytokines IL-1α, IL-1ß, IL-6, and MIP1-α induced by Tat and cocaine. Transcriptome profiling further determined that Nrf2 target genes such as NAD(P)H quinone oxidoreductase 1 (Nqo1), Glutathione S-transferase Pi (Gstp1), and glutamate cysteine ligase catalytic (Gclc), were most significantly activated in Tat-4OI treated cultures, relative to Tat alone. Further, genes associated with cytoskeleton dynamics in inflammatory microglia were downregulated by 4OI treatment. Together, the results strongly suggest 4-octyl-itaconate holds promise as a potential candidate for therapeutic development aimed at addressing HAND coupled with CUD comorbidities.

Quinacrine has anticancer activity in breast cancer cells through inhibition of topoisomerase activity.

The small molecule Quinacrine (QC, a derivative of 9-aminoacridine), an anti-malaria drug, displays activity against cancer cell lines and can simultaneously suppress nuclear factor-κB (NF-κB) and activate p53 signaling. In this study, we investigated the anticancer mechanism underlying these drug activities in breast cancer cell lines. QC caused a dose-dependent decrease of both anchorage dependent and independent growth of breast cancer cells (MCF-7 and MDA-MB-231) without affecting normal breast epithelial cells (MCF-10A), as evident from clonogenic cell survival, [3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide] viability, wound healing and soft agar growth. QC activated the proapoptotic marker Bax, PARP cleavage, p53 and its downstream target, p21 (Cip1/Waf1) and downregulated the antiapoptotic marker Bcl-xL and relative luciferase activity of NF-κB in MCF-7 cells. Results of DAPI nuclear staining and FACS analysis show that QC increased apoptosis in a dose-dependent manner. QC caused apoptosis by increasing the cell population in S-phase and simultaneously decreasing the G1 and G2/M populations. A dose-dependent increase of DNA damage as measured by the comet assay was seen in MCF-7 cells after exposure to QC. With regards to the mechanism of DNA damage, we found that QC inhibited topoisomerase activity in MCF-7 cells by increasing the unwinding of supercoiled DNA. Collectively, the results demonstrate that QC has efficient anticancer potential against breast cancer cells via not only an induction of p53 and p21 but also an induction of S phase arrest, DNA damage and inhibition of topoisomerase activity.

Quinacrine-mediated autophagy and apoptosis in colon cancer cells is through a p53- and p21-dependent mechanism.

We previously showed that quinacrine (QC), a small molecule antimalarial agent, also presented anticancer activity in breast cancer cells through activation of p53, p21, and inhibition of topoisomerase activity. Here we have systematically studied the detailed cell death mechanism of this drug using three colon cancer cell lines (HCT-116 parental, isogenic HCT-116 p53-/-, and HCT-116 p21-/- sublines). QC caused a dose-dependent reduction in cell viability in all three cell lines. However, the parental cells were more susceptible to QC-mediated cell death, suggesting that p53- and p21-dependent processes were involved. QC-mediated cell death was measured with the following endpoints: the Bax/Bcl-xL ratio, cleaved PARP, apoptotic nuclei visualized by DAPI staining, and COMET formation. In addition, markers of autophagy were measured. Acridine orange staining revealed increased accumulation of autophagic vacuoles (AVs) after QC treatment in a dose-dependent manner in parental cells, and decreased staining in isogenic HCT-116 p53-/- and HCT-116 p21-/- cells. Immunofluorescence of LC3B was significantly lowered in QC-treated cells lacking p53 or p21, compared to the parental cells. Interestingly, the expression of the autophagy marker LC3B-II after exposure to QC was decreased in either p53 or p21 null cells compared to parental cells. After deletion of p21 in HCT-116 p53-/- cells, no change in LC3B-II expression was noted following QC treatment. Collectively, the results suggest that QC-mediated autophagy and apoptosis dependent on p53 and p21.

Structure-activity and mechanistic studies of non-peptidic inhibitors of the PLK1 polo box domain identified through REPLACE.

Polo-like kinase 1 (PLK1) is a serine/threonine-protein kinase involved in cell cycle regulation and mitotic progression. Studies have shown that PLK1 is upregulated in many tumors and high levels are adversely related to a poor prognosis. Knocking down or inhibiting PLK1 results in synthetic lethality in PTEN deficient prostate tumors and Kras mutant colorectal tumors, further validating PLK1 as an oncotarget. Substrate recognition by PLK1 occurs through the Polo-Box Domain (PBD), which is a phospho-peptide binding site also responsible for subcellular localization. Much effort has been directed to target this kinase therapeutically through the ATP-binding site, and a few such inhibitors have advanced to clinical trials however with limited clinical efficacy. Moreover, it has been shown that a point mutation in PLK1 (C67V) confers dramatic cellular resistance to catalytic site inhibitors. An alternative approach to target PLK1 potently and selectively is through the PBD to block its protein-protein interactions. Through the REPLACE strategy, for converting peptide inhibitors into more drug-like non peptidic compounds, a PBD targeting compound series ("ABBAs"), has been identified and the key determinants of potency and selectivity elucidated through structure-activity relationship studies. In cellular experiments, the ABBAs were shown to lead to profound effects on the cell cycle, to inhibit tumor proliferation and overcome resistance of cells expressing the PLK1 C67V mutant to ATP-based inhibitors. These non-ATP competitive inhibitors of PLK1 were also used chemical biology probes to investigate the gene regulatory effects of PLK1, known to act on transcription factors such as p53.