Publications by authors named "Shizuko Sei"

22 Publications

  • Page 1 of 1

Optimization of Erlotinib Plus Sulindac Dosing Regimens for Intestinal Cancer Prevention in an Apc-Mutant Model of Familial Adenomatous Polyposis (FAP).

Cancer Prev Res (Phila) 2020 Dec 4. Epub 2020 Dec 4.

Center for Epigenetics and Disease Prevention, Texas A&M Health Science Center, Houston, Texas.

A clinical trial in patients with familial adenomatous polyposis (FAP) demonstrated that sulindac plus erlotinib (SUL+ERL) had good efficacy in the duodenum and colon, but toxicity issues raised concerns for long-term prevention. We performed a biomarker study in the polyposis in rat colon (Pirc) model, observing phosphorylated Erk inhibition in colon polyps for up to 10 days after discontinuing ERL+SUL administration. In a follow-up study lasting 16 weeks, significant reduction of colon and small intestine (SI) tumor burden was detected, especially in rats given 250 ppm SUL in the diet plus once-a-week intragastric dosing of ERL at 21 or 42 mg/kg body weight (BW). A long-term study further demonstrated antitumor efficacy in the colon and SI at 52 weeks, when 250 ppm SUL was combined with once-a-week intragastric administration of ERL at 10, 21, or 42 mg/kg BW. Tumor-associated (), tumor necrosis factor (), and early growth response 1 () were decreased at 16 weeks by ERL+SUL, and this was sustained in the long-term study for and . Based on the collective results, the optimal dose combination of ERL 10 mg/kg BW plus 250 ppm SUL lacked toxicity, inhibited molecular biomarkers, and exhibited effective antitumor activity. We conclude that switching from continuous to once-per-week ERL, given at one-quarter of the current therapeutic dose, will exert good efficacy with standard-of-care SUL against adenomatous polyps in the colon and SI, with clinical relevance for patients with FAP before or after colectomy. PREVENTION RELEVANCE: This investigation concludes that switching from continuous to once-per-week erlotinib, given at one-quarter of the current therapeutic dose, will exert good efficacy with standard-of-care sulindac against adenomatous polyps in the colon and small intestine, with clinical relevance for patients with FAP before or after colectomy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1940-6207.CAPR-20-0262DOI Listing
December 2020

Cancer Immunoprevention: Challenges and Potential Opportunities for Use of Immune Checkpoint Inhibitors.

Cancer Prev Res (Phila) 2020 Nov 18;13(11):897-900. Epub 2020 Sep 18.

Chemopreventive Agent Development Research Group, Division of Cancer Prevention, National Cancer Institute, Rockville, Maryland.

Cancer immunoprevention is achieved through promoting antitumor immune surveillance to block tumor formation and progression. Following the success of prophylactic vaccines against human papillomavirus (HPV) in preventing HPV-associated cancer, immunopreventive cancer vaccines targeting tumor antigens have been increasingly evaluated against cancers of noninfectious origin. While advances in cancer immunotherapy with immune checkpoint inhibitors (ICI) have clearly shown that the host immune system can mount effective antitumor immunity against tumor antigens when immune checkpoints are optimally blocked, the use of ICIs in the prevention setting has not been widely explored because of concerns of ICI-associated adverse events. In this issue of , Chung and colleagues demonstrate that the human cirrhotic liver harbors neoantigens, which accumulate further as the disease progresses to hepatocellular carcinoma (HCC), suggesting that cirrhotic liver may be susceptible to ICI therapy. Utilizing an established mouse model of carcinogen-induced liver fibrosis and HCC, they show that intermittent intervention by ICI, anti-mouse PD-1 (CD279) antibody, can prevent the progression of the precancerous stage of cirrhosis to HCC accompanied by increased T-cell infiltrates in the liver parenchyma. Importantly, there were no overt ICI-associated toxicities in the treated mice, indicating that safe dosing regimens could be established. This work is both significant and timely, opening the door to future studies, where the utility of ICI therapy can be further investigated not only in cirrhosis but other high-risk precancerous conditions. In this perspective, we discuss the implications of their findings, and the challenges and potential opportunities for use of ICIs for cancer immunoprevention..
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1940-6207.CAPR-20-0432DOI Listing
November 2020

Naproxen chemoprevention promotes immune activation in Lynch syndrome colorectal mucosa.

Gut 2021 Mar 8;70(3):555-566. Epub 2020 Jul 8.

Department of Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA

Objective: Patients with Lynch syndrome (LS) are at markedly increased risk for colorectal cancer. It is being increasingly recognised that the immune system plays an essential role in LS tumour development, thus making an ideal target for cancer prevention. Our objective was to evaluate the safety, assess the activity and discover novel molecular pathways involved in the activity of naproxen as primary and secondary chemoprevention in patients with LS.

Design: We conducted a Phase Ib, placebo-controlled, randomised clinical trial of two dose levels of naproxen sodium (440 and 220 mg) administered daily for 6 months to 80 participants with LS, and a co-clinical trial using a genetically engineered mouse model of LS and patient-derived organoids (PDOs).

Results: Overall, the total number of adverse events was not different across treatment arms with excellent tolerance of the intervention. The level of prostaglandin E2 in the colorectal mucosa was significantly decreased after treatment with naproxen when compared with placebo. Naproxen activated different resident immune cell types without any increase in lymphoid cellularity, and changed the expression patterns of the intestinal crypt towards epithelial differentiation and stem cell regulation. Naproxen demonstrated robust chemopreventive activity in a mouse co-clinical trial and gene expression profiles induced by naproxen in humans showed perfect discrimination of mice specimens with LS and PDOs treated with naproxen and control.

Conclusions: Naproxen is a promising strategy for immune interception in LS. We have discovered naproxen-induced gene expression profiles for their potential use as predictive biomarkers of drug activity.

Trial Registration Number: gov Identifier: NCT02052908.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/gutjnl-2020-320946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790993PMC
March 2021

Combination of Erlotinib and Naproxen Employing Pulsatile or Intermittent Dosing Profoundly Inhibits Urinary Bladder Cancers.

Cancer Prev Res (Phila) 2020 03 9;13(3):273-282. Epub 2019 Dec 9.

Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama.

Daily dosing of either NSAIDs or EGFR inhibitors has been shown to prevent bladder cancer development in a N-butyl-(4-hydroxybutyl)nitrosamine (OH-BBN)-induced rat model. However, these inhibitors cause gastrointestinal ulceration and acneiform rash, respectively, limiting their continuous use in a clinical prevention setting. We studied chemopreventive efficacy of pulsatile dosing of EGFR inhibitor erlotinib (42 mg/kg BW, once/week) combined with intermittent or continuous low doses of the NSAID naproxen (30 mg/kg BW/day, 3 weeks on/off or 128 ppm daily in diet) in the OH-BBN induced rat bladder cancer model. The interventions were started either at 1 or 4 weeks (early intervention) or 3 months (delayed intervention) after the last OH-BBN treatment, by which time the rats had developed microscopic bladder lesions. All combination regimens tested as early versus late intervention led to the reduction of the average bladder tumor weights (54%-82%; < 0.01 to < 0.0001), a decrease in tumor multiplicity (65%-85%; < 0.01 to < 0.0001), and a decrease in the number of rats with large palpable tumors (>200 mg; 83%-90%; < 0.01 to < 0.0001). Levels of signal transduction markers, Ki-67, cyclin D1, IL1β, pSTAT3, and pERK, were significantly ( < 0.05 to < 0.001) reduced in the treated tumors, demonstrating their potential utility as predictive markers for efficacy. These findings demonstrate that significant chemopreventive efficacy could be achieved with alternative intervention regimens designed to reduce the toxicity of agents, and that starting erlotinib and/or naproxen treatments at the time microscopic tumors were present still conferred the efficacy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1940-6207.CAPR-19-0339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060101PMC
March 2020

Potentiation of Kras peptide cancer vaccine by avasimibe, a cholesterol modulator.

EBioMedicine 2019 Nov 31;49:72-81. Epub 2019 Oct 31.

Center for Disease Prevention Research and Department of Pharmacology & Toxicology, Medical College of Wisconsin, Milwaukee, WI, USA. Electronic address:

Background: No effective approaches to target mutant Kras have yet been developed. Immunoprevention using KRAS-specific antigenic peptides to trigger T cells capable of targeting tumor cells relies heavily on lipid metabolism. To facilitate better TCR/peptide/MHC interactions that result in better cancer preventive efficacy, we combined KVax with avasimibe, a specific ACAT1 inhibitor, tested their anti-cancer efficacy in mouse lung cancer models, where Kras mutation was induced before vaccination.

Methods: Control of tumor growth utilizing a multi-peptide Kras vaccine was tested in combination with avasimibe in a syngeneic lung cancer mouse model and a genetically engineered mouse model (GEMM). Activation of immune responses after administration of Kras vaccine and avasimibe was also assessed by flow cytometry, ELISpot and IHC.

Findings: We found that Kras vaccine combined with avasimibe significantly decreased the presence of regulatory T cells in the tumor microenvironment and facilitated CD8+ T cell infiltration in tumor sites. Avasimibe also enhanced the efficacy of Kras vaccines target mutant Kras. Whereas the Kras vaccine significantly increased antigen-specific intracellular IFN-γ and granzyme B levels in CD8+ T cells, avasimibe significantly increased the number of tumor-infiltrating CD8+ T cells. Additionally, modulation of cholesterol metabolism was found to specifically impact in T cells, and not in cancer cells.

Interpretation: Avasimibe complements the efficacy of a multi-peptide Kras vaccine in controlling lung cancer development and growth. This treatment regimen represents a novel immunoprevention approach to prevent lung cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ebiom.2019.10.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6945201PMC
November 2019

Intermittent Dosing Regimens of Aspirin and Naproxen Inhibit Azoxymethane-Induced Colon Adenoma Progression to Adenocarcinoma and Invasive Carcinoma.

Cancer Prev Res (Phila) 2019 11 17;12(11):751-762. Epub 2019 Sep 17.

Center for Cancer Prevention and Drug Development, Department of Medicine, Hem-Onc Section, Stephenson Cancer Center and University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma.

Chronic use of aspirin and related drugs to reduce cancer risk is limited by unwanted side effects. Thus, we assessed the efficacy associated with different dosing regimens of aspirin and naproxen. Azoxymethane (AOM)-rat colon cancer model was used to establish the pharmacodynamic efficacy of aspirin and naproxen under different dosing regimens. Colon tumors were induced in rats (36/group) by two weekly doses of AOM. At the early adenoma stage, rats were fed diets containing aspirin (700 and 1,400 ppm) or naproxen (200 and 400 ppm), either continuously, 1 week on/1 week off, or 3 weeks on/3 weeks off, or aspirin (2,800 ppm) 3 weeks on/3 weeks off. All rats were euthanized 48 weeks after AOM treatment and assessed for efficacy and biomarkers in tumor tissues. Administration of aspirin and naproxen produced no overt toxicities. Administration of different treatment regimens of both agents had significant inhibitory effects with clear dose-response effects. Aspirin suppressed colon adenocarcinoma multiplicity (both invasive and noninvasive) by 41% ( < 0.003) to 72% ( < 0.0001) and invasive colon adenocarcinomas by 67%-91% ( < 0.0001), depending on the treatment regimen. Naproxen doses of 200 and 400 ppm inhibited invasive adenocarcinoma multiplicity by 53%-88% ( < 0.0001), depending on the dosing regimen. Colonic tumor biomarker analysis revealed that proliferation (proliferating cell nuclear antigen and p21), apoptosis (p53 and Caspase-3), and proinflammatory mediators (IL1β and prostaglandin E) were significantly correlated with the tumor inhibitory effects of aspirin and naproxen. Overall, our results suggest that intermittent dosing regimens with aspirin or naproxen demonstrated significant efficacy on the progression of adenomas to adenocarcinomas, without gastrointestinal toxicities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1940-6207.CAPR-19-0312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6849393PMC
November 2019

Proteomic characterization of outer membrane vesicles from gut mucosa-derived fusobacterium nucleatum.

J Proteomics 2019 03 8;195:125-137. Epub 2019 Jan 8.

Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, United States. Electronic address:

Fusobacterium nucleatum is a Gram-negative bacterium commonly found in the oral cavity and is often involved in periodontal diseases. Recent studies have shown increased F. nucleatum prevalence in colorectal cancer (CRC) tissues, and causal data has linked this bacterium to CRC tumorigenesis. Immune-based approaches to contain, reduce or eradicate its gut colonization may prevent CRC. Outer membrane vesicles (OMVs) are naturally produced by Gram-negative bacteria, typically contain multiple putative virulence factors and may elicit protective immune responses if used as vaccines. Here, OMVs were isolated from F. nucleatum cultures and purified using gradient centrifugation. Proteins contained within the OMVs were identified by nano LC/MS/MS analysis. Of 98 proteins consistently identified from duplicate analyses, 60 were predicted to localize to the outer membrane or periplasm via signal peptide driven translocation. Of these, six autotransporter proteins, which constitute the majority of protein mass of OMVs, were associated with Type V secretion system. In addition, other putative virulence factor proteins with functional domains, including FadA, MORN2 and YadA-like domain, were identified with multiple exposed epitope sites as determined by in silico analysis. Altogether, the non-replicative OMVs of F. nucleatum contain multiple antigenic virulence factors that may play important roles in the design and development of vaccines against F. nucleatum. SIGNIFICANCE: Fusobacterium nulceatum has been proved playing significant role in colorectal carcinogenesis. Outer membrane vesicles are nanoparticles that naturally secreted by Gram-negative bacterial containing various antigenic components, which provides new insight in vaccine development. Understanding the constituents of F. nucleatum OMVs will provide fundamental information and potential strategies for OMV-based F. nucleatum vaccines design. Based on our knowledge this is the first proteomic study of OMVs from F. nucleatum.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jprot.2018.12.029DOI Listing
March 2019

Immunoprevention of KRAS-driven lung adenocarcinoma by a multipeptide vaccine.

Oncotarget 2017 Oct 1;8(47):82689-82699. Epub 2017 Aug 1.

Cancer Center and Department of Pharmacology & Toxicology, Medical College of Wisconsin, Milwaukee, WI, USA.

Lung cancer remains the leading cause of cancer death worldwide. Mutations in KRAS are detected in up to 30% of lung cancer cases. No effective therapies specifically targeting mutant KRAS have been developed. Vaccination against KRAS mutants is one of the venues of active exploration. The present study evaluated both immunogenicity and antitumor efficacy of a newly formulated multipeptide vaccine targeting multiple epitopes of the KRAS molecule. The formulated vaccine contained top four peptides, which elicited the strongest immunologic response and showed 100% sequence homology between human and mouse. The multipeptide KRAS vaccine was tested in an inducible CCSP-TetO-KRAS mouse model, where the vaccine was administered prior to activating the mutant KRAS protein. The KRAS peptide vaccine exhibited striking efficacy, reducing tumor number and tumor burden by >80% when compared with adjuvant alone. Splenocytes collected from vaccinated animals showed a robust immunologic response to the immunizing peptides. Furthermore, stimulation of these splenocytes by the vaccinated peptides resulted in the secretion of cytokines indicative of Th1 responses but with minimal secretion of Th2-related cytokines. The multipeptide KRAS vaccine was immunogenic and efficacious in the primary prevention of KRAS-induced lung cancer, indicating that the approach potentially can be used to prevent other KRAS-driven cancers, either alone or in combination with other modalities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.19831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669920PMC
October 2017

Identification of a nucleoside analog active against adenosine kinase-expressing plasma cell malignancies.

J Clin Invest 2017 Jun 15;127(6):2066-2080. Epub 2017 May 15.

Department of Pathology and Laboratory Medicine.

Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase-inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI-sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI83936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451239PMC
June 2017

Synergistic antitumor activity of oncolytic reovirus and chemotherapeutic agents in non-small cell lung cancer cells.

Mol Cancer 2009 Jul 14;8:47. Epub 2009 Jul 14.

Laboratory of Human Toxicology and Pharmacology, SAIC-Frederick, Inc, NCI-Frederick, Frederick, Maryland, USA.

Background: Reovirus type 3 Dearing strain (ReoT3D) has an inherent propensity to preferentially infect and destroy cancer cells. The oncolytic activity of ReoT3D as a single agent has been demonstrated in vitro and in vivo against various cancers, including colon, pancreatic, ovarian and breast cancers. Its human safety and potential efficacy are currently being investigated in early clinical trials. In this study, we investigated the in vitro combination effects of ReoT3D and chemotherapeutic agents against human non-small cell lung cancer (NSCLC).

Results: ReoT3D alone exerted significant cytolytic activity in 7 of 9 NSCLC cell lines examined, with the 50% effective dose, defined as the initial virus dose to achieve 50% cell killing after 48 hours of infection, ranging from 1.46 +/- 0.12 approximately 2.68 +/- 0.25 (mean +/- SD) log10 pfu/cell. Chou-Talalay analysis of the combination of ReoT3D with cisplatin, gemcitabine, or vinblastine demonstrated strong synergistic effects on cell killing, but only in cell lines that were sensitive to these compounds. In contrast, the combination of ReoT3D and paclitaxel was invariably synergistic in all cell lines tested, regardless of their levels of sensitivity to either agent. Treatment of NSCLC cell lines with the ReoT3D-paclitaxel combination resulted in increased poly (ADP-ribose) polymerase cleavage and caspase activity compared to single therapy, indicating enhanced apoptosis induction in dually treated NSCLC cells. NSCLC cells treated with the ReoT3D-paclitaxel combination showed increased proportions of mitotic and apoptotic cells, and a more pronounced level of caspase-3 activation was demonstrated in mitotically arrested cells.

Conclusion: These data suggest that the oncolytic activity of ReoT3D can be potentiated by taxanes and other chemotherapeutic agents, and that the ReoT3D-taxane combination most effectively achieves synergy through accelerated apoptosis triggered by prolonged mitotic arrest.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1476-4598-8-47DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2723073PMC
July 2009

Rational strain selection and engineering creates a broad-spectrum, systemically effective oncolytic poxvirus, JX-963.

J Clin Invest 2007 Nov;117(11):3350-8

Department of Pediatrics and Bio-X Program, James H. Clark Center, Stanford University School of Medicine, Stanford, California, USA.

Replication-selective oncolytic viruses (virotherapeutics) are being developed as novel cancer therapies with unique mechanisms of action, but limitations in i.v. delivery to tumors and systemic efficacy have highlighted the need for improved agents for this therapeutic class to realize its potential. Here we describe the rational, stepwise design and evaluation of a systemically effective virotherapeutic (JX-963). We first identified a highly potent poxvirus strain that also trafficked efficiently to human tumors after i.v. administration. This strain was then engineered to target cancer cells with activation of the transcription factor E2F and the EGFR pathway by deletion of the thymidine kinase and vaccinia growth factor genes. For induction of tumor-specific cytotoxic T lymphocytes, we further engineered the virus to express human GM-CSF. JX-963 was more potent than the previously used virotherapeutic Onyx-015 adenovirus and as potent as wild-type vaccinia in all cancer cell lines tested. Significant cancer selectivity of JX-963 was demonstrated in vitro in human tumor cell lines, in vivo in tumor-bearing rabbits, and in primary human surgical samples ex vivo. Intravenous administration led to systemic efficacy against both primary carcinomas and widespread organ-based metastases in immunocompetent mice and rabbits. JX-963 therefore holds promise as a rationally designed, targeted virotherapeutic for the systemic treatment of cancer in humans and warrants clinical testing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI32727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2040321PMC
November 2007

The history of N-methanocarbathymidine: the investigation of a conformational concept leads to the discovery of a potent and selective nucleoside antiviral agent.

Antiviral Res 2006 Sep 6;71(2-3):268-75. Epub 2006 May 6.

Laboratory of Medicinal Chemistry, National Cancer Institute at Frederick, P.O. Box B, Building 539, Frederick, MD 21702, USA.

Conformationally locked (North)-methanocarbathymidine (N-MCT) and (South)-methanocarbathymidine (S-MCT) have been used to investigate the conformational preferences of kinases and polymerases. The herpes kinases show a distinct bias for S-MCT, while DNA polymerases almost exclusively incorporate the North 5'-triphosphate (N-MCT-TP). Only N-MCT demonstrated potent antiviral activity against herpes simplex viruses (HSV-1 and 2) and Kaposi's sarcoma-associated herpesvirus (KSHV). The activity of N-MCT depends on its metabolic transformation to N-MCT-TP by the herpes kinases (HSV-tk or KSHV-tk), which catalyze the mono and diphosphorylation steps; cellular kinases generate the triphosphate. N-MCT at a dose of 5.6 mg/kg was totally protective for mice inoculated intranasally with HSV-1. Tumor cells that are not responsive to antiviral therapy became sensitive to N-MCT if the cells expressed HSV-tk. N-MCT given twice daily (100 mg/kg) for 7 days completely inhibited the growth of MC38 tumors derived from cells that express HSV-tk in mice while exhibiting no effect on tumors derived from non-transduced cells. After i.p. administration, N-MCT was rapidly absorbed and distributed in all organs examined with slow penetration into brain and testes. N-MCT-TP was also a potent inhibitor of HIV replication in human osteosarcoma (HOS) cells expressing HSV-tk.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.antiviral.2006.04.012DOI Listing
September 2006

Chemical library screen for novel inhibitors of Kaposi's sarcoma-associated herpesvirus processive DNA synthesis.

Antiviral Res 2006 Jan 18;69(1):9-23. Epub 2005 Nov 18.

Laboratory of Antiviral Drug Mechanisms, SAIC-Frederick, Frederick, MD, USA.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma and certain lymphoproliferative disorders. The role of KSHV lytic replication has been implicated in the tumor pathogenesis. A highly specific molecular complex formed by the KSHV DNA polymerase (POL8) and processivity factor (PF8) is indispensable for lytic viral DNA synthesis and may serve as an excellent molecular anti-KSHV target. The majority of conventional nucleoside-based anti-herpetic DNA synthesis inhibitors require intracellular phosphorylation/activation before they can exert inhibitory activity as competitive substrates for viral DNA polymerases. Novel and more potent inhibitors of KSHV DNA synthesis may be discovered through POL8/PF8-targeted high throughput screening (HTS) of small molecule chemical libraries. We developed a microplate-based KSHV POL8/PF8-mediated DNA synthesis inhibition assay suitable for HTS and screened the NCI Diversity Set that comprised 1992 synthetic compounds. Twenty-eight compounds exhibited greater than 50% inhibition. The inhibitory activity was confirmed for 25 of the 26 hit compounds available for further testing, with the 50% inhibitory concentrations ranging from 0.12+/-0.07 microM (mean+/-S.D.) to 10.83+/-4.19 microM. Eighteen of the confirmed active compounds efficiently blocked KSHV processive DNA synthesis in vitro. One of the hit compounds, NSC 373989, a pyrimidoquinoline analog, was shown to dose-dependently reduce the levels of KSHV virion production and KSHV DNA in lytically induced KSHV-infected BCBL-1 cells, suggesting that the compound blocked lytic KSHV DNA synthesis. HTS for KSHV POL8/PF8 inhibitors is feasible and may lead to discovery of novel non-nucleoside KSHV DNA synthesis inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.antiviral.2005.09.005DOI Listing
January 2006

Potent antiviral activity of north-methanocarbathymidine against Kaposi's sarcoma-associated herpesvirus.

Antimicrob Agents Chemother 2005 Dec;49(12):4965-73

Laboratory of Antiviral Drug Mechanisms, SAIC-Frederick, Frederick, Maryland 21702, USA.

Kaposi's sarcoma-associated herpesvirus (KSHV) infection is a prerequisite for the development of Kaposi's sarcoma (KS). Blocking lytic KSHV replication may hinder KS tumorigenesis. Here, we report potent in vitro anti-KSHV activity of 2'-exo-methanocarbathymidine [North-methanocarbathymidine (N-MCT)], a thymidine analog with a pseudosugar ring locked in the northern conformation, which has previously been shown to block the replication of herpes simplex virus types 1 and 2. N-MCT inhibited KSHV virion production in lytically induced KSHV-infected BCBL-1 cells with a substantially lower 50% inhibitory concentration (IC50) than those of cidofovir (CDV) and ganciclovir (GCV) (IC50, mean +/- standard deviation: 0.08 +/- 0.03, 0.42 +/- 0.07, and 0.96 +/- 0.49 microM for N-MCT, CDV, and GCV, respectively). The reduction in KSHV virion production was accompanied by a corresponding decrease in KSHV DNA levels in the N-MCT-treated BCBL-1 cells, indicating that the compound blocked lytic KSHV DNA replication. A time- and dose-dependent accumulation of N-MCT-triphosphate (TP) was demonstrated in lytically induced BCBL-1 cells, while uninfected cells showed virtually no accumulation. The levels of N-MCT-TP were significantly decreased in the presence of 5'-ethynylthymidine, a potent inhibitor of herpesvirus thymidine kinase, resulting in the abrogation of anti-KSHV activity of N-MCT. N-MCT-TP more effectively blocked in vitro DNA synthesis by KSHV DNA polymerase with an IC50 of 6.24 +/- 0.08 microM (mean +/- standard deviation) compared to CDV-diphosphate (14.70 +/-2.47 microM) or GCV-TP (24.59 +/- 5.60 microM). Taken together, N-MCT is a highly potent and target-specific anti-KSHV agent which inhibits lytic KSHV DNA synthesis through its triphosphate metabolite produced in KSHV-infected cells expressing a virally encoded thymidine kinase.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.49.12.4965-4973.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1315933PMC
December 2005

Discovery of small-molecule human immunodeficiency virus type 1 entry inhibitors that target the gp120-binding domain of CD4.

J Virol 2005 May;79(10):6122-33

Laboratory of Antiviral Drug Mechanisms, Screening Technologies Branch, Developmental Therapeutics Program, SAIC-Frederick, NCI-Frederick, Bldg. 439, P.O. Box B, Frederick, MD 21702, USA.

The interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and the CD4 receptor is highly specific and involves relatively small contact surfaces on both proteins according to crystal structure analysis. This molecularly conserved interaction presents an excellent opportunity for antiviral targeting. Here we report a group of pentavalent antimony-containing small molecule compounds, NSC 13778 (molecular weight, 319) and its analogs, which exert a potent anti-HIV activity. These compounds block the entry of X4-, R5-, and X4/R5-tropic HIV-1 strains into CD4(+) cells but show little or no activity in CD4-negative cells or against vesicular stomatitis virus-G pseudotyped virions. The compounds compete with gp120 for binding to CD4: either immobilized on a solid phase (soluble CD4) or on the T-cell surface (native CD4 receptor) as determined by a competitive gp120 capture enzyme-linked immunosorbent assay or flow cytometry. NSC 13778 binds to an N-terminal two-domain CD4 protein, D1/D2 CD4, immobilized on a surface plasmon resonance sensor chip, and dose dependently reduces the emission intensity of intrinsic tryptophan fluorescence of D1/D2 CD4, which contains two of the three tryptophan residues in the gp120-binding domain. Furthermore, T cells incubated with the compounds alone show decreased reactivity to anti-CD4 monoclonal antibodies known to recognize the gp120-binding site. In contrast to gp120-binders that inhibit gp120-CD4 interaction by binding to gp120, these compounds appear to disrupt gp120-CD4 contact by targeting the specific gp120-binding domain of CD4. NSC 13778 may represent a prototype of a new class of HIV-1 entry inhibitors that can break into the gp120-CD4 interface and mask the gp120-binding site on the CD4 molecules, effectively repelling incoming virions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.79.10.6122-6133.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1091715PMC
May 2005

Beta-diketo acid pharmacophore hypothesis. 1. Discovery of a novel class of HIV-1 integrase inhibitors.

J Med Chem 2005 Jan;48(1):111-20

Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, California 90089, USA.

HIV-1 Integrase (IN) is an essential enzyme for viral replication. The discovery of beta-diketo acids was crucial in the validation of IN as a legitimate target in drug discovery against HIV infection. In this study, we discovered a novel class of IN inhibitors using a 3D pharmacophore guided database search. We used S-1360 (1), the first IN inhibitor to undergo clinical trials, and three other analogues to develop a common feature pharmacophore hypothesis. Testing this four-featured pharmacophore against a multiconformational database of 150,000 structurally diverse small molecules yielded 1,700 compounds that satisfied the 3D query. Subsequently, all 1,700 compounds were docked into the active site of IN. On the basis of docking scores, Lipinski's rule-of-five, and structural novelty, 110 compounds were selected for biological screening. We found that compounds that contain both salicylic acid and a 2-thioxo-4-thiazolidinone (rhodanine) group (e.g. 5-13) showed significant inhibitory potency against IN, while the presence of either salicylic acid or a rhodanine group alone did not. Although some of the compounds containing only a salicylic acid showed inhibitory potency against IN, none of the compounds containing only rhodanine exhibited considerable potency. Of the 52 compounds reported in this study, 11 compounds (5, 6, 8, 10-13, 32-33, 51, and 53) inhibited 3'-processing or strand transfer activities of IN with IC(50) < or = 25 microM. This is the first reported use of S-1360 and its analogues as leads in developing a pharmacophore hypothesis for IN inhibition and for identification of new compounds with potent inhibition of this enzyme.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm0496077DOI Listing
January 2005

Rapid screening of chemical inhibitors that block processive DNA synthesis of herpesviruses: potential application to high-throughput screening.

Methods Mol Biol 2005 ;292:481-92

Department of Microbiology, University of Pennsylvania, Philadelphia, PA, USA.

Processivity factors associate with DNA polymerases, enabling them to incorporate thousands of nucleotides without dissociating from the template. The processivity factors encoded by each of the herpesviruses are ideal targets for specifically blocking viral replication, particularly since they have unique primary amino acid sequences. Here we provide details of a rapid mechanistic plate assay and its potential application to high-throughput screening of libraries of tens of thousands of chemical compounds to identify inhibitors of processive DNA synthesis. Methods of validation testing are presented.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1385/1-59259-848-x:481DOI Listing
March 2005

Design and synthesis of novel indole beta-diketo acid derivatives as HIV-1 integrase inhibitors.

J Med Chem 2004 Oct;47(21):5298-310

Dipartimento Farmaco Chimico Tossicologico, Università di Sassari, Via Muroni 23/A, 07100 Sassari, Italy.

Diketo acids such as S-1360 (1A) and L-731,988 (2) are potent and selective inhibitors of HIV-1 integrase (IN). A plethora of diketo acid-containing compounds have been claimed in patent literature without disclosing much biological activities and synthetic details (reviewed in Neamati, N. Exp. Opin. Ther. Pat. 2002, 12, 709-724). To establish a coherent structure-activity relationship among the substituted indole nucleus bearing a beta-diketo acid moiety, a series of substituted indole-beta-diketo acids (4a-f and 5a-e) were synthesized. All compounds tested showed anti-IN activity at low micromolar concentrations with varied selectivity against the strand transfer process. Three compounds, the indole-3-beta-diketo acids 5a and 5c, and the parent ester 9c, have shown an antiviral activity in cell-based assays. We further confirmed a keto-enolic structure in the 2,3-position of the diketo acid moiety of a representative compound (4c) using NMR and X-ray crystallographic analysis. Using this structure as a lead for all of our computational studies, we found that the title compounds extensively interact with the essential amino acids on the active site of IN.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm049944fDOI Listing
October 2004

Rational design and synthesis of novel dimeric diketoacid-containing inhibitors of HIV-1 integrase: implication for binding to two metal ions on the active site of integrase.

J Med Chem 2004 May;47(10):2561-73

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, CAS, 555 Zuchongzhi Road, Shanghai 201203, China.

Discovery of diketoacid-containing compounds as HIV-1 integrase (IN) inhibitors played a major role in validating this enzyme as an important target for the development of therapeutics against HIV infection. In fact, S-1360, the first clinically used IN inhibitor containing a triazole ring as a bioisostere of a carboxylic acid moiety belongs to this class of compounds. To understand the role of divalent metal-chelating in the inhibition of IN (J. Med. Chem. 2002, 45, 5661-5670), we designed and synthesized a series of novel dimeric diketo-containing compounds with the notion that such dimeric compounds may simultaneously bind to two divalent metal ions on the active site of IN. We rationalized that the two diketo subunits separated by uniquely designed linkers can potentially chelate two metal ions that are either provided from one IN active site or two active sites juxtaposed together in a higher order tetramer. Herein, we show that all the new compounds are highly potent against purified IN with varied selectivity for strand transfer, and that some of the analogues exert potent inhibition of the cytopathic effect of HIV-1 in infected CEM cells. This study represents the first attempt to rationally target two divalent metal ions on the active site of IN and may have potential implications for the design of second generation diketoacid-containing class of inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm030559kDOI Listing
May 2004

Effects of CCR5-delta32 and CCR2-64I alleles on disease progression of perinatally HIV-1-infected children: an international meta-analysis.

AIDS 2003 Jul;17(11):1631-8

Clinical and Molecular Epidemiology Unit, Department of Hygiene and Epidemiology, University of Ioannina School of Medicine, Ioannina, Greece.

Objective: Among perinatally infected children, the effects of certain alleles of the CCR5 and CCR2 genes on the rate of disease progression remain unclear. We addressed the effects of CCR5-delta32 and CCR2-64I in an international meta-analysis.

Methods: Genotype data were contributed from 10 studies with 1317 HIV-1-infected children (7263 person-years of follow-up). Time-to-event analyses were performed stratified by study and racial group. Endpoints included progression to clinical AIDS, death, and death after the diagnosis of clinical AIDS. The time-dependence of the genetic effects was specifically investigated.

Results: There was large heterogeneity in the observed rates of disease progression between different cohorts. For progression to clinical AIDS, both CCR5-delta32 and CCR2-64I showed overall non-significant trends for protection [hazard ratios 0.84, 95% confidence interval (CI) 0.58-1.23; and 0.87, 95% CI 0.67-1.14, respectively]. However, analyses of survival showed statistically significant time-dependence. No deaths occurred among CCR5-delta32 carriers in the first 3 years of life, whereas there was no protective effect (hazard ratio 0.95; 95% CI 0.43-2.10) in later years (P=0.01 for the time-dependent model). For CCR2-64I, the hazard ratio for death was 0.69 (95% CI 0.39-1.21) in the first 6 years of life and 2.56 (95% CI 1.26-5.20) in subsequent years (P<0.01 for the time-dependent model). CCR5-delta32 and CCR2-64I offered no clear protection after clinical AIDS had developed.

Conclusion: The CCR5-delta32 and CCR2-64I alleles are associated with a decreased risk of death among perinatally infected children, but only for the first years of life.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/01.aids.0000060411.18106.0fDOI Listing
July 2003

Expression and purification of recombinant Kaposi's sarcoma-associated herpesvirus DNA polymerase using a Baculovirus vector system.

Protein Expr Purif 2003 May;29(1):42-50

Laboratory of Antiviral Drug Mechanisms, SAIC-Frederick, P.O. Box B, Frederick, MD 21702, USA.

The DNA polymerase (POL) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for viral DNA replication and, thus, may be considered as a viable target for anti-KSHV therapeutics. To produce large quantities of homogeneous and pure POL required for high-throughput screening (HTS) for inhibitors, we generated a recombinant baculovirus vector encoding a hexahistidine (His6)-tagged POL and infected Spodoptera frugiperda Sf-9 insect cells. High expression of recombinant POL (rPOL) was achieved for up to 72h post-infection. The rPOL was solubilized in lysis buffer containing 0.3% Cymal-5 detergent, purified by metal-chelating and dsDNA-cellulose affinity chromatography, and analyzed by anti-His antibody Western blot and mass spectrometry. The functionality of rPOL was confirmed by its DNA synthesis activity in vitro, which was effectively blocked by the anti-herpetic DNA polymerase inhibitors, foscarnet and cidofovir diphosphate, in a dose-dependent manner. The POL expressed and purified from the recombinant baculovirus-infected insect cells may be useful toward the development of HTS of large chemical libraries to identify novel KSHV DNA polymerase inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s1046-5928(03)00017-2DOI Listing
May 2003

Identification of HIV-1 nucleocapsid protein: nucleic acid antagonists with cellular anti-HIV activity.

Biochem Biophys Res Commun 2002 Sep;296(5):1228-37

Protein Chemistry Laboratory, SAIC-Frederick, Inc., NCI Frederick, 21702, Frederick, MD, USA.

The crucial functions of HIV-1 nucleocapsid-p7 protein (NC-p7) at different stages of HIV replication are dependent on its nucleic acid binding properties. In this study, a search has been made to identify antagonists of the interaction between NC-p7 and d(TG)(4). A chemical library of approximately 2000 small molecules (the NCI Diversity Set) was screened, of the 26 active inhibitors that were identified, five contained a xanthenyl ring structure. Further analysis of 63 structurally related compounds led to the identification of 2,3,4,5-tetrachloro-6-(4('),5('),6(')-trihydroxy-3(')-oxo-3H-xanthen-9(')-yl)benzoic acid, which binds to NC-p7 stoichiometrically. This compound exerted a significant anti-HIV activity in vitro with an IC(50) of 16.6+/-4.3 microM (means+/-SD). Synthetic variants lacking the two hydroxyls at positions 4(') and 5(') in the xanthenyl ring system failed to bind NC-p7 and showed significantly less protection against HIV infection. Molecular modeling predicts that these hydroxyl groups would bind to the amide nitrogen of Gly(35) with other contacts at the carbonyl oxygens of Gly(40) and Lys(33).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0006-291x(02)02063-6DOI Listing
September 2002