Robert Peralta

Description of Staphylococcus Serine Protease (ssp) Operon in Staphylococcus aureus and Nonpolar Inactivation of sspA-Encoded Serine Protease

ABSTRACT Signature tagged mutagenesis has recently revealed that the Ssp serine protease (V8 protease) contributes to in vivo growth and survival of Staphylococcus aureus in different infection models, and our previous work indicated that Ssp could play a role in controlling microbial adhesion. In this study, we describe an operon structure within the ssp locus of S. aureusRN6390. The ssp gene encoding V8 protease is designated assspA, and is followed by sspB, which encodes a 40.6-kDa cysteine protease, and sspC, which encodes a 12.9-kDa protein of unknown function. S. aureus SP6391 is an isogenic derivative of RN6390, in which specific loss of SspA function was achieved through a nonpolar allelic replacement mutation. In addition to losing SspA, the culture supernatant of SP6391 showed a loss of 22- to 23-kDa proteins and the appearance of a 40-kDa protein corresponding to SspB. Although the 40-kDa SspB protein could degrade denatured collagen, our data establish that this is a precursor form which is normally processed by SspA to form a mature cysteine protease. Culture supernatant of SP6391 also showed a new 42-kDa glucosaminidase and enhanced glucosaminidase activity in the 29 to 32 kDa range. Although nonpolar inactivation of sspA exerted a pleiotropic effect, S. aureus SP6391 exhibited enhanced virulence in a tissue abscess infection model relative to RN6390. Therefore, we conclude that SspA is required for maturation of SspB and plays a role in controlling autolytic activity but does not by itself exert a significant contribution to the development of tissue abscess infections.

Adhesion and Virulence Properties of Epidemic Canadian Methicillin-Resistant Staphylococcus aureus Strain 1: Identification of Novel Adhesion Functions Associated with Plasmin-Sensitive Surface Protein

Epidemic Canadian methicillin-resistant Staphylococcus aureus strain 1 (CMRSA-1) comprises related subtypes that differ in phenotype and prevalence, with subtypes 1A, 1B, and 1D representing 1%, 71%, and 18%, respectively, of total CMRSA-1 isolates. The predominant CMRSA-1B subtype possesses a variant of the staphylococcal cassette chromosome mec, harboring pls, which encodes plasmin-sensitive surface protein (Pls). CMRSA-1B cells that express Pls exhibited poor adhesion to keratinocyte extracellular matrix. However, CMRSA-1B and purified Pls adhered to cellular lipids and glycolipids, and Pls promoted bacterial cell-cell interactions. Although exoprotein expression was restricted to a precursor form of lipase in CMRSA-1B, it was not attenuated in virulence relative to CMRSA-1A, which exhibits normal exoprotein expression. In contrast, CMRSA-1D exhibited a pleiotropic defect in exoprotein expression and attenuated virulence relative to CMRSA-1A. These data indicate that the high transmissibility of CMRSA-1B was not achieved at the expense of attenuated virulence and that Pls confers a novel adhesion mechanism.

Synthetic Peptide Immunogens Elicit Polyclonal and Monoclonal Antibodies Specific for Linear Epitopes in the D Motifs of Staphylococcus aureus Fibronectin-Binding Protein, Which Are Composed of Amino Acids That Are Essential for Fibronectin Binding

ABSTRACT A fibronectin (Fn)-binding adhesin of Staphylococcus aureus contains three tandem 37- or 38-amino-acid motifs (D1, D2, and D3), which function to bind Fn. Plasma from patients with S. aureus infections contain antibodies that preferentially recognize ligand induced binding sites in the D motifs and do not inhibit Fn binding (F. Casolini, L. Visai, D. Joh, P. G. Conaldi, A. Toniolo, M. Höök, and P. Speziale, Infect. Immun. 66:5433–5442, 1998). To eliminate the influence of Fn binding on antibody development, we used synthetic peptide immunogens D121–34 and D320–33, which each contain a conserved pattern of amino acids that is essential for Fn binding but which cannot bind Fn without N- or C-terminal extensions. The D320–33 immunogen promoted the production of polyclonal antibodies that were 10-fold more effective as inhibitors of Fn-binding to the D3 motif than antibodies obtained by immunizing with an extended peptide D316–36, which exhibits functional Fn binding. The D320–33 immunogen also facilitated the production of a monoclonal antibody, 9C3, which was highly specific for the epitope SVDFEED, and abolished Fn binding by the D3 motif. When mixed with polyclonal anti-D121–34 immunoglobulin G, 70% inhibition of Fn binding to the three tandem D motifs was achieved compared to no more than 30% inhibition with either antibody preparation alone. Therefore, by immunizing with short synthetic peptides that are unable to bind Fn, we have effectively stimulated the production of antibodies specific for epitopes comprised of amino acids that are essential for Fn binding. Although these epitopes occur within a conserved pattern of amino acids that is required for Fn binding, the antibodies recognized specific linear epitope sequences and not a conserved structure common to all repeated motifs.

A novel small molecule with potent anticancer activity inhibits cell growth by modulating intracellular labile zinc homeostasis

ML-133 is a novel small molecule with potent antiproliferative activity, as shown in cancer cell lines and in a human colon tumor xenograft model. ML-133 reduces the concentration of intracellular labile zinc in HT-29 colon cancer cells, leading to induction of the Krüppel-like factor 4 transcription factor. Krüppel-like factor 4 displaces the positive regulator SP1 from the cyclin D1 promoter, thereby negatively regulating the expression of cyclin D1 and promoting the G1-S phase arrest of cell proliferation. The antiproliferative and antitumor activity of ML-133 described in the present study suggests modulation of intracellular zinc homeostasis as a potential strategy for the treatment of several cancer types, and ML-133 represents a promising new class of antitumor agents that deserves further development. [Mol Cancer Ther 2009;8(9):2586–96]

Potent antimicrobial activity of 3-(4,5-diaryl-1H-imidazol-2-yl)-1H-indole derivatives against methicillin-resistant Staphylococcus aureus

A new series of antimicrobial derivatives [3-(4,5-diaryl-1H-imidazol-2-yl)-1H-indole)] have been synthesized with potent activity against strains of Staphylococcus aureus, including methicillin-resistant strains (MRSA). Compound 17 [3-(4,5-bis(4-fluorophenyl)-1H-imidazol-2-yl)-5-bromo-1H-indole], the most active derivative was shown to inhibit the growth of all Gram-positive strains tested, including vancomycin resistant Enterococcus faecalis and Enterococcus faecium with no activity against Gram-negative bacteria.

Abstract 4544: Induction of KLF4 by LOR-253 as an innovative therapeutic approach to induce apoptosis in acute myeloid leukemia

LOR-253, a small molecule anticancer agent that acts through induction of the tumor suppressor Kruppel-like factor 4 (KLF4), has demonstrated antitumor activity as a single agent in a Phase I study in patients with advanced or metastatic solid tumors. Recently, the vast majority of patients with acute myeloid leukemia (AML) were shown to inappropriately express the embryonic CDX2 gene in bone marrow stem and progenitor cells, resulting in down-regulation of KLF4 expression as the leukemogenic event. Consequently, we examined the antitumor activity and mechanism of action for LOR-253 in AML cells and cells representing other hematological malignancies. Indeed, LOR-253 was found to inhibit proliferation of various human leukemia and lymphoma cell lines in vitro with low nM IC50 values. Further LOR-253 induced high levels of KLF4 mRNA expression in AML cells and a resultant significant increase in expression of p21, a cyclin-dependent kinase inhibitor that is transcriptionally regulated by KLF4. Consistent with these findings, in AML cells we found that LOR-253 induced G1/S cell cycle arrest and apoptosis, based on positive Annexin V staining, activated caspase-3, and increased BAX mRNA expression. Studies are underway to further characterize the pathway that mediates KLF4 induction by LOR-253, to characterize the effects of LOR-253 in combination with approved chemotherapies for AML, and to assess the efficacy of LOR-253 in animal models of AML. Induction of KLF4 represents a novel approach to the treatment of AML and other hematologic malignancies, and LOR-253 is the only clinical stage agent to act through this mechanism of action. Citation Format: Ronnie Lum, Mojib Javadi, Tiffany Cheng, Robert Peralta, Howard Cukier, Jeff Lightfoot, Yoon Lee, Aiping Young, William G. Rice. Induction of KLF4 by LOR-253 as an innovative therapeutic approach to induce apoptosis in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4544. doi:10.1158/1538-7445.AM2014-4544

Abstract 4649: Utilization of KLF-4 as a pharmacodynamic biomarker for in vivo anticancer activity of a novel small molecule drug LOR-253.

LOR-253 is a potent and selective growth inhibitor of several cancer types, including non-small cell lung cancer (NSCLC), colon cancer and leukemia. LOR-253 stimulates Kruppel Like Factor-4 (KLF-4), a tumor suppressor factor which is characteristically deficient in a variety of cancers, and so represents a new approach to cancer therapy. The current Phase I dose-escalation study is being conducted at Memorial Sloan-Kettering Cancer Center in New York and MD Anderson Cancer Center in Houston. In the present preclinical study, the changes in KLF-4 expression levels were evaluated in vivo to examine the utility of KLF-4 as a pharmacodynamic biomarker, aiming at linking the antitumor effects of LOR-253 to the induction of KLF-4. In the human H226 NSCLC xenograft mouse model, LOR-253 demonstrated dose-dependent antitumor activity when administered at 1, 5, and 15 mg/kg and mediated significant tumor growth inhibition of about 55% at the highest dose given by intravenous bolus injection (p=0.05). Tumors were harvested 24h after the last dose following the treatments at 1, 5, and 15 mg/kg and KLF-4 mRNA and protein levels were analyzed by real-time RT-PCR and Western blot, respectively. A dose-dependent induction of KLF-4 mRNA and protein levels was observed. Pharmacodynamic studies to characterize the effect of LOR-253 on intratumoral KLF-4 were also conducted in the H226 NSCLC xenograft mouse model to determine the optimal time point for tumor biopsy sampling, following the treatment schedule in the ongoing Phase 1 clinical trial (2 days dosing, 12 day break, 2 days of dosing, 12 day break (1 cycle)). Tumor samples were taken 24h after the second, fourth and eighth dose of LOR-253 and analyzed for KLF-4 expression. It was determined that tumor sampling after the second and eighth doses was optimal at demonstrating increased intratumoral KLF-4 levels. Furthermore, antitumor efficacy of LOR-253 was also evaluated in nude rats bearing established human H226 NSCLC tumors when LOR-253 was infused intravenously following the current Phase I clinical dosing schedule. A significant efficacy was observed at the end of the first cycle of treatment, demonstrating 44% decrease in mean tumor volume when compared with the control treatment (p=0.025). Inhibition of tumor growth was paralleled by the induction of KLF-4 in isolated tumors when analyzed by real-time RT-PCR and Western blot. Taken together, the in vivo data obtained in both mouse and rat models provide strong preclinical support for utilizing KLF-4 as a potential pharmacodynamic biomarker for LOR-253 treatment in clinic. Citation Format: Howard Cukier, Robert Peralta, Hongnan Jin, Yu Cheng, Venkata Nedunuri, Salah Salehi, Yoon S. Lee, Aiping Young. Utilization of KLF-4 as a pharmacodynamic biomarker for in vivo anticancer activity of a novel small molecule drug LOR-253. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4649. doi:10.1158/1538-7445.AM2013-4649

Abstract 3710: Preclinical dose scheduling studies of LOR-253, a novel anticancer drug, in combination with chemotherapeutics in lung and colon cancers

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL LOR-253 is an anticancer small molecule that is currently in a Phase I clinical study in patients with advanced or metastatic solid tumors. LOR-253 has a novel anticancer mechanism based on chelation of intracellular labile zinc, leading to inhibition of angiogenesis as well as G1/S cell cycle arrest due to induction of the tumor suppressor Kruppel-like factor 4 (KLF4). In preclinical studies, LOR-253 has shown potent antitumor activity against several cancers, including non-small cell lung cancer (NSCLC) and colon cancer, without significant toxicity. To support the clinical development of LOR-253 in combination with chemotherapeutics, we investigated the effect of dose scheduling of LOR-253 plus chemotherapy agents on anticancer activity in NSCLC (H226) and colon cancer (SW620) cell lines. Treatment of H226 cells in vitro with docetaxel, paclitaxel, or cisplatin for two days followed by increasing doses of LOR-253 resulted in significantly higher anti-proliferative activity than either agent alone. Assessment of drug interactions by determination of the combination index (D) (Berenbaum MC, Adv Cancer Res., 1981; 35:269-335) showed that the anticancer activities of the combination of LOR-253 with these chemotherapy agents were synergistic (D < 1). In studies with LOR-253 and docetaxel, synergistic anticancer activity against H226 was maintained with either concurrent or sequential treatments of each agent. Anticancer synergy was also seen in SW620 cells treated with LOR-253 in combination with oxaliplatin, CPT-11, or Fluorouracil. The effects of dose scheduling on activity of LOR-253 and docetaxel in vivo was also examined. Nude mice with H226 tumor xenografts were treated with repeat cycles of docetaxel once per week, followed two days later by treatment with LOR-253 for three consecutive days. Docetaxel was administered at an experimentally-determined ED50 dose level (defined as the effective dose at which 50% of mice showed tumor growth inhibition), while LOR-253 was given at its maximum efficacious dose in this model (10 mg/kg) or sub-optimal dose (5 mg/kg). Sequential administration of docetaxel followed by LOR-253 showed significant antitumor activity when docetaxel was given at either ED50 or sub-ED50 doses plus 10 mg/kg of LOR-253, compared to either agent alone given at the same dose, or with concurrent administration of LOR-253 and docetaxel at these dose levels. Preliminary results also show significant antitumor activity of LOR-253 plus docetaxel against H226 tumors in mice treated with repeat cycles of the reverse order of these treatments (LOR-253 for three days, followed by docetaxel 1X/week). In summary, dose scheduling studies with LOR-253 plus chemotherapy drugs demonstrate strong anticancer activities in NSCLC and colon cancer, providing support for the design of LOR-253 combination strategies for treatment of these cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3710. doi:1538-7445.AM2012-3710

Abstract 667: Anticancer activity and tumor selectivity of LOR-253, a novel drug candidate, in lung carcinoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL LOR-253 is a novel drug candidate entering a first in man dose escalation Phase I clinical trial in patients with advanced solid tumors. LOR-253 was well tolerated in single and repeat-dose GLP-toxicology studies conducted in rats and dogs at significantly higher doses than equivalent efficacious doses determined in xenograft models in mice, which indicates a potential wide therapeutic window. Previously we reported that LOR-253 reduced the levels of intracellular labile zinc leading to cell cycle arrest, through the down regulation of the metal-responsive transcription factor 1 (MTF-1) and the induction of the Kruppel like factor 4 (KLF4). KLF4 is a G1/S cell cycle checkpoint gene with tumor suppressor function in a variety of cancers. A recent study demonstrated dramatically decreased levels of KLF4 expression in most primary lung tumors (Hu et al. Clin Cancer Res 2009; 15 (18): 5688-95). Anti-tumor activity of KLF4 was demonstrated when KLF4 expression was enforced in cancer cell lines with low basal KLF4 expression levels. In addition, tumor growth in animals was highly compromised when KLF4 gene was stably transfected. The aim of this study was to investigate differential effects of LOR-253 on normal lung fibroblasts vs lung cancer cell lines with different basal levels of KLF4 expression. We found an inverse correlation between LOR-253 sensitivity and basal KLF4 expression. LOR-253 was highly active in three different NSCLC cell lines (H1299, H322 and H226) as shown by much lower IC50 values. KLF4 expression levels in these cell lines were significantly lower than that in normal cells. In contrast, tumor cell lines with KLF4 expression levels similar or higher than normal cells were less sensitive to LOR-253 treatment. Similar results were obtained in in vivo xenograft models, which showed higher in vivo efficacy of LOR-253 in treated mice harboring low basal KLF4-expressing tumors. A time-course gene and protein expression of KLF4 and cyclin D1 in normal lung fibroblast (HFL1) and in NSCLC H226 cells treated with LOR-253 demonstrated a differential effect between normal vs cancer cells. A maximum expression of KLF4 and suppression of cyclin D1 in H226 cells were observed between 16 and 24 hours of treatment while there was no significant effect on HFL1 cells. Based on these results the cell line H226 was selected for further LOR-253 target validation and efficacy biomarker expression studies. A dose-response effect of LOR-253 treatment on tumor growth and KLF4 and cyclin D1 expression was observed in the H226 xenograft model using RT-PCR, Western blot and immunohistochemical analysis. Studies are underway to determine the expression levels of other biomarkers such as MTF-1, HIF-1 a and b-catenin in response to LOR-253 treatment, as well as the effect of in vivo gene knock down and over-expression of MTF-1, KLF4 and other biomarkers on the antitumor activity of LOR-253. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 667. doi:10.1158/1538-7445.AM2011-667