Snehlata Tripathi

Anti-HIV-1 activity of anti-TAR polyamide nucleic acid conjugated with various membrane transducing peptides

The transactivator responsive region (TAR) present in the 5′-NTR of the HIV-1 genome represents a potential target for antiretroviral intervention and a model system for the development of specific inhibitors of RNA–protein interaction. Earlier, we have shown that an anti-TAR polyamide nucleotide analog (PNATAR) conjugated to a membrane transducing (MTD) peptide, transportan, is efficiently taken up by the cells and displays potent antiviral and virucidal activity [B. Chaubey, S. Tripathi, S. Ganguly, D. Harris, R. A. Casale and V. N. Pandey (2005) Virology, 331, 418–428]. In the present communication, we have conjugated five different MTD peptides, penetratin, tat peptide, transportan-27, and two of its truncated derivatives, transportan-21 and transportan-22, to a 16mer PNA targeted to the TAR region of the HIV-1 genome. The individual conjugates were examined for their uptake efficiency as judged by FACScan analysis, uptake kinetics using radiolabeled conjugate, virucidal activity and antiviral efficacy assessed by inhibition of HIV-1 infection/replication. While FACScan analysis revealed concentration-dependent cellular uptake of all the PNATAR–peptide conjugates where uptake of the PNATAR–penetratin conjugate was most efficient as >90% MTD was observed within 1 min at a concentration of 200 nM. The conjugates with penetratin, transportan-21 and tat-peptides were most effective as an anti-HIV virucidal agents with IC50 values in the range of 28–37 nM while IC50 for inhibition of HIV-1 replication was lowest with PNATAR–transportan-27 (0.4 μM) followed by PNATAR–tat (0.72 μM) and PNATAR–penetratin (0.8 μM). These results indicate that anti-HIV-1 PNA conjugated with MTD peptides are not only inhibitory to HIV-1 replication in vitro but are also potent virucidal agents which render HIV-1 virions non-infectious upon brief exposure.

Pharmacokinetic Analysis of Polyamide Nucleic-Acid-Cell Penetrating Peptide Conjugates Targeted against HIV-1 Transactivation Response Element

We have demonstrated that polyamide nucleic acids complementary to the transactivation response (TAR) element of HIV-1 LTR inhibit HIV-1 production when transfected in HIV-1 infected cells. We have further shown that anti-TAR PNA (PNA(TAR)) conjugated with cell-penetrating peptide (CPP) is rapidly taken up by cells and exhibits strong antiviral and anti-HIV-1 virucidal activities. Here, we pharmacokinetically analyzed (125)I-labeled PNA(TAR) conjugated with two CPPs: a 16-mer penetratin derived from antennapedia and a 13-mer Tat peptide derived from HIV-1 Tat. We administered the (125)I-labeled PNA(TAR)-CPP conjugates to male Balb/C mice through intraperitoneal or gavage routes. The naked (125)I-labeled PNA(TAR) was used as a control. Following a single administration of the labeled compounds, their distribution and retention in various organs were monitored at various time points. Regardless of the administration route, a significant accumulation of each PNA(TAR)-CPP conjugate was found in different mouse organs and tissues. The clearance profile of the accumulated radioactivity from different organs displayed a biphasic exponential pathway whereby part of the radioactivity cleared rapidly, but a significant portion of it was slowly released over a prolonged period. The kinetics of clearance of individual PNA(TAR)-CPP conjugates slightly varied in different organs, while the overall biphasic clearance pattern remained unaltered regardless of the administration route. Surprisingly, unconjugated naked PNA(TAR) displayed a similar distribution and clearance profile in most organs studied although extent of its uptake was lower than the PNA(TAR)-CPP conjugates.

STAT3 Inhibition in Prostate and Pancreatic Cancer Lines by STAT3 Binding Sequence Oligonucleotides: Differential Activity Between 5′ and 3′ Ends

Signal transducers and activators of transcription (STAT) were originally discovered as components of signal transduction pathways. Persistent aberrant activation of STAT3 is a feature of many malignancies including prostate cancer and pancreatic cancer. One consequence of persistently activated STAT3 in malignant cells is that they depend on it for survival; thus, STAT3 is an excellent molecular target for therapy. Previously, we reported that single-stranded oligonucleotides containing consensus STAT3 binding sequences (13410 and 13411) were more effective for inducing apoptosis in prostate cancer cells than antisense STAT3 oligonucleotides. Control oligonucleotides (scrambled sequences) had no effect. Here, we report that authentic STAT3 binding sequences, identified from published literature, were more effective for inducing apoptosis in prostate cancer cells and pancreatic cancer cells than was oligonucleotide 13410. Moreover, the authentic STAT3 binding sequences showed differing efficacies in the malignant cell lines depending on whether the canonical STAT3 binding sequence was truncated at the 5′ or the 3′ end. Finally, expression of one STAT3-regulated gene was decreased following treatment, suggesting that STAT3 may regulate the same set of genes in the two types of cancer. We conclude that truncating the 5′ end left intact enough of the canonical STAT3 binding site for effective hybridization to the genome, whereas truncation of the 3′ end, which is outside the canonical binding site, may have affected binding of required cofactors essential for STAT3 activity, thereby reducing the capacity of this modified oligonucleotide to induce apoptosis. Additional experiments to answer this hypothesis are under way. [Mol Cancer Ther 2008;7(6):1543–50]

Single Acute-Dose and Repeat-Doses Toxicity of anti-HIV-1 PNATAR–Penetratin Conjugate after Intraperitoneal Administration to Mice

Polyamide (peptide) nucleic acids conjugated with membrane-penetrating peptide are potential antisense therapeutic agents because of their unique chemical properties, high target specificity, and efficient cellular uptake. However, studies of their potential toxicity in animal models are lacking. In this study, we evaluated the toxicity of the response of Balb/C mice to anti-HIV-1 PNA TAR-penetratin conjugate targeted against the transactivation response (TAR) element of HIV-1 LTR. A single i.p. dose of 600 mg/kg of body weight was lethal, killing all mice within 72 hours. However, death did not occur after single doses of 100 and 300 mg/kg, although all mice experienced initial and transitory diarrhea and loss of agility. Repeated daily doses of 10, 30, and 100 mg/kg were well tolerated by mice during 8 days of treatment, although daily doses of 100 mg/kg caused diarrhea during the first 4 days of treatment. During 8 weeks of follow-up, mice fully recuperated. Serositis was observed in the spleens, livers, and kidneys at the ninth day of treatment, but not after the follow-up period. Necropsies, clinical chemistry studies, and hematological parameters demonstrated normal function of the major organs and no irreversible damage to the mice. These observations indicate that the PNA-peptide conjugate would be nontoxic at probable therapeutic doses and thus support its therapeutic potential as an antisense drug.

Structural Determinants of Slippage-mediated Mutations by Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Single-base deletions at nucleotide runs or -1 frameshifting by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) result from template slippage during polymerization. In crystal structures of HIV-1 RT complexed with DNA-DNA template-primer, the palm subdomain in the template cleft contacts the template backbone near the proposed site of slippage via the Glu89 side chain. We investigated the role of Glu89 in frameshifting by perturbing this interaction. Substitutions with Asp, Gly, Ala, Val, Ser, Thr, Asn, or Lys were created in recombinant HIV RT, and frameshift frequencies of the resulting mutant RTs were measured. All substitutions led to reduced -1 frameshifting by HIV-1 RT (2-40-fold). Interestingly, the suppression of -1 frameshifting frequently coincided with an enhancement of +1 frameshifting (3-47-fold) suggesting that Glu89 can influence the slippage of both strands. Glu89 substitutions also led to reduced rates of dNTP misincorporation that paralleled reductions in -1 frameshifting, suggesting a common structural mechanism for both classes of RT error. Our results reveal a major influence of Glu89 on slippage-mediated errors and dNTP incorporation fidelity. The crystal structure of HIV-1 RT reveals a salt bridge between Glu89 and Lys154, which may facilitate -1 frameshifting; this concept is supported by the observed reduction in -1 frameshifting for K154A and K154R mutants.

An Efficient Biodelivery System for Antisense Polyamide Nucleic Acid (PNA)

With the aim of developing a general and straightforward procedure for the intracellular delivery of naked peptide nucleic acids (PNAs), we designed an intracellularly biodegradable triphenylphosphonium (TPP) cation based transporter system. In this system, TPP is linked, via a biolabile disulfide bridge, to an activated mercaptoethoxycarbonyl moiety, allowing its direct coupling to the N-terminal extremity of a free PNA through a carbamate bond. We found that such TPP-PNA-carbamate conjugates were highly stable in a cell culture medium containing fetal calf serum. In a glutathione-containing medium mimicking the cytosol, the conjugates were rapidly degraded into an unstable intermediate, which spontaneously decomposed, releasing the free PNA. Using a fluorescence-labeled PNA-TPP conjugate, we demonstrated that conjugates were taken up by cells. Efficient cellular uptake and release of the PNA into the cytosol was further confirmed by the anti-HIV activity measured for the TPP-conjugate of a 16-mer PNA targeting the TAR region of the HIV-1 genome. This conjugate exhibited an IC(50) value of 1 microM, while the free 16-mer PNA did not inhibit replication of HIV in the same cellular test.

Abstract B176: In vitro and in vivo studies demonstrating sustained drug release for multiple anticancer payloads and improved anticancer effects of a cabazitaxel β-cyclodextrin-PEG copolymer-based nanoparticle-drug conjugate (NDC)

We have demonstrated the ability to generate nanoparticle-drug conjugates (NDCs) with tunable and diverse in vitro and in vivo drug release kinetics by the conjugation of multiple anticancer agents (docetaxel, cabazitaxel, and gemcitabine) to a β-cyclodextrin-PEG (CDP) copolymer through a variety of linker strategies. Linker molecules included glycinate, alaninate, hexanoate, and diester linker β-alanine-glycolate, which were conjugated via ester linkages to the anticancer payloads. In vitro release studies demonstrated that the β-alanine-glycolate linker released drug from the CDP copolymer relatively faster than the glycinate and alaninate linkers, while the hexanoate linker showed much slower drug release. Release studies in native and heat-inactivated plasma had no influence on the release kinetics, indicating that drug release was pH- but not enzyme-triggered. The in vitro release profiles for the respective linkers varied slightly (more so in vivo) depending on the API (Active Pharmaceutical Ingredient) payload, demonstrating that release kinetics can be varied through selection of linker molecules and that NDC chemistry is customizable with respect to API. In vivo PK studies with cabazitaxel (CBTX) NDCs demonstrated that the hexanoate-containing NDC (slower releasing linker) led to higher total (conjugated drug + released) drug levels and lower released drug levels compared to the glycinate NDC (faster releasing linker) and the separately dosed cabazitaxel. This likely led to the greater tolerability (i.e., higher MTD) observed for the CBTX-hexanoate NDC. Both the CBTX-hexanoate and -glycinate NDCs led to high and sustained levels of released drug in tumor tissues (>72 hrs). The maximum drug concentrations in the blood (Cmax) of released drug was lower for all NDCs tested compared to similarly dosed parent drug, thereby addressing Cmax-related toxicities. Two CBTX NDCs (hexanoate and glycinate) were chosen for in vivo efficacy studies in mouse tumor models. Both demonstrated vastly improved efficacy (and survival) over the parent drug, cabazitaxel, at similar doses including efficacy against docetaxel-resistant UISO-BCA-1 tumor cells. The CBTX-hexanoate NDC showed a greater therapeutic index (TI) compared to parent drug as well as the CBTX-glycinate NDC. Citation Format: Chester Metcalf, III, Donna Brown, Jungyeon Hwang, Sujan Kabir, Douglas Lazarus, Pochi Shum, Snehlata Tripathi, Scott Eliasof. In vitro and in vivo studies demonstrating sustained drug release for multiple anticancer payloads and improved anticancer effects of a cabazitaxel β-cyclodextrin-PEG copolymer-based nanoparticle-drug conjugate (NDC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B176.