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Epithelial cell adhesion molecule (EpCAM) is one of the critical bio-maker for circulating tumor cells (CTC) detection. For capturing CTC, antibody-antigen-based techniques have mainly been explored. However, the expensiveness and tedious manufacturing process have posed certain limitations for antibody-based techniques for its wide applications in cell capturing. On the other hand, peptides are inexpensive bimolecular probes with high specificity and tunability. Although there are few reports on EpCAM binding peptides are available in literature, those peptides were selected through random library screening. Interestingly, de-novo design of the peptides against EpCAM has not been reported till date. For the first time, we have developed a small peptide (Pep14) from the complementary derived region (CDRs) of antibody MOC31 through systematic virtual screening. Selected peptide has demonstrated good binding affinity towards EpCAM with dissociation constant (Kd) of 870 nM and found to be co-localized with the anti-EpCAM antibody in EpCAM expressing cancer cells (MCF-7). Therefore, the short peptide Pep14 hold promise for capturing circulatory tumor cells through EpCAM binding.

Introduction: The outburst of the novel coronavirus COVID-19, at the end of December 2019 has turned itself into a pandemic taking a heavy toll on human lives. The causal agent being SARS-CoV-2, a member of the long-known Coronaviridae family, is a positive sense single-stranded enveloped virus and quite closely related to SARS-CoV. It has become the need of the hour to understand the pathophysiology of this disease, so that drugs, vaccines, treatment regimens and plausible therapeutic agents can be produced. Methods: In this regard, recent studies uncovered the fact that the viral genome of SARS-CoV-2 encodes nonstructural proteins like RNA dependent RNA polymerase (RdRp) which is an important tool for its transcription and replication process. A large number of nucleic acid based anti-viral drugs are being repurposed for treating COVID-19 targeting RdRp. Few of them are in the advanced stage of clinical trials including Remdesivir. While performing close investigation of the large set of nucleic acid based drugs, we were surprised to find that the synthetic nucleic acid backbone is explored very little or rare. Results: We have designed scaffolds derived from peptide nucleic acid (PNA) and subjected them for in-silico screening systematically. These designed molecules have demonstrated excellent binding towards RdRp. Compound 12 was found to possess similar binding affinity as Remdesivir with comparable pharmacokinetics. However, the in-silico toxicity prediction indicates compound 12 may be a superior molecule which can be explored further due to its excellent safety-profile with LD50 (12,000mg/kg) as opposed to Remdesivir (LD50 =1000mg/kg). Conclusion: Compound 12 falls in the safe category of class 6. Synthetic feasibility, equipotent binding and very low toxicity of this peptide nucleic acid derived compounds can serve as a leading scaffold to design, synthesize and evaluate many of similar compounds for the treatment of COVID-19.

The global burden of neurological disorders has been increasing day by day which calls for immediate attention to the solutions. Novel drug delivery systems are one of the alternatives that we count on to counteract these disorders. As the blood-brain barrier creates a significant hindrance to the delivery of drugs across the endothelium lining of the brain, nose-to-brain delivery has been the favorite option to administer such drugs. In recent times, bioconjugation has been viewed as a rapidly growing area in the field of pharmaceuticals. The pharmaceutical industry and academic research are investing significantly in bioconjugated structures as an attractive and advantageous potential aid to nanoparticulate delivery systems, with all of its flexible benefits in terms of tailor grafting and custom design as well as overcoming the majority of their drawbacks. This review discusses drug delivery via the intranasal route and gives insight into bioconjugation systems for drug molecules, their chemistry, and benefits over other systems. Conjugation of drugs/macromolecules with peptides, carbohydrates, ligands, and nucleic acids has also been discussed in detail. The figure represents few types of novel drug delivery systems and molecules that have been attempted by researchers for nose-to-brain delivery through nasal (mucosal) route for the effective management of epilepsy, Alzheimer's disease, brain cancer, and other brain disorders.

Bone-specific functionalization strategies on liposomes are promising approaches to delivering the drug in osteoporotic conditions. This approach delivers the drug to the bone surface specifically, reduces the dose and off-target effects of the drug, and thereby reduces the toxicity of the drug. The purpose of the current research work was to fabricate the bone-specific peptide conjugated pegylated nanoliposomes to deliver anabolic drug and its physicochemical evaluations. For this, a bone-specific peptide (SDSSD) was synthesized, and the synthesized peptide was conjugated with a linker (DSPE-PEG2000-COOH) to obtain a bone-specific conjugate (SDSSD-DSPE). Purified SDSSD-DSPE was characterized by HPLC, Maldi-TOF, NMR, and Scanning Electron Microscope/Energy Dispersive Spectroscopy (SEM/EDS). Further, peptide-conjugated and anabolic drug-encapsulated liposomes (SDSSD-LPs) were developed using the ethanol injection method and optimized by Central Composite Design (CCD) using a statistical approach. Optimized SDSSD-LPs were evaluated for their physicochemical properties, including surface morphology, particle size, zeta potential, in vitro drug release, and bone mineral binding potential. The obtained results from these studies demonstrated that SDSSD-DSPE conjugate and SDSSD-LPs were optimized successfully. The particle size, % EE, and zeta potential of SDSSD-LPs were observed to be 183.07 ± 0.85 nm, 66.72 ± 4.22%, and −25.03 ± 0.21 mV, respectively. SDSSD-LPs demonstrated a sustained drug release profile. Further, the in vitro bone mineral binding assay demonstrated that SDSSD-LPs deliver the drug to the bone surface specifically. These results suggested that SDSSD-LPs could be a potential targeting approach to deliver the anabolic drug in osteoporotic conditions.

Because of their commanding properties, ultrashort and short peptides are gaining significance as viable candidates for molecular self-assembly, which is a naturally inspired approach for developing supramolecular structures and can be used to design various strategies of significance in the field of biomaterials. Self-assembly of biomolecules like proteins, lipids, and nucleic acids is observed in living organisms, various biological-process-based examples like amyloid-β plaque formation, lipid bilayer assembly, and the complementary binding of the nucleotide bases of nucleic acids involve self-assembly. Among all biomolecules, peptide-based self-assembly has the advantage of the availability of the source, peptides can be easily synthesized or obtained from the natural degradation process and can be engineered to modulate their action, making them an area of immense interest for research. Multiple modification options provide a wide area for the engineering of amino acid sequences. Understanding of the amino acid residues with their existing properties and modified properties is very helpful for further improvements. Computational approaches like molecular dynamics simulations provide atomistic-level insight into the self-assembly process, by which newer physical-chemical modifications can be planned. Virtual screening of the peptides on the basis of their properties and probability for the desired activity are helpful as well. Engineered and programmed peptides have been reported for various applications like drug delivery and target specific formulations. A combined approach of computational and experimental studies is helpful to understand and optimize the self-assembly process and mechanism at the atomic level. These self-assembled ultrashort peptides have been used in a wide range of applications from hydrogels to drug delivery agents, biosensors, emulsifiers, and so on.

Peptides are signaling epitopes that control many vital biological events. Increased specificity, synthetic feasibility with concomitant lack of toxicity, and immunogenicity make this emerging class of biomolecules suitable for different applications including therapeutics, diagnostics, and biomedical engineering. Further, chitosan, a naturally occurring linear polymer composed of d-glucosamine and N-acetyl-d-glucosamine units, possesses anti-microbial, muco-adhesive, and hemostatic properties along with excellent biocompatibility. As a result, chitosan finds application in drug/gene delivery, tissue engineering, and bioimaging. Despite these applications, chitosan demonstrates limited cell adhesion and lacks biosignaling. Therefore, peptide-chitosan hybrids have emerged as a new class of biomaterial with improved biosignaling properties and cell adhesion properties. As a result, recent studies encompass increased application of peptide-chitosan hybrids as composites or conjugates in drug delivery, cell therapy, and tissue engineering and as anti-microbial material. This review discusses the recent investigations involving chitosan-peptide materials and uncovers various aspects of these interesting hybrid materials for biomedical applications.

Chronic inflammation (CI) is a primary contributing factor involved in multiple diseases like cancer, stroke, diabetes, Alzheimer's disease, allergy, asthma, autoimmune diseases, coeliac disease, glomerulonephritis, sepsis, hepatitis, inflammatory bowel disease, reperfusion injury, and transplant rejections. Despite several expansions in our understanding of inflammatory disorders and their mediators, it seems clear that numerous proteins participate in the onset of CI. One crucial protein pyruvate kinase M2 (PKM2) much studied in cancer is also found to be inextricably woven in the onset of several CI's. It has been found that PKM2 plays a significant role in several disorders using a network of proteins that interact in multiple ways. For instance, PKM2 forms a close association with epidermal growth factor receptors (EGFRs) for uncontrolled growth and proliferation of tumor cells. In neurodegeneration, PKM2 interacts with apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) to onset Alzheimer's disease pathogenesis. The cross-talk of protein tyrosine phosphatase 1B (PTP1B) and PKM2 acts as stepping stones for the commencement of diabetes. Perhaps PKM2 stores the potential to unlock the pathophysiology of several diseases. Here we provide an overview of the notoriously convoluted biology of CI's and PKM2. The cross-talk of PKM2 with several proteins involved in stroke, Alzheimer's, cancer, and other diseases has also been discussed. We believe that considering the importance of PKM2 in inflammation-related diseases, new options for treating various disorders with the development of more selective agents targeting PKM2 may appear.

Alzheimer disease is multi-factorial and inflammation plays a major role in the disease progression and severity. Metals and reactive oxygen species (ROS) are the key mediators for inflammatory conditions associated with Alzheimer’s. Along multi-factorial nature, major challenge for developing new drug is the ability of the molecule to cross blood brain barrier (BBB). We have designed and synthesized multi-target directed hexafluorocarbinol containing triazoles to inhibit Amyloid β aggregation and simultaneously chelate the excess metals present in the extracellular space and scavenge the ROS thus reduce the inflammatory condition. From the screened compound library, compound 1c found to be potent and safe. It has demonstrated inhibition of Amyloid β aggregation (IC50 of 4.6 μM) through selective binding with Amyloid β at the nucleation site (evidenced from the molecular docking). It also chelate metals (Cu+2, Zn+2 and Fe+3) and scavenges ROS significantly. Due to the presence of hexafluorocarbinol moiety in the molecule it may assist to permeate BBB and improve the pharmacokinetic properties. The in-vitro results of compound 1c indicate the promiscuity for the development of hexafluorocarbinol containing triazoles amide scaffold as multi-target directed therapy against Alzheimer disease.

Peptide nucleic acids have emerged over the past two decades as a promising class of nucleic acid mimics because of their strong binding affinity and sequence selectivity toward DNA and RNA, and resistance to enzymatic degradation by proteases and nucleases. While they have been shown to be effective in regulation of gene expression in vitro, and to a small extent in vivo, their full potential for molecular therapy has not yet been fully realized due to poor cellular uptake. Herein, we report the development of cell-permeable, guanidine-based peptide nucleic acids targeting the epidermal growth factor receptor (EGFR) in preclinical models as therapeutic modality for head and neck squamous cell carcinoma (HNSCC) and nonsmall cell lung cancer (NSCLC). A GPNA oligomer, 16 nucleotides in length, designed to bind to EGFR gene transcript elicited potent antisense effects in HNSCC and NSCLC cells in preclinical models. When administered intraperitoneally in mice, EGFRAS-GPNA was taken-up by several tissues including the xenograft tumor. Systemic administration of EGFRAS-GPNA induced antitumor effects in HNSCC xenografts, with similar efficacies as the FDA-approved EGFR inhibitors: cetuximab and erlotinib. In addition to targeting wild-type EGFR, EGFRAS-GPNA is effective against the constitutively active EGFR vIII mutant implicated in cetuximab resistance. Our data reveals that GPNA is just as effective as a molecular platform for treating cetuximab resistant cells, demonstrating its utility in the treatment of cancer.

Chiral γPNAs containing miniPEG side-chains can invade any sequence of double helical B-form DNA, with the recognition occurring through direct Watson-Crick base-pairing.

Developed in the early 1990s, peptide nucleic acid (PNA) has emerged as a promising class of nucleic acid mimic because of its strong binding affinity and sequence selectivity toward DNA and RNA and resistance to enzymatic degradation by proteases and nucleases; however, the main drawbacks, as compared to other classes of oligonucleotides, are water solubility and biocompatibility. Herein we show that installation of a relatively small, hydrophilic (R)-diethylene glycol ("miniPEG", R-MP) unit at the γ-backbone transforms a randomly folded PNA into a right-handed helix. Synthesis of optically pure (R-MP)γPNA monomers is described, which can be accomplished in a few simple steps from a commercially available and relatively cheap Boc-l-serine. Once synthesized, (R-MP)γPNA oligomers are preorganized into a right-handed helix, hybridize to DNA and RNA with greater affinity and sequence selectivity, and are more water soluble and less aggregating than the parental PNA oligomers. The results presented herein have important implications for the future design and application of PNA in biology, biotechnology, and medicine, as well as in other disciplines, including drug discovery and molecular engineering.

Addition of bromoform to conjugated nitroalkenes in the presence of Mg provided beta-tribromomethyl nitroalkanes in good to excellent yields and diastereoselectivity. These novel Michael adducts, formed under radical conditions, underwent elimination of HBr in the same pot under reflux to afford beta-dibromomethylenated nitroalkanes in good yield. Alternatively, a one-pot high yielding synthesis of the dibromides was possible under anionic conditions via LDA mediated addition of bromoform to nitroalkenes.

A general method for preparing optically pure guanidine-based gamma-peptide nucleic acid (gammaGPNA) monomers for all four natural nucleobases (A, C, G, and T) is described. These second-generation gammaGPNAs differ from the first-generation GPNAs in that the guanidinium group is installed at the gamma- instead of the alpha-position of the N-(2-aminoethyl)glycine backbone unit. This positional switch enables GPNAs to be synthesized from relatively cheap L- as opposed to D-amino acids. Unlike their alpha-predecessors, which are randomly folded, gammaGPNAs prepared from L-amino acids are preorganized into a right-handed helix and bind to DNA and RNA with exceptionally high affinity and sequence selectivity and are readily taken up by mammalian cells.

Nucleic acids are an attractive platform for organizing molecular self-assembly because of their specific nucleobase interactions and defined length scale. Routinely employed in the organization and assembly of materials in vitro, however, they have rarely been exploited in vivo, due to the concerns for enzymatic degradation and cross-hybridization with the host's genetic materials. Herein we report the development of a tight-binding, orthogonal, synthetically versatile, and informationally interfaced nucleic acid platform for programming molecular interactions, with implications for in vivo molecular assembly and computing. The system consists of three molecular entities: the right-handed and left-handed conformers and a nonhelical domain. The first two are orthogonal to each other in recognition, while the third is capable of binding to both, providing a means for interfacing the two conformers as well as the natural nucleic acid biopolymers (i.e., DNA and RNA). The three molecular entities are prepared from the same monomeric chemical scaffold, with the exception of the stereochemistry or lack thereof at the γ-backbone that determines if the corresponding oligo adopts a right-handed or left-handed helix, or a nonhelical motif. These conformers hybridize to each other with exquisite affinity, sequence selectivity, and level of orthogonality. Recognition modules as short as five nucleotides in length are capable of organizing molecular assembly.

A convenient method for the two-step synthesis of arenediynes from 1,2-arenedialdehydes is reported. Dibromomethylenation of dialdehydes under Corey–Fuchs conditions (CBr4, Ph3P, Zn) provides the tetrabromides in excellent yields. Treatment of the tetrabromides with n-BuLi or LDA affords 3,4-unsaturated 1,5-diynes, the key structural moiety present in several naturally occurring antitumour antibiotics, in varying yields. The key intermediates in these transformations appear to be vinylidenecarbenes or carbenoids, generated in situ via metal–halogen exchange and elimination., © Elsevier

Background Lung cancer is the major cause of cancer-related deaths and many cases of Non Small Cell Lung Cancer (NSCLC), a common type of lung cancer, have frequent genetic/oncogenic activation of EGFR, KRAS, PIK3CA, BRAF, and others that drive tumor growth. Some patients though initially respond, but later develop resistance to erlotinib/gefitinib with no option except for cytotoxic therapy. Therefore, development of novel targeted therapeutics is imperative to provide improved survival benefit for NSCLC patients. The mTOR cell survival pathway is activated in naïve, or in response to targeted therapies in NSCLC. Methods We have discovered P7170, a small molecule inhibitor of mTORC1/mTORC2/ALK1 and investigated its antitumor efficacy using various in vitro and in vivo models of human NSCLC. Results P7170 inhibited the phosphorylation of AKT, S6 and 4EBP1 (substrates for mTORC2 and mTORC1) levels by 80-100% and growth of NSCLC cells. P7170 inhibited anchorage-independent colony formation of NSCLC patient tumor–derived cells subsistent of disease sub-types. The compound also induced apoptosis in NSCLC cell lines. P7170 at a well-tolerated daily dose of 20 mg/kg significantly inhibited the growth of NSCLC xenografts independent of different mutations (EGFR, KRAS, or PIK3CA) or sensitivity to erlotinib. Pharmacokinetic-pharmacodynamic (PK-PD) analysis showed sub-micro molar tumor concentrations along with mTORC1/C2 inhibition. Conclusions Our results provide evidence of antitumor activity of P7170 in the erlotinib –sensitive and –insensitive models of NSCLC. Electronic supplementary material The online version of this article (doi:10.1186/1476-4598-13-259) contains supplementary material, which is available to authorized users.

Peptide nucleic acids have emerged over the past two decades as a promising class of nucleic acid mimics because of their strong binding affinity and sequence selectivity toward DNA and RNA, and resistance to enzymatic degradation by proteases and nucleases. While they have been shown to be effective in regulation of gene expression in vitro, and to a small extent in vivo, their full potential for molecular therapy has not yet been fully realized due to poor cellular uptake. Herein, we report the development of cell-permeable, guanidine-based peptide nucleic acids targeting the epidermal growth factor receptor (EGFR) in preclinical models as therapeutic modality for head and neck squamous cell carcinoma (HNSCC) and nonsmall cell lung cancer (NSCLC). A GPNA oligomer, 16 nucleotides in length, designed to bind to EGFR gene transcript elicited potent antisense effects in HNSCC and NSCLC cells in preclinical models. When administered intraperitoneally in mice, EGFRAS-GPNA was taken-up by several tissues including the xenograft tumor. Systemic administration of EGFRAS-GPNA induced antitumor effects in HNSCC xenografts, with similar efficacies as the FDA-approved EGFR inhibitors: cetuximab and erlotinib. In addition to targeting wild-type EGFR, EGFRAS-GPNA is effective against the constitutively active EGFR vIII mutant implicated in cetuximab resistance. Our data reveals that GPNA is just as effective as a molecular platform for treating cetuximab resistant cells, demonstrating its utility in the treatment of cancer.

Pyridylpyrimidine based potent and selective inhibitors of HIF-1α are designed and synthesized.

Developed in the early 1990s, peptide nucleic acid (PNA) has emerged as a promising class of nucleic acid mimic because of its strong binding affinity and sequence selectivity toward DNA and RNA and resistance to enzymatic degradation by proteases and nucleases; however, the main drawbacks, as compared to other classes of oligonucleotides, are water solubility and biocompatibility. Herein we show that installation of a relatively small, hydrophilic (R)-diethylene glycol ("miniPEG", R-MP) unit at the γ-backbone transforms a randomly folded PNA into a right-handed helix. Synthesis of optically pure (R-MP)γPNA monomers is described, which can be accomplished in a few simple steps from a commercially available and relatively cheap Boc-l-serine. Once synthesized, (R-MP)γPNA oligomers are preorganized into a right-handed helix, hybridize to DNA and RNA with greater affinity and sequence selectivity, and are more water soluble and less aggregating than the parental PNA oligomers. The results presented herein have important implications for the future design and application of PNA in biology, biotechnology, and medicine, as well as in other disciplines, including drug discovery and molecular engineering.

Hypoxia-inducible factor-1α (HIF-1α) is a critical regulatory protein of cellular response to hypoxia, and regulates the transcription of many genes involved in key aspects of cancer biology, including immortalization, maintenance of stem cell pools, cellular dedifferentiation, vascularization, and invasion/metastasis. HIF-1α has been implicated in the regulation of genes involved in angiogenesis, for example, VEGF and is associated with tumor progression. In the last decade, over expression of HIF-1α has been demonstrated in many common human cancers and emerging as a validated target for anticancer drug discovery. Here we report the discovery of newly designed and synthesized pyridylpyrimidine based potent and selective inhibitors of HIF-1α. P2630 has been found as potent antiproliferative, antiangiogenic and orally efficacious compound in PC-3 xenograft mice model.

γ-PNA, a new class of peptide nucleic acids, promises to overcome previous sequence limitations of double-stranded DNA (dsDNA) targeting with PNA. To check the potential of γ-PNA, we have synthesized a biotinylated, pentadecameric γ-PNA of mixed sequence carrying three guanidinium G-clamp nucleobases. We have found that strand invasion reactions of the γ-PNA oligomer to its fully complementary target within dsDNA occurs with significantly higher binding rates than to targets containing single mismatches. Association of the PNA oligomer to mismatched targets does not go to completion but instead reaches a stationary level at or below 60%, even at conditions of very low ionic strength. Initial binding rates to both matched and mismatched targets experience a steep decrease with increasing salt concentration. We demonstrate that a linear DNA target fragment with the correct target sequence can be purified from DNA mixtures containing mismatched target or unrelated genomic DNA by affinity capture with streptavidin-coated magnetic beads. Similarly, supercoiled plasmid DNA is obtained with high purity from an initial sample mixture that included a linear DNA fragment with the fully complementary sequence. Based on the results obtained in this study we believe that γ-PNA has a great potential for specific targeting of chosen duplex DNA sites in a sequence-unrestricted fashion.

In this communication, we show that peptide nucleic acids (PNAs) with lengths of 15-20 nucleotides, when preorganized into a right-handed helix, can invade mixed-sequence double-helical B-form DNA (B-DNA). Strand invasion occurs in a highly sequence-specific manner through direct Watson-Crick base pairing. Unlike the previously developed double-duplex invasion strategy, which requires simultaneous binding of two strands of pseudocomplementary PNAs to DNA, only a single strand of gammaPNA is required for invasion in this case, and no nucleobase substitution is needed.

Addition of bromoform to conjugated nitroalkenes in the presence of Mg provided β-tribromomethyl nitroalkanes in good to excellent yields and diastereoselectivity. These novel Michael adducts, formed under radical conditions, underwent elimination of HBr in the same pot under reflux to afford β-dibromomethylenated nitroalkanes in good yield. Alternatively, a one-pot high yielding synthesis of the dibromides was possible under anionic conditions via LDA mediated addition of bromoform to nitroalkenes.

Nature uses Watson–Crick base pairings as a means to store and transmit genetic information because of their high fidelity. These specific A–T (or A–U) and G–C nucleobase interactions, in turn, provide chemists and biologists with a general paradigm for designing molecules to bind to DNA and RNA. With knowledge of the sequence information, one can design oligonucleotides to bind to just about any part of these biopolymeric targets simply by choosing the corresponding nucleobase sequence according to these digital base-pairing rules. Although conceptually simple, such principles in general can only be applied to the recognition of single-stranded DNA or RNA, but not the double-stranded form. The reason is that in double-helical DNA (or RNA) not only are the Watson–Crick faces of the nucleobases already occupied, they are buried within the double helix. [1] Such molecular encapsulation imposes a steep energetic barrier on the designer molecules. To establish binding, not only would they need to be able to gain access to the designated nucleobase targets, which are blocked by the existing base pairs, they would also need to be able to compete with the complementary DNA strand to prevent it from re-annealing with its partner—a task that has rarely been accomplished by any class of molecules. To circumvent this challenge, most of the research effort to date has been focused on establishing principles for recognizing chemical groups in the minor and major groove instead because they are more readily accessible and energetically less demanding. [2] While impressive progress has been made on this front, especially in the development of triplex-forming oligonucleotides, [3–5] polyamides, [6–8] and zinc-finger-binding pep

The synthesis of 3,4-ene-1,5-diynes, the key structural moiety present in several naturally occurring antitumor antibiotics, from 1,2-enedialdehydes under two different experimental conditions is reported. One method involves the dibromomethylenation of dialdehydes under Corey–Fuchs conditions (CBr4, Ph3P, and Zn) and treatment of the resulting tetrabromides with nBuLi or LDA to afford enediynes. The second method involves a base-mediated reaction of enedialdehydes with diethyl (1-diazo-2-oxopropyl)phosphonate (Bestmann–Ohira reagent) and subsequent transformation of the bis(diazo) compounds generated in situ to enediynes. While the transformation of bis(diazo) compounds to enediynes could take place exclusively through alkylidenecarbenes, generated in situ by geminal elimination of N2, an alternative pathway, involving the vicinal elimination of HBr to afford an intermediate bromoalkyne and its subsequent metal-halogen exchange and protonation during workup, exists for the bis(dibromoalkylidenes). However, our deuterium- labeling experiments with a model substrate, deuterated p-methoxybenzylidene dibromide, established the predominance of the alkylidenecarbenes, generated in situ by metal-halogen exchange and elimination, for this substrate and, by analogy, for the tetrabromides as well. The scope of this novel methodology was extended to the synthesis of various heteroatom-based (S, Se, and P) enediynes by quenching the acetylides with suitable electrophiles., © WILEY-VCH Verlag GmbH & Co. KGaA

The epidermal growth factor receptor (EGFR) is upregulated in majority of head and neck squamous cell carcinoma (HNSCC) tumors. In preclinical models inhibition of EGFR using several strategies results in tumor growth inhibition. However, clinical trials using agents that inhibit EGFR activation and signaling demonstrated limited antitumor efficacy. Another approach involving downmodulation of EGFR protein levels using antisense DNA sequences has been reported to be highly effective in inhibiting HNSCC growth in preclinical models. However, DNA based agents are prone to nuclease degradation necessitating intratumoral delivery of the agent. To facilitate systemic delivery of EGFR antisense oligonucleotides, we modified the DNA backbone to a pseudo-peptide moiety containing guanidinium groups called guanidinium-peptide nucleic acids (GPNA). This novel class of molecules binds the target DNA or RNA in a highly sequence specific manner, is resistant to nulceases and proteases, has a strong affinity for complementary DNA and RNA sequences. Experimental procedures: We designed a GPNA antisense oligomer complementary to 16 bases on the EGFR mRNA (EGFRAS GPNA). Transfection efficiency of the GPNA oligomer was determined using GFP expressing vectors and confocal imaging. Localization of the EGFRAS GPNA within the endoplasmic reticulum (ER) was determined using an ER specific dye from Molecular Probes. Immunoblotting and RT-PCR determinations were carried out on EGFR levels in HNSCC cells treated with EGFRAS GPNA. In vitro antitumor effects were assessed using the Cell Titre Glo assay. Antitumor effects of EGFRAS GPNA (5 mg/kg daily injections) were examined on local intratumoral and systemic intraperitoneal delivery in HNSCC subcutaneous tumor bearing athymic mice. In order to compare the antitumor efficacy of the EGFRAS GPNA to EGFR inhibitors currently in clinical use, we treated HNSCC xenograft tumor bearing mice with either EGFRAS GPNA (5 mg/kg body weight daily), the scrambled control GPNA (5 mg/kg body weight daily), cetuximab (0.8 mg/mouse thrice a week) or erlotinib (90 mg/kg daily via oral gavage). Results: Our data demonstrate that EGFRAS GPNA can be successfully introduced into HNSCC cells in the presence of serum containing medium with almost 100 % transfection efficiency. Further, EGFRAS GPNA treatment results in anti-tumor effects in vitro by down modulating EGFR mRNA levels. Our data demonstrate that EGFRAS GPNA has similar antitumor effects on HNSCC xenografts when delivered systemically via intraperitoneal injections or locally via intratumoral injections. A scrambled control GPNA had no antitumor effects in vivo demonstrating that EGFRAS GPNA specifically inhibited HNSCC growth. Our data demonstrate that EGFRAS GPNA has comparable antitumor effects to FDA approved EGFR inhibitors. Further, EGFRAS GPNA downmodulated EGFR mRNA levels in the HNSCC xenografts. Conclusions: EGFRAS GPNA has high cellular uptake and specifically downmodulates EGFR levels in HNSCC cells. When delivered systemically, EGFRAS GPNA has antitumor efficacy comparable to that of erlotinib and cetuximab. A novel strategy to target EGFR when systemically administered has immense potential to improve current therapeutic approaches for head and neck cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C175.