Robert P. Hammer

Selective Modulation of Matrix Metalloproteinase 9 (MMP-9) Functions via Exosite Inhibition

Unregulated activities of the matrix metalloproteinase (MMP) family have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix components, such as collagen and laminin. However, clinical trials with small molecule MMP inhibitors have been largely unsuccessful, with a lack of selectivity considered particularly problematic. Enhanced selectivity could be achieved by taking advantage of differences in substrate secondary binding sites (exosites) within the MMP family. In this study, triple-helical substrates and triple-helical transition state analog inhibitors have been utilized to dissect the roles of potential exosites in MMP-9 collagenolytic behavior. Substrate and inhibitor sequences were based on either the α1(V)436–450 collagen region, which is hydrolyzed at the Gly ↓ Val bond selectively by MMP-2 and MMP-9, or the Gly ↓ Leu cleavage site within the consensus interstitial collagen sequence α1(I–III)769–783, which is hydrolyzed by MMP-1, MMP-2, MMP-8, MMP-9, MMP-13, and MT1-MMP. Exosites within the MMP-9 fibronectin II inserts were found to be critical for interactions with type V collagen model substrates and inhibitors and to participate in interactions with an interstitial (types I–III) collagen model inhibitor. A triple-helical peptide incorporating a fibronectin II insert-binding sequence was constructed and found to selectively inhibit MMP-9 type V collagen-based activities compared with interstitial collagen-based activities. This represents the first example of differential inhibition of collagenolytic activities and was achieved via an exosite-binding triple-helical peptide.

Peptide targeting of platinum anti-cancer drugs.

Besides various side effects caused by platinum anticancer drugs, they are not efficiently absorbed by the tumor cells. Two Pt-peptide conjugates; cyclic mPeg-CNGRC-Pt (7) and cyclic mPeg-CNGRC-Pten (8) bearing the Asn-Gly-Arg (NGR) targeting sequence, a malonoyl linker, and low molecular weight miniPEG groups have been synthesized. The platinum ligand was attached to the peptide via the carboxylic end of the malonate group at the end of the peptide. The pegylated peptide is nontoxic and highly soluble in water. Platinum conjugates synthesized using the pegylated peptides are also water-soluble with reduced or eliminated peptide immunogenicity. The choice of carboplatin as our untargeted platinum complex was due to the fact that the malonate linker chelates platinum in a manner similar to that of carboplatin. Cell toxicity assay and competition assay on the PC-3 cells (CD13 positive receptors) revealed selective delivery and destruction of PC-3 cells using targeted Pt-peptide conjugates 7 and 8 significantly more than untargeted carboplatin. Platinum uptake on PC-3 cells was 12-fold more for conjugate 7 and 3-fold more for conjugate 8 compared to that of the untargeted carboplatin, indicating selective activation of the CD13 receptors and delivery of the conjugates to CD13 positive cells. Further analysis on effects of conjugates 7 and 8 on PC-3 cells using caspase-3/7, fluorescence microscopy, and DNA fragmentation confirmed that the cells were dying by apoptosis.

Fully 2′-Deoxy-2′-Fluoro Substituted Nucleic Acids Induce RNA Interference in Mammalian Cell Culture

RNA interference is a phenomenon in which RNA molecules elicit potent and sequence‐specific post‐transcriptional gene silencing. Recent studies have shown that small interfering RNA containing pyrimidine 2′‐fluoro modifications elicit RNAi. In this study, we demonstrate that fully‐2′‐fluorinated nucleic acids can be generated for RNAi studies through either custom solid‐phase synthesis or in vitro transcription using a mutated polymerase and fluorinated nucleoside triphosphates. Single‐stranded and hybridized fully‐2′‐fluorinated nucleic acids were subjected to a ribonuclease to assess their resistance to digestion. Duplex siFNA and antisense fully‐2′‐fluorinated nucleic acids were evaluated for their ability to knockdown green fluorescent protein expression in mammalian cell culture. Based on the results, fully‐2′‐fluorinated nucleic acids can be successfully generated, and fully‐2′‐fluorinated nucleic acids products show superior resistance to digestion over native RNA. Melt curve analysis suggests that transcribed fully‐2′‐fluorinated nucleic acids may contain base miscoding errors or early termination products. Small interfering fluoronucleic acid can induce RNAi and the silencing efficiency is nearly equivalent to the unmodified small interfering RNA species. Silencing from antisense fully‐2′‐fluorinated nucleic acids was greatly reduced relative to the duplex form. The lack of silencing activity from single‐stranded fully‐2′‐fluorinated nucleic acids, combined with reverse transcription polymerase chain reaction data showing that mRNA decreases following siFNA treatment, suggests that knockdown from siFNA is likely enzymatically driven as opposed to simple translational arrest.

[DLys(6)]-luteinizing hormone releasing hormone-curcumin conjugate inhibits pancreatic cancer cell growth in vitro and in vivo.

Pancreatic ductal adenocarcinomas are invariably lethal, and developing effective treatments that have minimal side effects is a challenge. Previous studies from our laboratory have shown that conjugates of cell membrane disrupting lytic peptides and luteinizing hormone releasing hormone (LHRH) target and destroy human prostate and breast cancer cells in xenografts in the nude mouse model (Hansel et al., Mol Cell Endocrinol 2007;260–262:183–9; Hansel et al., Mol Cell Endocrinol 2007;269:26–33), which express LHRH receptors. The objectives of our study were to synthesize a bioconjugate of LHRH analog ([DLys6]‐LHRH) and a dietary microchemical (curcumin) and test the hypothesis that [DLys6]‐LHRH–curcumin targets and inhibits pancreatic cancer cell growth in vitro and in vivo. In in vitro studies, we determined by confocal microscopy, flow cytometry analysis and reverse transcriptase‐polymerase chain reaction that MIAPaCa‐2, Panc‐1 and BxPC‐3 pancreatic cancer cell lines express LHRH receptors. [DLys6]‐LHRH–curcumin inhibited cell proliferation of pancreatic cancer cell lines and induced apoptotic cell death (p < 0.05). Apoptosis was induced by cleavage of polyadenosine‐5′‐diphosphate‐ribose‐polymerase and caspase‐3. The activity of [DLys6]‐LHRH–curcumin was equal to free curcumin at equimolar concentrations in vitro. Unlike curcumin itself, the [DLys6]‐LHRH–curcumin conjugate is water soluble which allows its intravenous administration. In two in vivo studies, [DLys6]‐LHRH–curcumin given intravenously caused a significant (p < 0.01) reduction in tumor weights and volumes, and free curcumin given by gavage at an equal dose failed to cause a significant reduction in tumor weights and volumes in the nude mouse pancreatic cancer model. [DLys6]‐LHRH–curcumin treatment enhanced apoptosis compared to [DLys6]‐LHRH and vehicle‐treated controls in tumor tissue. In conclusion, [DLys6]‐LHRH–curcumin may be useful in treating pancreatic cancer.

Side chain and flexibility contributions to the Raman optical activity spectra of a model cyclic hexapeptide

A model peptide, cyclo-(Phe-d-Pro-Gly-Arg-Gly-Asp), with a distinct folded structure containing short beta-hairpin and beta-sheet patterns was studied by Raman and Raman optical activity (ROA) spectroscopies. Unlike for previously analyzed vibrational circular dichroism of the same compound (Chirality 2008, 20, 1104), the Raman spectrum is dominated by side chain contributions and is more sensitive to their geometry fluctuations. The spectra and molecular motion were analyzed with the aid of the density functional theory simulations combined with molecular dynamics (MD). The side chain geometry fluctuations were found to significantly contribute to the broadening of the spectral bands, while dynamics of the backbone is rather restricted. According to our MD results, the side chains do not move freely but largely oscillate around preferred conformations. Averaging of computed spectra for many structures derived from the MD trajectories provided better spectral profiles than did a fixed geometry. The Raman and ROA scattering is dominated by the more polarizable phenylalanine and proline groups, as could be verified both by the computations and by comparison to experiments with a model Phe-d-Pro dipeptide. Computational analyses suggest that the ROA spectrum mostly senses local side chain conformation, whereas a vibrational coupling between different side chains contributes less. The coupling is mostly mediated by the peptide backbone and is restricted to specific vibrational region. The ROA spectroscopic technique thus provides important local structural information that needs, however, to be extracted by multiscale (QM/MM) simulation techniques.

Near-infrared, laser-induced fluorescence detection for DNA sequencing applications

Laser-induced fluorescence detection has become the detection strategy of choice in many large-scale DNA sequencing applications due to its ease of Implementation, sensitivity and the ability to identify the constituent bases of DNA in a single separation lane when the probes used have a distinct spectral characteristic. While the common strategy is to use fluorescent dyes which show absorption and emission properties in the visible region (400-600 nm) of the electromagnetic spectrum, our efforts have been directed toward developing near-IR (700-1000 nm) fluorescence as a viable detection strategy for DNA sequencing. In this paper, we discuss our results concerning the use of near-IR fluorescence detection for DNA sequencing carried out in a capillary gel column, where the capillary column has an internal diameter of 75 /spl mu/m, and the loading level of DNA onto this column is in the nL regime, requiring ultra-sensitive detection. In addition, we discuss our efforts toward the development of a highly efficient, single lane, single fluor, base-calling strategy using lifetime discrimination of heavy-atom modified near-IR dyes. The dyes developed for this application contain an intramolecular heavy atom (halogen) on a remote section of the chromophore, resulting in a perturbation in the fluorescence lifetime without altering the absorption or emission maximum of the base chromophore. This will allow the dye series to be excited with a single laser with the fluorescence processed on a single detector and the identity of the terminal base accomplished via lifetime discrimination. In order to effectively carry out lifetime measurements during capillary electrophoretic separation of the oligonucleotides, a simple solid-state time-correlated single photon counting instrument was constructed.

Mono-amine Functionalized Phthalocyanines: Microwave-Assisted Solid-Phase Synthesis and Bioconjugation Strategies

Phthalocyanines (Pcs) are excellent candidates for use as fluors for near-infrared (near-IR) fluorescent tagging of biomolecules for a wide variety of bioanalytical applications. Monofunctionalized Pcs, having two different types of peripheral substitutents, one for covalent conjugation of the Pc to biomolecules and others to improve the solubility of the macrocycle, are ideally suited for the desired applications. To date, difficulties faced during the purification of monofunctionalized Pcs limited their usage in various types of applications. Herein are reported a new synthetic method for rapid synthesis of the target Pcs and bioconjugation techniques for labeling of the oligonucleotides with the near-IR fluors. A novel synthetic route was developed utilizing a hydrophilic, poly(ethylene glycol) (PEG)-based support with an acid-labile Rink Amide linker. The Pcs were functionalized with an amine group for covalent conjugation purposes and were decorated with short PEG chains, serving as solubilizing groups. Microwave-assisted solid-phase synthetic method was successfully applied to obtain pure asymmetrically substituted monoamine functionalized Pcs in a short period of time. Three different bioconjugation techniques, reductive amination, amidation, and Huisgen cycloaddition, were employed for covalent conjugation of Pcs to oligonucleotides. The described microwave-assisted bioconjugation methods give an opportunity to synthesize and isolate the Pc-oligonucleotide conjugate in a few hours.

Effects of Peptides Derived from Terminal Modifications of the Aβ Central Hydrophobic Core on Aβ Fibrillization

Considerable research effort has focused on the discovery of mitigators that block the toxicity of the β-amyloid peptide (Aβ) by targeting a specific step involved in Aβ fibrillogenesis and subsequent aggregation. Given that aggregation intermediates are hypothesized to be responsible for Aβ toxicity, such compounds could likely prevent or mitigate aggregation, or alternatively cause further association of toxic oligomers into larger nontoxic aggregates. Herein we investigate the effect of modifications of the KLVFF hydrophobic core of Aβ by replacing N- and C-terminal groups with various polar moieties. Several of these terminal modifications were found to disrupt the formation of amyloid fibrils and in some cases induced the disassembly of preformed fibrils. Significantly, mitigators that incorporate MiniPEG polar groups were found to be effective against Aβ(1-40) fibrilligonesis. Previously, we have shown that mitigators incorporating alpha,alpha-disubstituted amino acids (ααAAs) were effective in disrupting fibril formation as well as inducing fibril disassembly. In this work, we further disclose that the number of polar residues (six) and ααAAs (three) in the original mitigator can be reduced without dramatically changing the ability to disrupt Aβ(1-40) fibrillization in vitro.

Near-IR single fluorophore quenching system based on phthalocyanine (Pc) aggregation and its application for monitoring inhibitor/activator action on a therapeutic target: L1-EN

Controlled H-aggregation of single Pc-labeled oligonucleotides is utilized as a fluorescence quenching system to discern changes in enzyme activity for the discovery of inhibitors for Long Interspersed Element 1 endonuclease (L1-EN), which is involved in genome instability and implicated in many different diseases.

Optimization of sequencing conditions using near-infrared lifetime identification methods in capillary gel electrophoresis

We have investigated the sample preparation and electrophoresis conditions necessary to prepare DNA sequencing samples appropriate for use with near‐infrared (IR) fluorescent labels with dye identification accomplished via lifetime techniques. It was found that several sample preparation protocols required attention to maximize the fluorescence yields of the labeling dyes, such as thermal cycling conditions, choice of counter ion used for the ethanol precipitation step and also, dye‐primer versus dye‐terminator chemistries. In addition, several different sieving matrices were investigated for their effects on both the fluorescence properties of the labeling dyes and electrophoretic resolution. Extended times used for the high temperature denaturing of duplexed DNA fragments during cycle sequencing produced cleavage products, in which the covalently attached dye to the sequencing primer was released through attack by dithiothreitol (DTT). Even under optimized thermal cycling conditions, free dye was generated that masked readable data from the sequencing traces. Ethanol precipitation was necessary to remove this free dye with the proper choice of counter ion (sodium). The results using different sieving matrices indicated that linear polyacrylamides (LPAs) were appropriate for any fluorescence measurement, since they could readily be replaced between runs minimizing deleterious memory effects associated with cross‐linked polyacrylamide gels. After investigation of several different sieving LPAs, the commercially available POP6 was found to be particularly attractive, since it produced good electrophoretic resolution, single exponential behavior for the near‐IR dye series investigated herein, and also, discernible lifetime differences within the dye set. Finally, dye‐terminator chemistry was also found to minimize bleeding in the gel matrix produced by large amounts of unextended dye‐primer within the gel lane.