Mechanically interlocked functionalization of monoclonal antibodies.

Because monoclonal antibodies (mAbs) have exceptional specificity and favorable pharmacology, substantial efforts have been made to functionalize them, either with potent cytotoxins, biologics, radionuclides, or fluorescent groups for therapeutic benefit and/or use as theranostic agents. To exploit our recently discovered meditope-Fab interaction as an alternative means to efficiently functionalize mAbs, we used insights from the structure to enhance the affinity and lifetime of the interaction by four orders of magnitude. To further extend the lifetime of the complex, we created a mechanical bond by incorporating an azide on the meditope, threading the azide through the Fab, and using click chemistry to add a steric group. The mechanically interlocked, meditope-Fab complex retains antigen specificity and is capable of imaging tumors in mice. These studies indicate it is possible to "snap" functionality onto mAbs, opening the possibility of rapidly creating unique combinations of mAbs with an array of cytotoxins, biologics, and imaging agents.

Stalled DNA Replication Forks at the Endogenous GAA Repeats Drive Repeat Expansion in Friedreich's Ataxia Cells.

Friedreich's ataxia (FRDA) is caused by the expansion of GAA repeats located in the Frataxin (FXN) gene. The GAA repeats continue to expand in FRDA patients, aggravating symptoms and contributing to disease progression. The mechanism leading to repeat expansion and decreased FXN transcription remains unclear. Using single-molecule analysis of replicated DNA, we detected that expanded GAA repeats present a substantial obstacle for the replication machinery at the FXN locus in FRDA cells. Furthermore, aberrant origin activation and lack of a proper stress response to rescue the stalled forks in FRDA cells cause an increase in 3'-5' progressing forks, which could enhance repeat expansion and hinder FXN transcription by head-on collision with RNA polymerases. Treatment of FRDA cells with GAA-specific polyamides rescues DNA replication fork stalling and alleviates expansion of the GAA repeats, implicating DNA triplexes as a replication impediment and suggesting that fork stalling might be a therapeutic target for FRDA.

A C-14 labeled Py-Im polyamide localizes to a subcutaneous prostate cancer tumor.

In an effort to quantitate Py-Im polyamide concentrations in vivo, we synthesized the C-14 radioactively labeled compounds 1-3, and investigated their tumor localization in a subcutaneous xenograft model of prostate cancer (LNCaP). Tumor concentrations were compared with representative host tissues, and exhibited a certain degree of preferential localization to the xenograft. Compound accumulation upon repeated administration was measured. Py-Im polyamide 1 was found to accumulate in LNCaP tumors at concentrations similar to the IC50 value for this compound in cell culture experiments.

Synthesis of cyclic Py-Im polyamide libraries.

Cyclic Py-Im polyamides containing two GABA turn units exhibit enhanced DNA binding affinity, but extensive studies of their biological properties have been hindered due to synthetic inaccessibility. A facile modular approach toward cyclic polyamides has been developed via microwave-assisted solid-phase synthesis of hairpin amino acid oligomer intermediates followed by macrocyclization. A focused library of cyclic polyamides 1-7 targeted to the androgen response element (ARE) and the estrogen response element (ERE) were synthesized in 12-17% overall yield. The Fmoc protection strategy also allows for selective modifications on the GABA turn units that have been shown to improve cellular uptake properties. The DNA binding affinities of a library of cyclic polyamides were measured by DNA thermal denaturation assays and compared to the corresponding hairpin polyamides. Fluorescein-labeled cyclic polyamides have been synthesized and imaged via confocal microscopy in A549 and T47D cell lines. The IC(50) values of compounds 1-7 and 9-11 were determined, revealing remarkably varying levels of cytotoxicity.

Role of mismatch repair enzymes in GAA·TTC triplet-repeat expansion in Friedreich ataxia induced pluripotent stem cells.

The genetic mutation in Friedreich ataxia (FRDA) is a hyperexpansion of the triplet-repeat sequence GAA·TTC within the first intron of the FXN gene. Although yeast and reporter construct models for GAA·TTC triplet-repeat expansion have been reported, studies on FRDA pathogenesis and therapeutic development are limited by the availability of an appropriate cell model in which to study the mechanism of instability of the GAA·TTC triplet repeats in the human genome. Herein, induced pluripotent stem cells (iPSCs) were generated from FRDA patient fibroblasts after transduction with the four transcription factors Oct4, Sox2, Klf4, and c-Myc. These cells were differentiated into neurospheres and neuronal precursors in vitro, providing a valuable cell model for FRDA. During propagation of the iPSCs, GAA·TTC triplet repeats expanded at a rate of about two GAA·TTC triplet repeats/replication. However, GAA·TTC triplet repeats were stable in FRDA fibroblasts and neuronal stem cells. The mismatch repair enzymes MSH2, MSH3, and MSH6, implicated in repeat instability in other triplet-repeat diseases, were highly expressed in pluripotent stem cells compared with fibroblasts and neuronal stem cells and occupied FXN intron 1. In addition, shRNA silencing of MSH2 and MSH6 impeded GAA·TTC triplet-repeat expansion. A specific pyrrole-imidazole polyamide targeting GAA·TTC triplet-repeat DNA partially blocked repeat expansion by displacing MSH2 from FXN intron 1 in FRDA iPSCs. These studies suggest that in FRDA, GAA·TTC triplet-repeat instability occurs in embryonic cells and involves the highly active mismatch repair system.

Microwave assisted synthesis of Py-Im polyamides.

Microwave synthesis was utilized to rapidly build Py-Im polyamides in high yields and purity using Boc-protection chemistry on Kaiser oxime resin. A representative polyamide targeting the 5'-WGWWCW-3' (W = A or T) subset of the consensus Androgen and Glucocorticoid Response Elements was synthesized in 56% yield after 20 linear steps and HPLC purification. It was confirmed by Mosher amide derivatization of the polyamide that a chiral α-amino acid does not racemize after several additional coupling steps.

Modulation of NF-κB-dependent gene transcription using programmable DNA minor groove binders.

Nuclear factor κB (NF-κB) is a transcription factor that regulates various aspects of immune response, cell death, and differentiation as well as cancer. In this study we introduce the Py-Im polyamide 1 that binds preferentially to the sequences 5'-WGGWWW-3' and 5'GGGWWW-3'. The compound is capable of binding to κB sites and reducing the expression of various NF-κB-driven genes including IL6 and IL8 by qRT-PCR. Chromatin immunoprecipitation experiments demonstrate a reduction of p65 occupancy within the proximal promoters of those genes. Genome-wide expression analysis by RNA-seq compares the DNA-binding polyamide with the well-characterized NF-κB inhibitor PS1145, identifies overlaps and differences in affected gene groups, and shows that both affect comparable numbers of TNF-α-inducible genes. Inhibition of NF-κB DNA binding via direct displacement of the transcription factor is a potential alternative to the existing antagonists.

Quantitative microarray profiling of DNA-binding molecules.

A high-throughput Cognate Site Identity (CSI) microarray platform interrogating all 524 800 10-base pair variable sites is correlated to quantitative DNase I footprinting data of DNA binding pyrrole-imidazole polyamides. An eight-ring hairpin polyamide programmed to target the 5 bp sequence 5'-TACGT-3' within the hypoxia response element (HRE) yielded a CSI microarray-derived sequence motif of 5'-WWACGT-3' (W = A,T). A linear beta-linked polyamide programmed to target a (GAA)3 repeat yielded a CSI microarray-derived sequence motif of 5'-AARAARWWG-3' (R = G,A). Quantitative DNase I footprinting of selected sequences from each microarray experiment enabled quantitative prediction of Ka values across the microarray intensity spectrum.

Completion of a Programmable DNA-Binding Small Molecule Library.

Hairpin pyrrole-imidazole (Py-Im) polyamides are programmable oligomers that bind the DNA minor groove in a sequence-specific manner with affinities comparable to those of natural DNA-binding proteins. These cell-permeable small molecules have been shown to enter the nuclei of live cells and downregulate endogenous gene expression. We complete here a library of 27 hairpin Py-Im polyamides which bind 7-base-pair sequences of the general form 5'-WWGNNNW-3' (where W = A or T, N = W, G, or C). Their equilibrium association constants (K(a)) range from K(a) = 1×10(8) M(-1) to 4×10(10) M(-1) with good sequence specificity. A table of binding affinities and sequence contexts for this completed 27-member library has been assembled for the benefit of the chemical biology community interested in molecular control of transcription.

DNA sequence-specific polyamides alleviate transcription inhibition associated with long GAA.TTC repeats in Friedreich's ataxia.

The DNA abnormality found in 98% of Friedreich's ataxia (FRDA) patients is the unstable hyperexpansion of a GAA.TTC triplet repeat in the first intron of the frataxin gene. Expanded GAA.TTC repeats result in decreased transcription and reduced levels of frataxin protein in affected individuals. Beta-alanine-linked pyrrole-imidazole polyamides bind GAA.TTC tracts with high affinity and disrupt the intramolecular DNA.DNA-associated region of the sticky-DNA conformation formed by long GAA.TTC repeats. Fluorescent polyamide-Bodipy conjugates localize in the nucleus of a lymphoid cell line derived from a FRDA patient. The synthetic ligands increase transcription of the frataxin gene in cell culture, resulting in increased levels of frataxin protein. DNA microarray analyses indicate that a limited number of genes are significantly affected in FRDA cells. Polyamides may increase transcription by altering the DNA conformation of genes harboring long GAA.TTC repeats or by chromatin opening.