ß-Alanine and N-terminal cationic substituents affect polyamide-DNA binding.

Minor-groove binding hairpin polyamides (PAs) bind specific DNA sequences. Synthetic modifications can improve PA-DNA binding affinity and include flexible modules, such as ß-alanine (ß) motifs to replace pyrroles (Py), and increasing compound charge using N-terminal cationic substituents. To better understand the variations in kinetics and affinities caused by these modifications on PA-DNA interactions, a comprehensive set of PAs with different numbers and positions of ß and different types of N-cationic groups was systematically designed and synthesized to bind their cognate sequence, the λB motif. The λB motif is also a strong binding promoter site of the major groove targeting transcription factor PU.1. The PA binding affinities and kinetics were evaluated using a spectrum of powerful biophysical methods: thermal melting, biosensor surface plasmon resonance and circular dichroism. The results show that ß inserts affect PA-DNA interactions in a number and position dependent manner. Specifically, a ß replacement between two imidazole heterocycles (ImßIm) generally strengthens binding. In addition, N-terminal cationic groups can accelerate the association between PA and DNA, but the bulky size of TMG can cause steric hindrance and unfavourable repulsive electrostatic interactions in some PAs. The future design of stronger binding PA requires careful combination of ßs and cationic substituents.

Modulation of DNA-polyamide interaction by ß-alanine substitutions: a study of positional effects on binding affinity, kinetics and thermodynamics.

Hairpin polyamides (PAs) are an important class of sequence-specific DNA minor groove binders, and frequently employ a flexible motif, ß-alanine (ß), to reduce the molecular rigidity to maintain the DNA recognition register. To better understand the diverse effects that ß can have on DNA-PA binding affinity, selectivity, and especially kinetics, which have rarely been reported, we have initiated a detailed study for an eight-heterocyclic hairpin PA and its ß derivatives with their cognate and mutant sequences. With these derivatives, all internal pyrroles of the parent PA are systematically substituted with single or double ßs. A set of complementary experiments have been conducted to evaluate the molecular interactions in detail: UV-melting, biosensor-surface plasmon resonance, circular dichroism and isothermal titration calorimetry. The ß substitutions generally weaken the binding affinities of these PAs with cognate DNA, and have large and diverse influences on PA binding kinetics in a position- and number-dependent manner. The DNA base mutations have also shown positional effects on the binding of a single PA. Besides the ß substitutions, the monocationic Dp group [3-(dimethylamino)propylamine] in parent PA has been modified into a dicationic Ta group (3,3'-diamino-N-methyldipropylamine) to minimize the frequently observed PA aggregation with ITC experiments. The results clearly show that the Ta modification not only maintains the DNA binding mode and affinity of PA, but also significantly reduces PA aggregation and allows the complete thermodynamic signature of eight-ring hairpin PA to be determined for the first time. This combined set of results significantly extends our understanding of the energetic basis of specific DNA recognition by PAs.

DNA Binding Polyamides and the Importance of DNA Recognition in their use as Gene-Specific and Antiviral Agents.

There is a long history for the bioorganic and biomedical use of N-methyl-pyrrole-derived polyamides (PAs) that are higher homologs of natural products such as distamycin A and netropsin. This work has been pursued by many groups, with the Dervan and Sugiyama groups responsible for many breakthroughs. We have studied PAs since about 1999, partly in industry and partly in academia. Early in this program, we reported methods to control cellular uptake of polyamides in cancer cell lines and other cells likely to have multidrug resistance efflux pumps induced. We went on to discover antiviral polyamides active against HPV31, where SAR showed that a minimum binding size of about 10 bp of DNA was necessary for activity. Subsequently we discovered polyamides active against two additional high-risk HPVs, HPV16 and 18, a subset of which showed broad spectrum activity against HPV16, 18 and 31. Aspects of our results presented here are incompatible with reported DNA recognition rules. For example, molecules with the same cognate DNA recognition properties varied from active to inactive against HPVs. We have since pursued the mechanism of action of antiviral polyamides, and polyamides in general, with collaborators at NanoVir, the University of Missouri-St. Louis, and Georgia State University. We describe dramatic consequences of ß-alanine positioning even in relatively small, 8-ring polyamides; these results contrast sharply with prior reports. This paper was originally presented by JKB as a Keynote Lecture in the 2nd International Conference on Medicinal Chemistry and Computer Aided Drug Design Conference in Las Vegas, NV, October 2013.

Different thermodynamic signatures for DNA minor groove binding with changes in salt concentration and temperature.

The effects of salt concentration and temperature on the thermodynamics of DNA minor groove binding have quite different signatures: binding enthalpy is salt concentration independent but temperature dependent. Conversely, binding free energy is salt dependent but essentially temperature independent through enthalpy-entropy compensation.

Promoter scanning of the human COX-2 gene with 8-ring polyamides: unexpected weakening of polyamide-DNA binding and selectivity by replacing an internal N-Me-pyrrole with ß-alanine.

Rules for polyamide-DNA recognition have proved invaluable for the design of sequence-selective DNA binding agents in cell-free systems. However, these rules are not fully transferrable to predicting activity in cells, tissues or animals, and additional refinements to our understanding of DNA recognition would help biomedical studies. Similar complexities are encountered when using internal ß-alanines as polyamide building blocks in place of N-methylpyrrole; ß-alanines were introduced in polyamide designs to maintain good hydrogen bonding registry with the target DNA, especially for long polyamides or those with several GC bp (P.B. Dervan, A.R. Urbach, Essays Contemp. Chem. (2001) 327-339). Thus, to clarify important subtleties of molecular recognition, we studied the effects of replacing a single pyrrole with ß-alanine in 8-ring polyamides designed against the Ets-1 transcription factor. Replacement of a single internal N-methylpyrrole with ß-alanine to generate a ß/Im pairing in two 8-ring polyamides causes a decrease in DNA binding affinity by two orders of magnitude and decreases DNA binding selectivity, contrary to expectations based on the literature. Measurements were made by fluorescence spectroscopy, quantitative DNA footprinting and surface plasmon resonance, with these vastly different techniques showing excellent agreement. Furthermore, results were validated for a range of DNA substrates from small hairpins to long dsDNA sequences. Docking studies helped show that ß-alanine does not make efficient hydrophobic contacts with the rest of the polyamide or nearby DNA, in contrast to pyrrole. These results help refine design principles and expectations for polyamide-DNA recognition.

Correlation of local effects of DNA sequence and position of ß-alanine inserts with polyamide-DNA complex binding affinities and kinetics.

To improve our understanding of the effects of ß-alanine (ß) substitution and the number of heterocycles on DNA binding affinity and selectivity, we investigated the interactions of an eight-ring hairpin polyamide (PA) and two ß derivatives as well as a six-heterocycle analogue with their cognate DNA sequence, 5'-TGGCTT-3'. Binding selectivity and the effects of ß have been investigated with the cognate and five mutant DNAs. A set of powerful and complementary methods have been employed for both energetic and structural evaluations: UV melting, biosensor surface plasmon resonance, isothermal titration calorimetry, circular dichroism, and a DNA ligation ladder global structure assay. The reduced number of heterocycles in the six-ring PA weakens the binding affinity; however, the smaller PA aggregates significantly less than the larger PAs and allows us to obtain the binding thermodynamics. The PA-DNA binding enthalpy is large and negative with a large negative ΔC(p) and is the primary driving component of the Gibbs free energy. The complete SPR binding results clearly show that ß substitutions can substantially weaken the binding affinity of hairpin PAs in a position-dependent manner. More importantly, the changes in the binding of PA to the mutant DNAs further confirm the position-dependent effects on the PA-DNA interaction affinity. Comparison of mutant DNA sequences also shows a different effect in recognition of T·A versus A·T base pairs. The effects of DNA mutations on binding of a single PA as well as the effects of the position of ß substitution on binding tell a clear and very important story about sequence-dependent binding of PAs to DNA.

Fluorescence assay of polyamide-DNA interactions.

Polyamides (PAs) are distamycin-type ligands of DNA that bind the minor groove and are capable of sequence selective recognition. This capability provides a viable route to their development as therapeutics. Presented here is a simple and convenient fluorescence assay for PA-DNA binding. PAs are titrated into a sample of a hairpin DNA featuring a TAMRA dye attached to an internal dU near the PA binding site. In a study of 6 PAs, PA binding leads to a steady reproducible decrease in fluorescence intensity that can be used to generate binding isotherms. The assay works equally well with both short (6- to 8-ring) and long (14-ring) PAs, and K(d) values ranging from approximately 1 nM to at least 140 nM were readily obtained using a simple monochromator or filter configuration. Competition assays provide a means to assessing possible dye interference, which can be negligible. The assay can also be used to determine PA extinction coefficients and to measure binding kinetics; thus, it is an accessible and versatile tool for the study of PA properties and PA-DNA interactions.

The novel benzopyran class of selective cyclooxygenase-2 inhibitors. Part III: the three microdose candidates.

In this manuscript, we report the discovery of the substituted 2-trifluoromethyl-2H-benzopyran-3-carboxylic acids as a novel series of potent and selective cyclooxygenase-2 (COX-2) inhibitors. We provide the structure-activity relationships, optimization of design, testing criteria, and human half-life data. The challenge of a surprisingly long half-life (t(1/2)=360 h) of the first clinical candidate 1 and human t(1/2) had been difficult to predict based on allometric scaling for this class of highly ppb compounds. We used a microdose strategy which led to the discovery of clinical agents 18c-(S), 29b-(S), and 34b-(S) with human half-life of 57, 13, and 11 h.

Discovery of (pyridin-4-yl)-2H-tetrazole as a novel scaffold to identify highly selective matrix metalloproteinase-13 inhibitors for the treatment of osteoarthritis.

Potent, highly selective and orally-bioavailable MMP-13 inhibitors have been identified based upon a (pyridin-4-yl)-2H-tetrazole scaffold. Co-crystal structure analysis revealed that the inhibitors bind at the S(1)(') active site pocket and are not ligands for the catalytic zinc atom. Compound 29b demonstrated reduction of cartilage degradation biomarker (TIINE) levels associated with cartilage protection in a preclinical rat osteoarthritis model.

Computer-Aided Design (CAD) of Synzymes: Use of Molecular Mechanics (MM) for the Rational Design of Superoxide Dismutase Mimics.

Mn(II) complexes of C-substituted macrocyclic 1,4,7,10,13-pentaazacyclopentadecane ligands have been shown to be excellent functional mimics (Synzymes) of the native enzyme manganese superoxide dismutase (Mn SOD). To better understand the profound effects that substituents exert on the SOD catalytic activity, we have utilized molecular mechanics (MM) calculations employing the CAChe system. Such a conformational analysis has made it possible to develop a consistent model that correlates catalytic rate with the ability of the ligand to adopt a folded geometry about the high-spin d(5) spherically symmetrical Mn(II) ion, thus affording a six-coordinate pseudo-octahedral geometry (the geometry required by Mn(III)). This conformational analysis is consistent with the model that one of the nitrogen donors of the pentaaza crown ligand folds to occupy a pseudo-axial coordination position of an octahedron. The DeltaE between the lowest energy folded ligand structure about Mn(II) and its corresponding Mn(III) structure correlates with catalytic activity; i.e., for a large series of complexes an excellent correlation is obtained for both the inner-sphere and outer-sphere rate constants for oxidation of Mn(II)-the rate-determining step in the catalytic cycle for these SOD mimics. From single-crystal X-ray structure determinations on several different members of this class of 7-coordinate dichloro(pentaaza crown) Mn(II) complexes, we have observed that the arrangement of NH's of the secondary amine donors is such that they alternate in their relative orientation to the plane generated by the five nitrogens and the Mn; i.e., the NH's are arranged in an up-down-up-down-up stereochemistry. Thus, one side of the plane of the macrocyclic ring possesses two nonadjacent NH's, while the opposite side has three NH's. Two unique folding motifs generated from MM calculations are found to correlate with the two pathways for Mn(II) oxidation: (1) the inner-sphere path correlates with an NH from the side of the two NH's folding into the axial octahedral coordination site, and (2) the outer-sphere path correlates with an NH from the side of the three NH's folded into an axial O(h) site. MM calculations allow one to probe the effect that substituents on the macrocyclic ring carbons have on the relative energies of the Mn(II) and Mn(III) complexes with these ligands in the various potential folded geometries. The details of this modeling paradigm and the results of MM calculations utilizing the folding motif for a large number complexes are described. Of particular significance is the ability of the MM tool to predict correctly that certain substituent patterns and substituents enhance or reduce the contribution of one or the other pathway to the overall catalytic rate. The syntheses of several new complexes are reported and the rate constants for the two pathways of Mn(II) oxidation have been measured and found to correlate with the predictions arising from the energetics of folding as calculated by MM calculations.