Testing the Predicted Enhancement in DNA Sequence Specificity and Binding Affinity of Formamido-pentaamides over their Formamido-triamide Counterparts

Polyamides that contain imidazole and pyrrole heterocycles are known to target the minor groove of DNA in sequence specific interactions. Such molecules have the ability to alter gene expression. Analogs of the naturally occurring product distamycin have been developed and shown to bind in stacked anti-parallel dimers, but range in their affinity and specificity towards their target sequences. Although the formamido group is usually omitted from these analogs, it has been found to be important in increasing affinity and binding site size. Various formamido triamides have been synthesized, with f-ImPyIm showing the greatest affinity in targeting its cognate DNA sequence, 5’-d(ACGCGT)-3’. The established pairing rules for minor groove recognition indicate that the formamido-pentaamide, f-ImPyImPyIm, should bind with greater specificity to its cognate sequence without sacrificing affinity. This also calls for the synthesis of f-PyPyPyPyPy, which would target the control sequence 5’-d(AAATTT)-3’. The focus of this research is directed towards the synthesis of f-ImPyImPyIm and f-PyPyPyPyPy and testing their affinity and specificity in comparison with their triamide counterparts

Biochemical and Biological Properties of Fluorescent Polyamides

Polyamides containing imidazole and pyrrole units target the minor groove of DNA by making specific sequence specific interactions and hydrogen bonds with the four base pairs of DNA in stacked, anti-parallel dimers. Other groups, such as phenyl and formyl, have been added to these polyamides with the goal of increasing sequence specificity, binding affinity, solubility, as well as to enhance the convenience in synthesis. However, in order to track the movement and localization of these compounds in cells, the need for polyamides that both fluoresce and bind to DNA in a sequence selective manner was realized. Ligands (Hx) of Hoechst 33258 (bisbenzimidazole), a small fluorescent molecule that acts like two consecutive pyrroles in targeting A/T rich sequences of DNA, were incorporated into polyamides. One such polyamide, Hx-IP was found to bind a sequence 5’-TACGAT-’3 present in the 5-flank of the ICB2 element in the promoter of topoisomerase II, thus blocking the repressor transcriptional factor NF-Y. A variety of DNA binding studies have been conducted on Hx-IP. Results from these studies will be presente

Synthesis, Cytotoxicity, And Structure-activity Insight Of Nh- And N-methyl-3,5-bis-(arylidenyl)-4-piperidones

Twenty-one NH- and N-methyl-3,5-bis-(arylidenyl)-4-piperidone analogs of curcumin, 12 of which are novel, were synthesized and evaluated for their cytotoxicity against B16 (murine melanoma) and L1210 (murine lymphoma) cells grown in culture. These curcumin analogs are related to a known anticancer STAT3 inhibitor 3,5-bis-(4-fluorobenzyl)-4-piperidone (3). The compounds showed remarkable cytotoxicity, especially against B16 cells. The dimethoxy substituted analogs 4e and 4f and dihydroxy analog 4i emerged as the most active compounds with IC50 values in the range of 0.2-2.3 mu M. 4e, f, and i were about 10-times more cytotoxic against both cell lines than 3. Analysis of the results demonstrates that the position of the hydroxyl group is crucial for cytotoxicity. Amino-containing analogs are generally less active than their halogenated and oxygen-containing analogs, and N-substitution in the 4-piperidone moiety adds value to the cytotoxicity of the compounds

Novel diamino imidazole and pyrrole-containing polyamides: Synthesis and DNA binding studies of mono- and diamino-phenyl-ImPy*Im polyamides designed to target 5 \u27-ACGCGT-3 \u27

Pyrrole- and imidazole-containing polyamides are widely investigated as DNA sequence selective binding agents that have potential use as gene control agents. The key challenges that must be overcome to realize this goal is the development of polyamides with low molar mass so the molecules can readily diffuse into cells and concentrate in the nucleus. In addition, the molecules must have appreciable water solubility, bind DNA sequence specifically, and with high affinity. It is on this basis that the orthogonally positioned diamino/dicationic polyamide Ph-ImPy*Im 5 was designed to target the sequence 5\u27-ACGCGT-3\u27. Py* denotes the pyrrole unit that contains a N-substituted aminopropyl pendant group. The DNA binding properties of diamino polyamide 5 were determined using a number of techniques including CD, Delta T(M), DNase I footprinting, SPR and ITC studies. The effects of the second amino moiety in Py* on DNA binding affinity over its monoamino counterpart Ph-ImPylm 3 were assessed by conducting DNA binding studies of 3 in parallel with 5. The results confirmed the minor groove binding and selectivity of both polyamides for the cognate sequence 5\u27-ACGCGT-3\u27. The diamino/dicationic polyamide 5 showed enhanced binding affinity and higher solubility in aqueous media over its monoamino/monocationic counterpart Ph-ImPylm 3. The binding constant of 5, determined from SPR studies, was found to be 1.5 x 10(7) M(-1), which is similar to 3 times higher than that for its monoamino analog 3 (4.8 x 10(6) M(-1)). The affinity of 5 is now approaching that of the parent compound f-ImPyIm 1 and its diamino equivalent 4. The advantages of the design of diamino polyamide 5 over 1 and 4 are its sequence specificity and the ease of synthesis compared to the N-terminus pyrrole analog 2. (C) 2011 Elsevier Ltd. All rights reserved

Synthesis and DNA Binding Properties of 1-(3-aminopropyl)-imidazole-containing Triamide f-Im*PyIm: A Novel Diamino Polyamide Designed to Target 5 \u27-ACGCGT-3 \u27

A novel diamino/dicationic polyamide f-Im*PyIm (5) that contains an orthogonally positioned aminopropyl chain on an imidazole (Im*) moiety was designed to target 5\u27-ACGCGT-3\u27. The DNA binding properties of the diamino polyamide 5, determined by CD, Delta T-M, DNase I footprinting, SPR, and ITC studies, were compared with those of its monoamino/monocationic counterpart f-ImPyIm (1) and its diamino/dicationic isomer f-ImPy*Im (2), which has the aminopropyl group attached to the central pyrrole unit (Py*). The results gave evidence for the minor groove binding and selectivity of polyamide 5 for the cognate sequence 5\u27-ACGCGT-3\u27, and with strong affinity (K-eq = 2.3 x 10(7) M-1). However, the binding affinities varied according to the order: f-ImPy*Im (2) \u3e f-ImPyIm (1) \u3e= f-Im*PyIm (5) confirming that the second amino group can improve affinity, but its position within the polyamide can affect affinity. (C) 2012 Elsevier Ltd. All rights reserved

Corrigendum to “Novel diamino imidazole and pyrrole-containing polyamides: Synthesis and DNA binding studies of mono- and diamino-phenyl-ImPy*Im polyamides designed to target 5′-ACGCGT-3′” [Bioorg. Med. Chem. 20 (2012) 693–701]

Pyrrole- and imidazole-containing polyamides are widely investigated as DNA sequence selective binding agents that have potential use as gene control agents. The key challenges that must be overcome to realize this goal is the development of polyamides with low molar mass so the molecules can readily diffuse into cells and concentrate in the nucleus. In addition, the molecules must have appreciable water solubility, bind DNA sequence specifically, and with high affinity. It is on this basis that the orthogonally positioned diamino/dicationic polyamide Ph-ImPy*Im 5 was designed to target the sequence 5′-ACGCGT-3′. Py* denotes the pyrrole unit that contains a N-substituted aminopropyl pendant group. The DNA binding properties of diamino polyamide 5 were determined using a number of techniques including CD, ΔTM, DNase I footprinting, SPR and ITC studies. The effects of the second amino moiety in Py* on DNA binding affinity over its monoamino counterpart Ph-ImPyIm 3 were assessed by conducting DNA binding studies of 3 in parallel with 5. The results confirmed the minor groove binding and selectivity of both polyamides for the cognate sequence 5′-ACGCGT-3′. The diamino/dicationic polyamide 5 showed enhanced binding affinity and higher solubility in aqueous media over its monoamino/monocationic counterpart Ph-ImPyIm 3. The binding constant of 5, determined from SPR studies, was found to be 1.5 × 107 M−1, which is ∼3 times higher than that for its monoamino analog 3 (4.8 × 106 M−1). The affinity of 5 is now approaching that of the parent compound f-ImPyIm 1 and its diamino equivalent 4. The advantages of the design of diamino polyamide 5over 1 and 4 are its sequence specificity and the ease of synthesis compared to the N-terminus pyrrole analog 2

Design, Synthesis and Cytotoxicity of Novel Chalcone Analogs Derived From 1-Cyclohexylpyrrolidin-2-one and 2,3-Dihydrobenzo[f]chromen-1-one

Two divergent series of novel chalcone analogs, one derived from 1-cyclohexylpyrrolidin-2-one and the other derived from 1-benzo[f]chromanone, were designed, synthesized and evaluated for cytotoxicity against two murine cancer cell lines. Two 1-benzo[f]chromanone analogs, 4g and 4j yielded moderate toxicity against both melanoma B16 and lymphoma L1210 cell lines with IC50 values between the range of 5 and 6?mu M. With an IC50 value of 3.4?mu M, compound 4g was also active against human MDA-MB-435 melanoma cells. X-ray structures of the beta-hydroxy ketone product (4a) and the a,beta-unsaturated ketone (4h) were collected, and confirm the syn-configuration between the carbonyl moiety and the beta-vinylic proton in 4h. X-ray structures of two 1-cyclohexylpyrrolidin-2-one derivatives were also obtained, and both showed an E-configuration for the double bond

Hx-amides: DNA Sequence Recognition by the Fluorescent Hx (p-anisylbenzimidazole)•Pyrrole and Hx•Imidazole Pairings

Hx-amides are fluorescent hybrids of imidazole (1)- and pyrrole (P)-containing polyamides and Hoechst 33258, and they bind in the minor groove of specific DNA sequences. Synthesis and DNA binding studies of HxII (5) complete our studies on the first set of Hx-amides: Hx-I/P-I/P. HxPP (2), HxIP (3) and HxPI (4) were reported earlier. Results from DNase I footprinting, biosensor-SPR, CD and Delta T-M studies showed that Hx-amides interacted with DNA via the anti-parallel and stacked, side-by-side motif. Hx was found to mimic the DNA recognition properties of two consecutive pyrrole units (PP) in polyamides. Accordingly, the stacked Hx/PP pairing binds preferentially to two consecutive AT base pairs, A/T-A/T; Hx/IP prefers C-A/T; Hx/PI prefers A/T-C; and Hx/II prefers C-C. The results also showed that Hx-amides bound their cognate sequence at a higher affinity than their formamido-triamide counterparts. (C) 2013 Elsevier Ltd. All rights reserved

Affinity And Kinetic Modulation Of Polyamide–DNA Interactions By N-modification Of The Heterocycles

Synthetic N-methyl imidazole and N-pyrrole containing polyamides (PAs) that can form stacked dimers can be programmed to target and bind to specific DNA sequences and control gene expression. To accomplish this goal, the development of PAs with lower molecular mass which allows for the molecules to rapidly penetrate cells and localize in the nucleus, along with increased water solubility, while maintaining DNA binding sequence specificity and high binding affinity is key. To meet these challenges, six novel f-ImPy*Im PA derivatives that contain different orthogonally positioned moieties were designed to target 5-ACGCGT-3. The synthesis and biophysical characterization of six f-ImPy*Im were determined by CD, TM, DNase I footprinting, SPR, and ITC studies, and were compared with those of their parent compound, f-ImPyIm. The results gave evidence for the minor groove binding and selectivity of PAs 1 and 6 for the cognate sequence 5-ACGCGT-3, and with strong affinity, Keq = 2.8 x 108 M1 and Keq = 6.2 x 107 M1, respectively. The six novel PAs presented in this study demonstrated increased water solubility, while maintaining low molecular mass, sequence specificity, and binding affinity, addressing key issues in therapeutic development. (c) 2013 Wiley Periodicals, Inc. Biopolymers 99: 497507, 2013