Konstantinos Kiakos

Publications (30) View all

• Article: Design, synthesis, and DNA binding characteristics of a group of orthogonally positioned diamino, N-formamido, pyrrole- and imidazole-containing polyamides.

  Sameer Chavda, Balaji Babu, Pravin Patil, Adam Plaunt, Amanda Ferguson, Megan Lee, Samuel Tzou, Robert Sjoholm, Toni Rice, Hilary Mackay, Joseph Ramos, Shuo Wang, Shicai Lin, Konstantinos Kiakos, W David Wilson, John A Hartley, Moses Lee
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  ABSTRACT: Orthogonally positioned diamino/dicationic polyamides (PAs) have good water solubility and enhanced binding affinity, whilst retaining DNA minor groove and sequence specificity compared to their monoamino/monocationic counterparts. The synthesis and DNA binding properties of the following diamino PAs: f-IPI (3a), f-IPP (4), f-PIP (5), and f-PPP (6) are described. P denotes the site where a 1-propylamino group is attached to the N1-position of the heterocycle. Binding of the diamino PAs to DNA was assessed by DNase I footprinting, thermal denaturation, circular dichroism titration, biosensor surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) studies. According to SPR studies, f-IPI (3a) bound more strongly (Keq=2.4×10(8)M(-1)) and with comparable sequence selectivity to its cognate sequence 5'-ACGCGT-3' when compared to its monoamino analog f-IPI (1). The binding of f-IPI (3a) to 5'-ACGCGT-3' via the stacked dimer motif was balanced between enthalpy and entropy, and that was quite different from the enthalpy-driven binding of its monoamino parent f-IPI (1). f-IPP (4) also bound more strongly to its cognate sequence 5'-ATGCAT-3' (Keq=7.4×10(6)M(-1)) via the side-by-side stacked motif than its monoamino analog f-IPP (2a). Although f-PPP (6) bound via a 1:1 motif, it bound strongly to its cognate sequence 5'-AAATTT-3' (Keq=4.8×10(7)M(-1)), 15-times higher than the binding of its monoamino analog f-PPP (2c), albeit f-PPP bound via the stacked motif. Finally, f-PIP (5) bound to its target sequence 5'-ATCGAT-3' as a stacked dimer and it has the lowest affinity among the diamino PAs tested (Keq <1×10(5)M(-1)). This was about two times lower in affinity than the binding of its monoamino analog f-PIP (2b). The results further demonstrated that the 'core rules' of DNA recognition by monoamino PAs also apply to their diamino analogs. Specifically, PAs that contain a stacked IP core structure bind most strongly (highest binding constants) to their cognate GC doublet, followed by the binding of PAs with a stacked PP structure to two degenerate AT base pairs, and finally the binding of PAs with a PI core to their cognate CG doublet.
  Bioorganic & medicinal chemistry 04/2013; · 2.82 Impact Factor
• Article: Evidence for different mechanisms of 'unhooking' for melphalan and cisplatin-induced DNA interstrand cross-links in vitro and in clinical acquired resistant tumour samples.

  Victoria J Spanswick, Helen L Lowe, Claire Newton, John P Bingham, Alessia Bagnobianci, Konstantinos Kiakos, Charles Craddock, Jonathan A Ledermann, Daniel Hochhauser, John A Hartley
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  ABSTRACT: BACKGROUND: DNA interstrand crosslinks (ICLs) are critical lesions produced by several cancer chemotherapy agents including platinum drugs and nitrogen mustards. We have previously shown in haematological (multiple myeloma) and solid tumours (ovarian cancer) that clinical sensitivity to such agents can result from a defect in DNA ICL processing leading to their persistence. Conversely, enhanced repair can result in clinical acquired resistance following chemotherapy. The repair of ICLs is complex but it is assumed that the 'unhooking' step is common to all ICLs. METHODS: Using a modification of the single cell gel electrophoresis (Comet) assay we measured the formation and unhooking of melphalan and cisplatin-induced ICLs in cell lines and clinical samples. DNA damage response in the form of gamma-H2AX foci formation and the formation of Rad51 foci as a marker of homologous recombination were also determined. Real-time PCR of 84 genes involved in DNA damage signalling pathways was also examined pre- and post-treatment. RESULTS: Plasma cells from multiple myeloma patients known to be clinically resistant to melphalan showed significant unhooking of melphalan-induced ICLs at 48 hours, but did not unhook cisplatin-induced ICLs. In ovarian cancer cells obtained from patients following platinum-based chemotherapy, unhooking of cisplatin-induced ICLs was observed at 48 hours, but no unhooking of melphalan-induced ICLs. In vitro, A549 cells were proficient at unhooking both melphalan and cisplatin-induced ICLs. gamma-H2AX foci formation closely followed the formation of ICLs for both drugs, and rapidly declined following the peak of formation. RPMI8226 cells unhooked melphalan, but not cisplatin-induced ICLs. In these cells, although cross-links form with cisplatin, the gamma-H2AX response is weak. In A549 cells, addition of 3nM gemcitabine resulted in complete inhibition of cisplatin-induced ICL unhooking but no effect on repair of melphalan ICLs. The Rad51 foci response was both drug and cell line specific. Real time PCR studies highlighted differences in the damage response to melphalan and cisplatin following equi-ICL forming doses. CONCLUSIONS: These data suggest that the mechanisms by which melphalan and cisplatin-induced ICLs are 'unhooked' in vitro are distinct, and the mechanisms of clinical acquired resistance involving repair of ICLs, are drug specific.
  BMC Cancer 09/2012; 12(1):436. · 3.01 Impact Factor
• Article: Characterization of the human SNM1A and SNM1B/Apollo DNA repair exonucleases.

  Blanka Sengerová, Charles K Allerston, Mika Abu, Sook Y Lee, Janet Hartley, Konstantinos Kiakos, Christopher J Schofield, John A Hartley, Opher Gileadi, Peter J McHugh
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  ABSTRACT: Human SNM1A and SNM1B/Apollo have both been implicated in the repair of DNA interstrand cross-links (ICLs) by cellular studies, and SNM1B is also required for telomere protection. Here, we describe studies on the biochemical characterization of the SNM1A and SNM1B proteins. The results reveal some fundamental differences in the mechanisms of the two proteins. Both SNM1A and SNM1B digest double-stranded and single-stranded DNA with a 5'-to-3' directionality in a reaction that is stimulated by divalent cations, and both nucleases are inhibited by the zinc chelator o-phenanthroline. We find that SNM1A has greater affinity for single-stranded DNA over double-stranded DNA that is not observed with SNM1B. Although both proteins demonstrate a low level of processivity on low molecular weight DNA oligonucleotide substrates, when presented with high molecular weight DNA, SNM1A alone is rendered much more active, being capable of digesting kilobase-long stretches of DNA. Both proteins can digest past ICLs induced by the non-distorting minor groove cross-linking agent SJG-136, albeit with SNM1A showing a greater capacity to achieve this. This is consistent with the proposal that SNM1A and SNM1B might exhibit some redundancy in ICL repair. Together, our work establishes differences in the substrate selectivities of SNM1A and SNM1B that are likely to be relevant to their in vivo roles and which might be exploited in the development of selective inhibitors.
  Journal of Biological Chemistry 06/2012; 287(31):26254-67. · 4.77 Impact Factor
• Article: Minor structural modifications to alchemix influence mechanism of action and pharmacological activity.

  Qasem M A Abdallah, Roger M Phillips, Fredrik Johansson, Thomas Helleday, Laura Cosentino, Hamdy Abdel-Rahman, Jasarat Etzad, Richard T Wheelhouse, Konstantinos Kiakos, John P Bingham, John A Hartley, Laurence H Patterson, Klaus Pors
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  ABSTRACT: Alchemix is an exemplar of a class of anthraquinone with efficacy against multidrug resistant tumours. We have explored further the mechanism of action of alchemix and investigated the effect of extending its side arm bearing the alkylating functionality with regard to DNA binding and activity against multidrug resistant cancer cells. Increasing the distance between the intercalating chromophore and the alkylating functionality of ICT2901 (propyl), ICT2902 (butyl) and ICT2903 (pentyl), led to a higher number of DNA alkylation sites, more potent topoisomerase II inhibition and generated more apoptotic and necrotic cells when analysed in p53-proficient HCT116 cells. Intriguingly, alchemix, the compound with the shortest distance between its intercalative chromophore and alkylating functionality (ethyl), did not conform to this SAR. A different toxicity pattern against DNA repair defective CHO cell lines as well as arrest of cells in G1 supports a somewhat distinct mode of action by alchemix compared with its analogues. Importantly, both alchemix and ICT2901 demonstrated greater cytotoxic activity against anthraquinone-resistant MCF-7/adr cells than wild-type MCF-7 cells. Subtle synthetic modification in this anthraquinone series has led to significant changes to the stability of DNA-compound complexes and cellular activity. Given that the failure of chemotherapy in the clinic is often associated with MDR, the results of both alchemix and ICT2901 represent important advances towards improved therapies.
  Biochemical pharmacology 03/2012; 83(11):1514-22. · 4.25 Impact Factor
• Article: 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'.

  Vijay Satam, Balaji Babu, Sameer Chavda, Mia Savagian, Robert Sjoholm, Samuel Tzou, Joseph Ramos, Yang Liu, Konstantinos Kiakos, Shicai Lin, W David Wilson, John A Hartley, Moses Lee
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  ABSTRACT: 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, ΔT(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-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 × 10(7)M(-1), which is ∼3 times higher than that for its monoamino analog 3 (4.8 × 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.
  Bioorganic & medicinal chemistry 01/2012; 20(2):693-701. · 2.82 Impact Factor