Integrin-αvß6 targeted peptide-toxin therapy in a novel αvß6-expressing immunocompetent model of pancreatic cancer.

Previously we reported that a novel αvß6-specific peptide-drug conjugate (SG3299) could eliminate established human pancreatic ductal adenocarcinoma (PDAC) xenografts. However the development of effective therapies for PDAC, which is an essential need, must show efficacy in relevant immunocompetent animals. Previously we reported that the KPC mouse transgenic PDAC model that closely recapitulates most stages of development of human PDAC, unlike in humans, failed to express αvß6 on their tumours or metastases. In this study we have taken the KPC-derived PDAC line TB32043 and engineered a variant line (TB32043mb6S2) that expresses mouse integrin αvß6. We report that orthotopic implantation of the αvß6 over-expressing TB32043mb6S2 cells promotes shorter overall survival and increase in metastases. Moreover, systemic treatment of mice with established TB32043mb6S2 tumours in the pancreas with SG2399 lived significantly longer (p < 0.001; mean OS 48d) compared with PBS or control SG3511 (mean OS 25.5d and 26d, respectively). Thus SG3299 is confirmed as a promising candidate therapeutic for the therapy of PDAC.

PANTHER: AZD8931, inhibitor of EGFR, ERBB2 and ERBB3 signalling, combined with FOLFIRI: a Phase I/II study to determine the importance of schedule and activity in colorectal cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) is a therapeutic target to which HER2/HER3 activation may contribute resistance. This Phase I/II study examined the toxicity and efficacy of high-dose pulsed AZD8931, an EGFR/HER2/HER3 inhibitor, combined with chemotherapy, in metastatic colorectal cancer (CRC). METHODS: Treatment-naive patients received 4-day pulses of AZD8931 with irinotecan/5-FU (FOLFIRI) in a Phase I/II single-arm trial. Primary endpoint for Phase I was dose limiting toxicity (DLT); for Phase II best overall response. Samples were analysed for pharmacokinetics, EGFR dimers in circulating exosomes and Comet assay quantitating DNA damage. RESULTS: Eighteen patients received FOLFIRI and AZD8931. At 160 mg bd, 1 patient experienced G3 DLT; 160 mg bd was used for cohort expansion. No grade 5 adverse events (AE) reported. Seven (39%) and 1 (6%) patients experienced grade 3 and grade 4 AEs, respectively. Of 12 patients receiving 160 mg bd, best overall response rate was 25%, median PFS and OS were 8.7 and 21.2 months, respectively. A reduction in circulating HER2/3 dimer in the two responding patients after 12 weeks treatment was observed. CONCLUSIONS: The combination of pulsed high-dose AZD8931 with FOLFIRI has acceptable toxicity. Further studies of TKI sequencing may establish a role for pulsed use of such agents rather than continuous exposure. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov number: NCT01862003.

Phase 0 Study of Vandetanib-Eluting Radiopaque Embolics as a Preoperative Embolization Treatment in Patients with Resectable Liver Malignancies.

PURPOSE: To assess the safety and tolerability of a vandetanib-eluting radiopaque embolic (BTG-002814) for transarterial chemoembolization (TACE) in patients with resectable liver malignancies. MATERIALS AND METHODS: The VEROnA clinical trial was a first-in-human, phase 0, single-arm, window-of-opportunity study. Eligible patients were aged ≥18 years and had resectable hepatocellular carcinoma (HCC) (Child-Pugh A) or metastatic colorectal cancer (mCRC). Patients received 1 mL of BTG-002814 transarterially (containing 100 mg of vandetanib) 7-21 days prior to surgery. The primary objectives were to establish the safety and tolerability of BTG-002814 and determine the concentrations of vandetanib and the N-desmethyl vandetanib metabolite in the plasma and resected liver after treatment. Biomarker studies included circulating proangiogenic factors, perfusion computed tomography, and dynamic contrast-enhanced magnetic resonance imaging. RESULTS: Eight patients were enrolled: 2 with HCC and 6 with mCRC. There was 1 grade 3 adverse event (AE) before surgery and 18 after surgery; 6 AEs were deemed to be related to BTG-002814. Surgical resection was not delayed. Vandetanib was present in the plasma of all patients 12 days after treatment, with a mean maximum concentration of 24.3 ng/mL (standard deviation ± 13.94 ng/mL), and in resected liver tissue up to 32 days after treatment (441-404,000 ng/g). The median percentage of tumor necrosis was 92.5% (range, 5%-100%). There were no significant changes in perfusion imaging parameters after TACE. CONCLUSIONS: BTG-002814 has an acceptable safety profile in patients before surgery. The presence of vandetanib in the tumor specimens up to 32 days after treatment suggests sustained anticancer activity, while the low vandetanib levels in the plasma suggest minimal release into the systemic circulation. Further evaluation of this TACE combination is warranted in dose-finding and efficacy studies.

Effects of N-terminus modified Hx-amides on DNA binding affinity, sequence specificity, cellular uptake, and gene expression.

Five X-HxIP (Hx-amides) 6a-e, in which the N-terminus p-anisyl moiety is modified, were designed and synthesised with the purpose of optimising DNA binding, improving cellular uptake/nuclear penetration, and enhancing the modulation of the topoisomerase IIα (TOP2A) gene expression. The modifications include a fluorophenyl group and other heterocycles bearing different molecular shapes, size, and polarity. Like their parent compound HxIP 3, all five X-HxIP analogues bind preferentially to their cognate sequence 5'-TACGAT-3', which is found embedded on the 5' flank of the inverted CCAAT box-2 (ICB2) site in the TOP2A gene promoter, and inhibit protein complex binding. Interestingly, the 4-pyridyl analog 6a exhibits greater binding affinity for the target DNA sequence and abolishes the protein:ICB2 interaction in vitro, at a lower concentration, compared to the prototypical compound HxIP 3. Analogues 6b-e, display improved DNA sequence specificity, but reduced binding affinity for the cognate sequence, relative to the unmodified HxIP 3, with polyamides 6b and 6e being the most sequence selective. However, unlike 3 and 6b, 6a was unable to enter cells, access the nucleus and thereby affect TOP2A gene expression in confluent human lung cancer cells. These results show that while DNA binding affinity and sequence selectivity are important, consideration of cellular uptake and concentration in the nucleus are critical when exerting biological activity is the desired outcome. By characterising the DNA binding, cellular uptake and gene regulatory properties of these small molecules, we can elucidate the determinants of the elicited biological activity, which can be impacted by even small structural modifications in the polyamide molecular design.

ADCT-402, a PBD dimer-containing antibody drug conjugate targeting CD19-expressing malignancies.

Human CD19 antigen is a 95-kDa type I membrane glycoprotein in the immunoglobulin superfamily whose expression is limited to the various stages of B-cell development and differentiation and is maintained in the majority of B-cell malignancies, including leukemias and non-Hodgkin lymphomas of B-cell origin. Coupled with its differential and favorable expression profile, CD19 has rapid internalization kinetics and is not shed into the circulation, making it an ideal target for the development of antibody-drug conjugates (ADCs) to treat B-cell malignancies. ADCT-402 (loncastuximab tesirine) is a novel CD19-targeted ADC delivering SG3199, a highly cytotoxic DNA minor groove interstrand crosslinking pyrrolobenzodiazepine (PDB) dimer warhead. It showed potent and highly targeted in vitro cytotoxicity in CD19-expressing human cell lines. ADCT-402 was specifically bound, internalized, and trafficked to lysosomes in CD19-expressing cells and, following release of the PBD warhead, resulted in formation of DNA crosslinks that persisted for 36 hours. Bystander killing of CD19- cells by ADCT-402 was also observed. In vivo, single doses of ADCT-402 resulted in highly potent, dose-dependent antitumor activity in several subcutaneous and disseminated human tumor models with marked superiority to comparator ADCs delivering tubulin inhibitors. Dose-dependent DNA crosslinks and γ-H2AX DNA damage response were measured in tumors by 24 hours after single dose administration, whereas matched peripheral blood mononuclear cells showed no evidence of DNA damage. Pharmacokinetic analysis in rat and cynomolgus monkey showed excellent stability and tolerability of ADCT-402 in vivo. Together, these impressive data were used to support the clinical testing of this novel ADC in patients with CD19-expressing B-cell malignancies.

Circulating tumour cells and their association with bone metastases in patients with neuroendocrine tumours.

BACKGROUND: Bone metastases are associated with a worse outcome in patients with neuroendocrine tumours (NETs). Tumour overexpression of C-X-C chemokine receptor 4 (CXCR4) appears predictive of skeletal involvement. We investigated the role of circulating tumour cells (CTCs) and CXCR4 expression on CTCs as potential predictors of skeleton invasion. METHODS: Blood from patients with metastatic bronchial, midgut or pancreatic NET (pNET) was analysed by CellSearch. CXCR4 immunohistochemistry was performed on matched formalin-fixed paraffin-embedded (FFPE) samples. RESULTS: Two hundred and fifty-four patients were recruited with 121 midgut and 119 pNETs, of which 51 and 36% had detectable CTCs, respectively. Bone metastases were reported in 30% of midgut and 23% of pNET patients and were significantly associated with CTC presence (p = 0.003 and p < 0.0001). In a subgroup of 40 patients, 85% patients with CTCs had CTCs positive for CXCR4 expression. The proportion of CXCR4-positive CTCs in patients with bone metastases was 56% compared to 35% in those without (p = 0.18) it. Staining for CXCR4 on matched FFPE tissue showed a trend towards a correlation with CXCR4 expression on CTCs (p = 0.08). CONCLUSIONS: CTC presence is associated with bone metastases in NETs. CXCR4 may be involved in CTC osteotropism and present a therapeutic target to reduce skeletal morbidity.

Human SNM1A and XPF-ERCC1 collaborate to initiate DNA interstrand cross-link repair.

One of the major DNA interstrand cross-link (ICL) repair pathways in mammalian cells is coupled to replication, but the mechanistic roles of the critical factors involved remain largely elusive. Here, we show that purified human SNM1A (hSNM1A), which exhibits a 5'-3' exonuclease activity, can load from a single DNA nick and digest past an ICL on its substrate strand. hSNM1A-depleted cells are ICL-sensitive and accumulate replication-associated DNA double-strand breaks (DSBs), akin to ERCC1-depleted cells. These DSBs are Mus81-induced, indicating that replication fork cleavage by Mus81 results from the failure of the hSNM1A- and XPF-ERCC1-dependent ICL repair pathway. Our results reveal how collaboration between hSNM1A and XPF-ERCC1 is necessary to initiate ICL repair in replicating human cells.

DNA-Binding Properties of New Fluorescent AzaHx Amides: Methoxypyridylazabenzimidazolepyrroleimidazole/pyrrole.

DNA minor groove binding polyamides have been extensively developed to control abnormal gene expression. The establishment of novel, inherently fluorescent 2-(p-anisyl)benzimidazole (Hx) amides has provided an alternative path for studying DNA binding in cells by direct observation of cell localization. Because of the 2:1 antiparallel stacking homodimer binding mode of these molecules to DNA, modification of Hx amides to 2-(p-anisyl)-4-azabenzimidazole (AzaHx) amides has successfully extended the DNA-recognition repertoire from central CG [recognized by Hx-I (I=N-methylimidazole)] to central GC [recognized by AzaHx-P (P=N-methylpyrrole)] recognition. For potential targeting of two consecutive GG bases, modification of the AzaHx moiety to 2- and 3-pyridyl-aza-benzimidazole (Pyr-AzaHx) moieties was explored. The newly designed molecules are also small-sized, fluorescent amides with the Pyr-AzaHx moiety connected to two conventional five-membered heterocycles. Complementary biophysical methods were performed to investigate the DNA-binding properties of these molecules. The results showed that neither 3-Pyr-AzaHx nor 2-Pyr-AzaHx was able to mimic I-I=N-methylimidazole-N-methylimidazole to target GG dinucleotides specifically. Rather, 3-Pyr-AzaHx was found to function like AzaHx, f-I (f=formamide), or P-I as an antiparallel stacked dimer. 3-Pyr-AzaHx-PI (2) binds 5'-ACGCGT'-3' with improved binding affinity and high sequence specificity in comparison to its parent molecule AzaHx-PI (1). However, 2-Pyr-AzaHx is detrimental to DNA binding because of an unfavorable steric clash upon stacking in the minor groove.

Design, synthesis, nuclear localization, and biological activity of a fluorescent duocarmycin analog, HxTfA.

HxTfA 4 is a fluorescent analog of a potent cytotoxic and antimalarial agent, TfA 3, which is currently being investigated for the development of an antimalarial vaccine, PlasProtect®. HxTfA contains a p-anisylbenzimidazole or Hx moiety, which is endowed with a blue emission upon excitation at 318 nm; thus enabling it to be used as a surrogate for probing the cellular fate of TfA using confocal microscopy, and addressing the question of nuclear localization. HxTfA exhibits similar selectivity to TfA for A-tract sequences of DNA, alkylating adenine-N3, albeit at 10-fold higher concentrations. It also possesses in vitro cytotoxicity against A549 human lung carcinoma cells and Plasmodium falciparum. Confocal microscopy studies showed for the first time that HxTfA, and by inference TfA, entered A549 cells and localized in the nucleus to exert its biological activity. At biologically relevant concentrations, HxTfA elicits DNA damage response as evidenced by a marked increase in the levels of γH2AX observed by confocal microscopy and immunoblotting studies, and ultimately induces apoptosis.

The emerging role of anti-CD25 directed therapies as both immune modulators and targeted agents in cancer.

CD25 (also termed IL2RA) forms one component of the high-affinity heterotrimeric interleukin 2 (IL2) receptor on activated T cells. Its affinity for IL2 and cellular function are tightly regulated and vary in different cell types. The high frequency of CD25 on the surface of many different haematological tumour cells is now well established and, apart from its prognostic significance, CD25 may be present on leukaemic stem cells and enable oncogenic signalling pathways in leukaemic cells. Additionally, high CD25 expression in activated circulating immune cells and Tregs is a factor that has already been exploited by IL2 immunotherapies for treatment of tumours and autoimmune disease. The relative clinical safety and efficacy of administering anti-CD25 radioimmunoconjugates and immunotoxins in various haematological tumour indications has been established and clinical trials of a novel CD25-directed antibody drug conjugate are underway.

Synthesis and in vitro evaluation of SG3227, a pyrrolobenzodiazepine dimer antibody-drug conjugate payload based on sibiromycin.

A novel pyrrolobenzodiazepine dimer payload, SG3227, was rationally designed based on the naturally occurring antitumour compound sibiromycin. SG3227 was synthesized from a dimeric core in an efficient fashion. An unexpected room temperature Diels-Alder reaction occurred during the final step of the synthesis and was circumvented by use of an iodoacetamide conjugation moiety in place of a maleimide. The payload was successfully conjugated to trastuzumab and the resulting ADC exhibited potent activity against a HER2-expressing human cancer cell line in vitro.

Expression of somatostatin receptors 2 and 5 in circulating tumour cells from patients with neuroendocrine tumours.

BACKGROUND: Neuroendocrine tumours (NET) overexpress somatostatin receptors (SSTR) that can be targeted for therapy. Somatostatin receptor expression is routinely measured by molecular imaging but the resolution is insufficient to define heterogeneity. We hypothesised that SSTR expression could be measured on circulating tumour cells (CTCs) and used to investigate heterogeneity of expression and track changes during therapy. METHODS: MCF-7 cells were transfected with SSTR2 or 5 and spiked into donor blood for analysis by CellSearch. Optimum anti-SSTR antibody concentration and exposure time were determined, and flow cytometry was used to evaluate assay sensitivity. For clinical evaluation, blood was analysed by CellSearch, and SSTR2/5 immunohistochemistry was performed on matched tissue samples. RESULTS: Flow cytometry confirmed CellSearch was sensitive and that detection of SSTR was unaffected by the presence of somatostatin analogue up to a concentration of 100 ng ml-l. Thirty-one NET patients were recruited: grade; G1 (29%), G2 (45%), G3 (13%), primary site; midgut (58%), pancreatic (39%). Overall, 87% had SSTR-positive tumours according to somatostatin receptor scintigraphy or 68-Ga-DOTATE PET/CT. Circulating tumour cells were detected in 21 out of 31 patients (68%), of which 33% had evidence of heterogeneous expression of either SSTR2 (n=5) or SSTR5 (n=2). CONCLUSIONS: Somatostatin receptors 2 and 5 are detectable on CTCs from NET patients and may be a useful biomarker for evaluating SSTR-targeted therapies and this is being prospectively evaluated in the Phase IV CALMNET trial (NCT02075606).

Genetically Encoded Azide Containing Amino Acid in Mammalian Cells Enables Site-Specific Antibody-Drug Conjugates Using Click Cycloaddition Chemistry.

Antibody-drug conjugates (ADC) have emerged as potent antitumor drugs that provide increased efficacy, specificity, and tolerability over chemotherapy for the treatment of cancer. ADCs generated by targeting cysteines and lysines on the antibody have shown efficacy, but these products are heterogeneous, and instability may limit their dosing. Here, a novel technology is described that enables site-specific conjugation of toxins to antibodies using chemistry to produce homogeneous, potent, and highly stable conjugates. We have developed a cell-based mammalian expression system capable of site-specific integration of a non-natural amino acid containing an azide moiety. The azide group enables click cycloaddition chemistry that generates a stable heterocyclic triazole linkage. Antibodies to Her2/neu were expressed to contain N6-((2-azidoethoxy)carbonyl)-l-lysine at four different positions. Each site allowed over 95% conjugation efficacy with the toxins auristatin F or a pyrrolobenzodiazepine (PBD) dimer to generate ADCs with a drug to antibody ratio of >1.9. The ADCs were potent and specific in in vitro cytotoxicity assays. An anti Her2/neu conjugate demonstrated stability in vivo and a PBD containing ADC showed potent efficacy in a mouse tumor xenograph model. This technology was extended to generate fully functional ADCs with four toxins per antibody. The high stability of the azide-alkyne linkage, combined with the site-specific nature of the expression system, provides a means for the generation of ADCs with optimized pharmacokinetic, biological, and biophysical properties.

AzaHx, a novel fluorescent, DNA minor groove and G·C recognition element: Synthesis and DNA binding properties of a p-anisyl-4-aza-benzimidazole-pyrrole-imidazole (azaHx-PI) polyamide.

The design, synthesis, and DNA binding properties of azaHx-PI or p-anisyl-4-aza-benzimidazole-pyrrole-imidazole (5) are described. AzaHx, 2-(p-anisyl)-4-aza-benzimidazole-5-carboxamide, is a novel, fluorescent DNA recognition element, derived from Hoechst 33258 to recognize G·C base pairs. Supported by theoretical data, the results from DNase I footprinting, CD, ΔT(M), and SPR studies provided evidence that an azaHx/IP pairing, formed from antiparallel stacking of two azaHx-PI molecules in a side-by-side manner in the minor groove, selectively recognized a C-G doublet. AzaHx-PI was found to target 5'-ACGCGT-3', the Mlu1 Cell Cycle Box (MCB) promoter sequence with specificity and significant affinity (K(eq) 4.0±0.2×10(7) M(-1)).

Activity of the DNA minor groove cross-linking agent SG2000 (SJG-136) against canine tumours.

BACKGROUND: Cancer is the leading cause of death in older dogs and its prevalence is increasing. There is clearly a need to develop more effective anti-cancer drugs in dogs. SG2000 (SJG-136) is a sequence selective DNA minor groove cross-linking agent. Based on its in vitro potency, the spectrum of in vivo and clinical activity against human tumours, and its tolerability in human patients, SG2000 has potential as a novel therapeutic against spontaneously occurring canine malignancies. RESULTS: In vitro cytotoxicity was assessed using SRB and MTT assays, and in vivo activity was assessed using canine tumour xenografts. DNA interstrand cross-linking (ICL) was determined using a modification of the single cell gel electrophoresis (comet) assay. Effects on cell cycle distribution were assessed by flow cytometry and measurement of γ-H2AX by immunofluorescence and immunohistochemistry. SG2000 had a multi-log differential cytotoxic profile against a panel of 12 canine tumour cell lines representing a range of common tumour types in dogs. In the CMeC-1 melanoma cell line, DNA ICLs increased linearly with dose following a 1 h treatment. Peak ICL was achieved within 1 h and no removal was observed over 48 h. A relationship between DNA ICL formation and cytotoxicity was observed across cell lines. The formation of γ-H2AX foci was slow, becoming evident after 4 h and reaching a peak at 24 h. SG2000 exhibited significant anti-tumour activity against two canine melanoma tumour models in vivo. Anti-tumour activity was observed at 0.15 and 0.3 mg/kg given i.v. either once, or weekly x 3. Dose-dependent DNA ICL was observed in tumours (and to a lower level in peripheral blood mononuclear cells) at 2 h and persisted at 24 h. ICL increased following the second and third doses in a repeated dose schedule. At 24 h, dose dependent γ-H2AX foci were more numerous than at 2 h, and greater in tumours than in peripheral blood mononuclear cells. SG2000-induced H2AX phosphorylation measured by immunohistochemistry showed good correspondence, but less sensitivity, than measurement of foci. CONCLUSIONS: SG2000 displayed potent activity in vitro against canine cancer cell lines as a result of the formation and persistence of DNA ICLs. SG2000 also had significant in vivo antitumour activity against canine melanoma xenografts, and the comet and γ-H2AX foci methods were relevant pharmacodynamic assays. The clinical testing of SG2000 against spontaneous canine cancer is warranted.

ADCT-602, a Novel PBD Dimer-containing Antibody-Drug Conjugate for Treating CD22-positive Hematologic Malignancies.

Relapsed or refractory B-cell acute lymphoblastic leukemia (R/R B-ALL) and lymphomas have poor patient outcomes; novel therapies are needed. CD22 is an attractive target for antibody-drug conjugates (ADCs), being highly expressed in R/R B-ALL with rapid internalization kinetics. ADCT-602 is a novel CD22-targeting ADC, consisting of humanized mAb hLL2-C220, site specifically conjugated to the pyrrolobenzodiazepine dimer-based payload tesirine. In preclinical studies, ADCT-602 demonstrated potent, specific cytotoxicity in CD22-positive lymphomas and leukemias. ADCT-602 was specifically bound, internalized, and trafficked to lysosomes in CD22-positive tumor cells; after cytotoxin release, DNA interstrand crosslink formation persisted for 48 hours. In the presence of CD22-positive tumor cells, ADCT-602 caused bystander killing of CD22-negative tumor cells. A single ADCT-602 dose led to potent, dose-dependent, in vivo antitumor activity in subcutaneous and disseminated human lymphoma/leukemia models. Pharmacokinetic analyses (rat and cynomolgus monkey) showed excellent stability and tolerability of ADCT-602. Cynomolgus monkey B cells were efficiently depleted from circulation after one dose. Gene signature association analysis revealed IRAK1 as a potential marker for ADCT-602 resistance. Combining ADCT-602 + pacritinib was beneficial in ADCT-602-resistant cells. Chidamide increased CD22 expression on B-cell tumor surfaces, increasing ADCT-602 activity. These data support clinical testing of ADCT-602 in R/R B-ALL (NCT03698552) and CD22-positive hematologic cancers.

Characterization of the human SNM1A and SNM1B/Apollo DNA repair exonucleases.

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.

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, ΔTM, 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 × 10(8) M(-1) and Keq = 6.2 × 10(7) M(-1), 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.

Hx-amides: DNA sequence recognition by the fluorescent Hx (p-anisylbenzimidazole)•pyrrole and Hx•imidazole pairings.

Hx-amides are fluorescent hybrids of imidazole (I)- 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 ΔTM 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.

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

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 (K(eq)=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' (K(eq)=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' (K(eq)=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.