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PDF file, 227K, SFT xenotransplanted in SCID mice pathologic evaluation before and after treatment. A and B Pre-treatment samples: high-grade sarcoma aspects mixed with cartilaginous (A) and osseous component (B). C and D Post-sunitinib, post-bevacizumab and post-pazopanib samples. 120 days after the end of treatment with sunitinib (C), bevacizumab (D) xenografts were sacrificed. The tumor samples were all made of hypercellular viable, mainly spindle tumor cells, intermingled with osteoid matrix. Pazopanib (E) xenograft was instead sacrificed soon after treatment end, Even in this case tumor sample appeared to be completely vital.

Purpose: To explore the value of triazines in solitary fibrous tumor (SFT).Experimental Design: We retrospectively reviewed 8 cases of patients with SFT treated with dacarbazine (1,200 mg/m2 every 3 weeks) as from January 2012. Then, we studied a dedifferentiated-SFT subcutaneously xenotransplanted into severe combined immunodeficient (SCID) mice. Dacarbazine, temozolomide, sunitinib, bevacizumab, and pazopanib were administered at their reported optimal doses for the mouse model when mean tumor volume (TV) was about 80 mm3; each experimental groups included 6 mice. Drug activity was assessed as tumor volume inhibition percentage (TVI%). Dacarbazine was tested according to two different schedules of administration. One hunded twenty days after treatment interruption, mouse tumor samples were analyzed.Results: Among the eight patients treated with dacarbazine, best response evaluation criteria in solid tumors responses (RECIST) were three partial responses, 4 stable disease, 1 progression. Two responsive patients had paraneoplastic hypoglycemia that disappeared after 10 days from starting dacarbazine. In the dedifferentiated-SFT xenograft model, dacarbazine and temozolomide showed the highest antitumor activity (about 95% TVI), confirmed pathologically. Sunitinib and pazopanib were only marginally active (52% and 41% TVI, respectively), whereas bevacizumab caused a 78% TVI. No tumor regrowth was observed up to 100 days from end of treatment with temozolomide and dacarbazine, whereas secondary progression followed sunitinib, pazopanib, and bevacizumab interruption.Conclusions: Dacarbazine as single agent has antitumor activity in SFT. Our preclinical results suggest a cytotoxic effect of temozolomide and dacarbazine, as compared with a cytostatic role for sunitinib, pazopanib, and bevacizumab. A phase II study on dacarbazine in advanced SFT is planned. Clin Cancer Res; 19(18); 5192–201. ©2013 AACR.

PDF file, 256K, A Human primary tumor: morphology (i.e., patternless growth, collagen deposition, moderate cellularity, mitotic index (>4X10 High Power Field (HPF), as shown in panel 1) and immunophenotype (i.e., diffuse positivity for CD34, as shown in panel 1/a) were consistent with a malignant SFT. Immunohistochemistry was positive for PDGFRB expression (panel 1/b). B Human relapsed tumor: morphology was consistent with a dedifferentiated SFT (DSFT), being marked by the abrupt transition from areas with MSFT aspects similar to what observed in the primary tumor (panel 2) to areas with high-grade sarcoma features (panel 3). High-grade areas showed hypercellularity (panel 3/a), chondroid (panel 3/b) and osteoid differentiation (panel 3/c). C Xenograft tumor: morphology was consistent with a high-grade DSFT (panel 4), superimposable to what observed in panel B, 3a/3b/3c, again with presence of mineralized trabeculaes and osteoid (panel 4/a). Immunohistochemistry was positive for PDGFRB expression (panel 4/b). Phospho receptor tyrosine kinase (pRTK) array (ARY001 Proteome ProfilerTM Array, R&D Systems) showed similar profiles for human relapsed tumor (panel B, 3/d) and xenograft (panel C, 4/c) with the activation of PDGFRA (green boxes), PDGFRB (red boxes) and VEGFR1 (yellow boxes). White boxes are the proteome profiler negative controls.

PDF file, 110K, The xenografts were sacrificed five days after starting sunitinib, bevacizumab and pazopanib. One sample was obtained also 4 weeks after the end of treatment with pazopanib. PDGFRB and VEGFR2 activation was analyzed trough immunoprecipitation (IP) and western blotting (WB). PDGFRB activation was reduced by sunitinib and pazopanib, while VEGFR2 activation was decreased by bevacizumab and pazopanib. A re-activation of both PDGFRB and VEGFR2 was detected after 4 weeks of treatment with pazopanib.

The P2X4 receptor is a ligand-gated ion channel activated by extracellular ATP. P2X4 activity is associated with neuropathic pain, vasodilation, and pulmonary secretion and is therefore of therapeutic interest. The structure-activity relationship of P2X4 antagonists is poorly understood. Here we elucidate the structure-activity of 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) at human P2X4 by combining pharmacology, electrophysiology, molecular modeling, and medicinal chemistry. 5-BDBD antagonized P2X4 in a noncompetitive manner but lacked effect at human P2X2. Molecular modeling and site-directed mutagenesis suggested an allosteric binding site for 5-BDBD located between two subunits in the body region of P2X4, with M109, F178, Y300, and I312 on one subunit and R301 on the neighboring subunit as key residues involved in antagonist binding. The bromine group of 5-BDBD was redundant for the antagonist activity of 5-BDBD, although an interaction between the carbonyl group of 5-BDBD and R301 in P2X4 was associated with 5-BDBD activity. 5-BDBD could inhibit the closed channel but poorly inhibited the channel in the open/desensitizing state. We hypothesize that this is due to constriction of the allosteric site after transition from closed to open channel state. We propose that M109, F178, Y300, R301, and I312 are key residues for 5-BDBD binding; provide a structural explanation of how they contribute to 5-BDBD antagonism; and highlight that the limited action of 5-BDBD on open versus closed channels is due to a conformational change in the allosteric site. SIGNIFICANCE STATEMENT: Activity of P2X4 receptor is associated with neuropathic pain, inflammation, and vasodilatation. Molecular information regarding small-molecule interaction with P2X4 is very limited. Here, this study provides a structural explanation for the action of the small-molecule antagonist 5-BDBD at the human P2X4 receptor.

Solid phase synthesis allowed the rapid generation of a peptide-drug conjugate. A peptide targeting the Thomsen-Friedenreich antigen (TFα) was conjugated to the alkylating subunit of the potent cytotoxin duocarmycin SA. The compound, containing a cathepsin B cleavable linker, was shown to be active and selective against TFα expressing tumour cell lines.

In a proof-of-concept study, solid phase synthesis allowed the rapid generation of a small molecule drug conjugate in which the glutamate carboxypeptidase II (GCPII) targeting small molecule DUPA was conjugated to the alkylating subunit of the potent cytotoxin duocarmycin SA. The targeted SMDC contained a cathepsin B cleavable linker, which was shown to be active and selective against cathepsin B over-expressing and GCPII-expressing tumour cell lines.

The duocarmycins are a family of natural products first described in 1978 with the discovery of CC-1065. These DNA alkylating spirocyclopropyl-cyclohexadienones demonstrate ultrapotent cytotoxic activity, provided by the sequence-selective alkylation of the N3 of adenine. The medicinal chemistry community immediately saw great potential in the picomolar potency of the duocarmycins in cell lines, but inherent toxicity in vivo has hindered their progression through to clinical use. Consequently, a variety of strategies have been developed to harness the power of the duocarmycins and to begin to realise the potential of their highly interesting mode of action, the most exciting of which is the development of antibody–drug conjugates (ADCs). This chapter will present the most recent understanding of the mechanism of action of the duocarmycins and the downstream effects of DNA alkylation. Innovative approaches to the synthesis of the duocarmycins, including stereoselective synthesis and new approaches for solid-phase synthesis, are discussed. This chapter also highlights the approaches of medicinal chemists to harness the duocarmycins for clinical use and an overview of prodrug strategies is presented, emphasising the most effective and creative methods to release the duocarmycins at the desired site of action. Finally, the use of duocarmycins as ADCs is reviewed, underlining the inventive chemical approaches to direct and deliver this ultrapotent payload.

The development of protein-protein interaction (PPI) inhibitors with therapeutic value is of increasing importance as the first clinical agent has now been approved, but PPIs remain difficult targets for the development of small molecule ligands. This article describes a highly efficient approach to the development of inhibitors of the p53/

We report the chemical synthesis and conformational analysis of a collection of 2-, 6- and 8-substituted derivatives of β-NAD(+) and AMP, and their biochemical evaluation against NAD(+)-dependent DNA ligases from Escherichia coli and Mycobacterium tuberculosis. Bacterial DNA ligases are validated anti-microbial targets, and new strategies for their inhibition are therefore of considerable scientific and practical interest. Our study includes several pairs of β-NAD(+) and AMP derivatives with the same substitution pattern at the adenine base. This has enabled the first direct comparison of co-substrate and inhibitor behaviour against bacterial DNA ligases. Our results suggest that an additional substituent in position 6 or 8 of the adenine base in β-NAD(+) is detrimental for activity as either co-substrate or inhibitor. In contrast, substituents in position 2 are not only tolerated, but appear to give rise to a new mode of inhibition, which targets the conformational changes these DNA ligases undergo during catalysis. Using a molecular modelling approach, we highlight that these findings have important implications for our understanding of ligase mechanism and inhibition, and may provide a promising starting point for the rational design of a new class of inhibitors against NAD(+)-dependent DNA ligases.

The synthesis of a series of cyclometalated gold(III) complexes supported by pyrazine-based (C^N^C)-type pincer ligands is reported, including the crystal structure of a cationic example. The compounds provide a new platform for the study of antiproliferative properties of gold(III) complexes. Seven complexes were tested: the neutral series (C^Npz^C)AuX [X = Cl (1), 6-thioguanine (4), C≡CPh (5), SPh (6)] and an ionic series that included the N-methyl complex [(C^NpzMe^C)AuCl]BF4 (7) and the N-heterocyclic carbene complexes [(C^Npz^C)AuL]+ with L = 1,3-dimethylbenzimidazol-2-ylidene (2) or 1,3,7,9-tetramethylxanthin-8-ylidene (3). Tests against human leukemia cells identified 1, 2, 3, and 4 as particularly promising, whereas protecting the noncoordinated N atom on the pyrazine ring by methylation (as in 7) reduced the cytotoxicity. Complex 2 proved to be the most effective of the entire series against the HL60 leukemia, MCF-7 breast cancer, and A549 lung cancer cell lines, with IC50 values down to submicromolar levels, associated with a lower toxicity toward healthy human lung fibroblast cells. The benzimidazolylidene complex 2 accumulated more effectively in human lung cancer cells than its caffeine-based analogue 3 and the gold(III) chloride 1. Compound 2 proved to be unaffected by glutathione under physiological conditions for periods of up to 6 days and stabilizes the DNA G-quadruplex and i-motif structures; the latter is the first such report for gold compounds. We also show the first evidence of inhibition of MDM2–p53 protein–protein interactions by a gold-based compound and identified the binding mode of the compound with MDM2 using saturation transfer difference NMR spectroscopy combined with docking calculations., We synthesized three new (C^Npz^C)Au(III) complexes and screened them along with four other complexes as potential anticancer agents against leukemia cells. We tested the cellular uptake, the interaction with G4 and i-motif DNA structures, and the interaction with MDM2 protein. We highlight the very different biological behaviors of the compounds due to the different ancillary ligands.

A new family of multivalent ligand platforms, the open-resorcinarenes, has been prepared in a straightforward two-step reaction. Modification of the core gives a range of topologically diverse scaffolds; functionalisation confirms the versatility of this approach, as shown through the formation of an octacalixarene array.

Previous studies on the natural product chlorofusin have shown that the full peptide and azaphilone structure are required for inhibition of the interaction between MDM2 and p53. In the current work, we utilized the cyclic peptide as a template and introduced an azidonorvaline amino acid in place of the ornithine/azaphilone of the natural product and carried out click chemistry with the resulting peptide. From this small library the first ever non-azaphilone containing chlorofusin analog with MDM2/p53 activity was identified. Further studies then suggested that the simple structure of the Fmoc-norvaline amino acid that had undergone a click reaction was also able to inhibit MDM2/p53 interaction. This is an example where studies of a natural product have led to the serendipitous identification of a new small molecule inhibitor of a protein–protein interaction.