Summary Non-small cell lung carcinoma (NSCLC) is the leading cause of cancer-related death worldwide, with an overall 5-year survival rate of only 10–15% 1. Deregulation of the Ras pathway is a frequent hallmark of NSCLC, often through mutations that directly activate Kras 2. p53 is also frequently inactivated in NSCLC and, since oncogenic Ras can be a potent trigger of p53 3, it seems likely that oncogenic Ras signalling plays a major and persistent part in driving the selection against p53. Hence, pharmacological restoration of p53 is an appealing therapeutic strategy for treating this disease 4. Here, we model the likely therapeutic impact of p53 restoration in a spontaneously evolving mouse model of NSCLC initiated by sporadic oncogenic activation of endogenous Kras 5. Surprisingly, p53 restoration failed to induce significant regression of established tumours although it did result in a significant decrease in the relative proportion of tumours classed as high grade. This is due to selective activation of p53 only in the more aggressive tumour cells within each tumour. Such selective activation of p53 correlates with marked up regulation in Ras signal intensity and induction of the oncogenic signalling sensor p19ARF 6. Our data indicate that p53-mediated tumour suppression is triggered only when oncogenic Ras signal flux exceeds a critical threshold. Importantly, the failure of low-level oncogenic Kras to engage p53 reveals inherent limits in the capacity of p53 to restrain early tumour evolution and to the efficacy of therapeutic p53 restoration to eradicate cancers.

The synthesis and structural characterization of a new pro-chelating agent, isonicotinic acid [2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzylidene]-hydrazide (BSIH), are presented. BSIH only weakly interacts with iron unless hydrogen peroxide (H2O2) is present to remove the boronic ester protecting group to reveal a phenol that is a key metal-binding group of tridentate salicylaldehyde isonicotinoyl hydrazone (SIH). BSIH prevents deoxyribose degradation caused by hydroxyl radicals that are generated from H2O2 and redox-active iron by sequestering Fe3+ and preventing iron-promoted hydroxyl radical formation. The rate-determining step for iron sequestration is conversion of BSIH to SIH, followed by rapid Fe3+ complexation. The pro-chelate approach of BSIH represents a promising strategy for chelating a specific pool of detrimental metal ions without disturbing healthy metal ion distribution.

Manipulation of weak intermolecular interactions guides the rational design [1] of sensors, drugs, and foldamers-synthetic, [2] nonnatural backbones that fold into an ordered, biomimetic array. 1,4-Substituted 1,2,3-triazoles, which are readily accessible through the Cu I -catalyzed Huisgen 1,3-dipolar cycloaddition of azides and alkynes, [3] are seemingly universal ligation tools [4] whose capacity for independent function has received far less attention. Recent reports, however, indicate that the size and dipole moment ( % 5 D) of triazoles make them interesting candidates for amide bond surrogates, [5] and Arora and co-workers have reported the contributions of triazoles to the conformational preferences of peptidotriazole oligomers. [6] We hypothesized that oligomer 1 would fold in a manner similar to other linear, flexible oligomers [7] to provide a model cavity by which to explore the intermolecular interactions between the electropositive CH side of 1,4-triazoles and electron-rich guests such as anions ( Figure 1). Our expectations were buoyed by previous reports of anion-induced folding, [8,9] in particular by a recent demonstration by Jeong and co-workers [8] that the folding of oligoindoles can be directed through a helical arrangement of NH···anion hydrogen bonds. Herein, we report 1) that 1:1 interactions between diaryl triazoles and chloride ions in acetone are directional and sufficiently strong as to be observable by 1 H NMR spectroscopy, 2) that the strength of the interaction increases with the generation of triazole-containing oligomer, and 3) that CH···anion contacts guide the folding of aryl triazole oligomers in solution and in the solid state.While the "click" coupling of alkyl azides with alkynes is highly efficient, [10] the formation of diaryl triazoles has, until recently, been relatively more difficult and less efficient. [11] Nonetheless, under modified conditions, the Cu I -catalyzed cycloaddition produces acceptable yields of the desired 1,4-diaryl-1,2,3-triazole-containing compounds 1-3. A tetraethylene glycol unit was introduced outside of the cavity for solubility.Oligomer 1 has appreciable conformational freedom only around the arene-triazole single bonds. Molecular modeling [12] suggested no significant preference for a particular rotamer, a prediction that is supported by NOESY spectra of 1 in [D 6 ]acetone (Figure 2 a). Modeling studies also show that complexation of 1 with Cl À aligns the electropositive triazole CH units toward the interior of a helix, within which the Cl À is bound.The computer modeling holds true in solution, where the chloride-induced folding of 1 is revealed by 1 H NMR spectroscopy. The 1 H NMR spectrum of 1 changes considerably upon the addition of tetrabutylammonium chloride

Background & Aims Although proteases control inflammation and pain, the identity, cellular origin, mechanism of action, and causative role of proteases that are activated during disease are not defined. We investigated the activation and function of cysteine cathepsins (Cat) in colitis. Methods Since protease activity, rather than expression, is regulated, we treated mice with fluorescent activity-based probes that covalently modify activated cathepsins. Activated proteases were localized by tomographic imaging of intact mice and confocal imaging of tissues, and were identified by electrophoresis and immunoprecipitation. We examined the effects of activated cathepsins on excitability of colonic nociceptors and on colonic pain, and determined their role in colonic inflammatory pain by gene deletion. Results Tomography and magnetic resonance imaging localized activated cathepsins to the inflamed colon of piroxicam-treated il10−/− mice. Confocal imaging detected activated cathepsins in colonic macrophages and spinal neurons and microglial cells of mice with colitis. Gel electrophoresis and immunoprecipitation identified activated Cat-B, Cat-L and Cat-S in colon and spinal cord, and Cat-S was preferentially secreted into the colonic lumen. Intraluminal Cat-S amplified visceromotor responses to colorectal distension and induced hyperexcitability of colonic nociceptors, which required expression of protease-activated receptor-2. Cat-S deletion attenuated colonic inflammatory pain induced with trinitrobenzene sulfonic acid. Conclusions Activity-based probes enable non-invasive detection, cellular localization and proteomic identification of proteases activated during colitis and are potential diagnostic tools for detection of predictive disease biomarkers. Macrophage cathepsins are activated during colitis, and Cat-S activates nociceptors to induce visceral pain via protease-activated receptor-2. Cat-S mediates colitis pain and is a potential therapeutic target.

Prostate-specific membrane antigen (PSMA) is a transmembrane protein commonly found on the surface of late-stage and metastatic prostate cancer and a well-known imaging biomarker for staging and monitoring therapy. Although 111In-labeled caprop-mab pendetide is the only approved agent available for PSMA imaging, its clinical use is limited because of its slow distribution and clearance that leads to challenging image interpretation. A small-molecule approach using radiolabeled urea-based PSMA inhibitors as imaging agents has shown promise for prostate cancer imaging. The motivation of this work is to explore phosphoramidates as a new class of potent PSMA inhibitors to develop more effective prostate cancer imaging agents with improved specificity and clearance properties. Methods N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) was conjugated to S-2-((2-(S-4-amino-4-carboxybutanamido)-S-2-carboxyethoxy)-hydroxyphosphorylamino)-pentanedioic acid (Phosphoramidate (1)), yielding S-2-((2-(S-4-(4-18F-fluorobenzamido)-4-carboxybutanamido)-S-2-carboxyethoxy)hydroxyphosphorylamino)-pentanedioic acid (3). In vivo studies were conducted in mice bearing either LNCaP (PSMA-positive) or PC-3 (PSMA-negative) tumors. PET images were acquired at 1 and 2 h with or without a preinjection of a nonradioactive version of the fluorophosphoramidate. Tissue distribution studies were performed at the end of the 2 h imaging sessions. Results Phosphoramidate (1) and its fluorobenzamido conjugate (2) were potent inhibitors of PSMA (inhibitory concentration of 50% [IC50], 14 and 0.68 nM, respectively). PSMA-mediated tumor accumulation was noted in the LNCaP versus the PC-3 tumor xenografts. The LNCaP tumor uptake was also blocked by the administration of nonradioactive (2) prior to imaging studies. With the exception of the kidneys, tumor-to-tissue and tumor-to-blood ratios were greater than 5:1 at 2 h. The strong kidney uptake may be due to the known PSMA expression in the mouse kidney, because significant reduction (>6-fold) in kidney activity was seen in mice injected with (2). Conclusion 18F-labeled phosphoramidate (3) is a representative of a new class of PSMA targeting peptidomimetic molecules that shows great promise as imaging agents for detecting PSMA+ prostate tumors.

Several new analogs of salicylaldehyde isonicotinoyl hydrazone (SIH) and salicylaldehyde benzoyl hydrazone (SBH) that contain an aryl boronic ester (BSIH, BSBH) or acid (BASIH) in place of an aryl hydroxide have been synthesized and characterized as masked metal ion chelators. These pro-chelators show negligible interaction with iron(III), although the boronic acid versions exhibit some interaction with copper(II), zinc(II) and nickel(II). Hydrogen peroxide oxidizes the aryl boronate to phenol, thus converting the pro-chelators to tridentate ligands with high affinity metal binding properties. An X-ray crystal structure of a bis-ligated iron(III) complex, [Fe(SBH(m-OMe)(3))(2)]NO(3), confirms the meridonal binding mode of these ligands. Modifications of the aroyl ring of the chelators tune their iron affinity, whereas modifications on the boron-containing ring of the pro-chelators attenuate their reaction rates with hydrogen peroxide. Thus, the methoxy derivative pro-chelator (p-OMe)BASIH reacts with hydrogen peroxide nearly 5 times faster than the chloro derivative (m-Cl)BASIH. Both the rate of pro-chelator to chelator conversion as well as the metal binding affinity of the chelator influence the overall ability of these molecules to inhibit hydroxyl radical formation catalyzed by iron or copper in the presence of hydrogen peroxide and ascorbic acid. This pro-chelator strategy has the potential to improve the efficacy of medicinal chelators for inhibiting metal-promoted oxidative stress.

Crystals of C 60 ؒPt II (OEP)ؒ2(C 6 H 6 ), TCNQؒCu II (OEP), TCNQؒH 2 (OEP), TCNQؒ2Cu II (OEP), TCNQؒ2Zn II (OEP) and TNFMؒCo II (OEP) [OEP is the dianion of octaethylporphyrin, TCNQ is 7,7,8,8-tetracyanoquinodimethane, TNFM is (2,4,7-trinitrofluorenylidene)malonitrile] have been obtained by diffusion of a solution of the porphyrin as donor into a solution of the respective acceptor molecule. The structure of C 60 ؒPt II (OEP)ؒ2(C 6 H 6 ) consists of an ordered C 60 cage nestled against the platinum porphyrin which makes close face-to-face contact with another Pt II (OEP) molecule. In contrast, there are no close face-to-face contacts between porphyrins in the crystal structures of TCNQؒCu II (OEP), TCNQؒH 2 (OEP), and TNFMؒCo II (OEP). These compounds consist of classical donoracceptor stacks of interleaved porphyrin and TCNQ or TNFM molecules with separations of ca. 3.3 Å between adjacent molecules. However with TCNQؒ2Cu II (OEP) and TCNQؒ2Zn II (OEP) the structures involve TCNQ (A) and M II (OEP) (D) molecules that crystallize in stacks with a DDA(DDA) n DDA arrangement. Within these stacks there are pairwise contacts between M II (OEP) molecules and these pairs are compared to those found in C 60 ؒPt II (OEP)ؒ2(C 6 H 6 ) and related fullerene-containing crystals. ResultsCo-crystallizations of M II (OEP) or H 2 OEP with TCNQ, TNFM, and C 60 were performed by layering saturated solutions

Crystallographic examination of [mu3-S(AuCNC7H13)3](SbF6) shows that it undergoes a reversible phase change from orthorhombic to monoclinic upon cooling. At 190 K, the structure shows that two cations self-associate to form a pseudo-octahedral array of six gold atoms connected by both intra- and interionic aurophilic interactions. On cooling, the clusters become less symmetric, and in one, the interionic Au...Au separations increase, while they decrease in the second cluster. The luminescence of crystalline [mu3-S(AuCNC7H13)3](SbF6) shows corresponding changes in emission, with two emissions of similar lifetimes but with different excitations at 77 K, but only a single emission at 298 K. In contrast, [mu3-S(AuCNC6H11)3](PF6), which has a similar structure to that of the high-temperature form of [mu3-S(AuCNC7H13)3](SbF6), does not undergo a phase change or change in its luminescence upon cooling.