Stapled α−helical peptides have emerged as a promising new modality for a wide range of therapeutic targets. Here, we report a potent and selective dual inhibitor of MDM2 and MDMX, ATSP-7041, which effectively activates the p53 pathway in tumors in vitro and in vivo. Specifically, ATSP-7041 binds both MDM2 and MDMX with nanomolar affinities, shows submicromolar cellular activities in cancer cell lines in the presence of serum, and demonstrates highly specific, on-target mechanism of action. A high resolution (1.7-Å) X-ray crystal structure reveals its molecular interactions with the target protein MDMX, including multiple contacts with key amino acids as well as a role for the hydrocarbon staple itself in target engagement. Most importantly, ATSP-7041 demonstrates robust p53-dependent tumor growth suppression in MDM2/MDMX-overexpressing xenograft cancer models, with a high correlation to on-target pharmacodynamic activity, and possesses favorable pharmacokinetic and tissue distribution properties. Overall, ATSP-7041 demonstrates in vitro and in vivo proofof-concept that stapled peptides can be developed as therapeutically relevant inhibitors of protein-protein interaction and may offer a viable modality for cancer therapy.T he human transcription factor protein p53 induces cell-cycle arrest and apoptosis in response to DNA damage and cellular stress and thereby plays a critical role in protecting cells from malignant transformation (1, 2). Inactivation of this guardian of the genome either by deletion or mutation or through overexpression of inhibitory proteins is the most common defect in human cancers (1, 2). Cancers that overexpress the inhibitory proteins MDM2 and MDMX also possess wild-type p53 (p53WT), and thus pharmacological disruption of the interactions between p53 and MDM2 and MDMX offers the opportunity to restore p53-dependent cell-cycle arrest and apoptosis in this important class of tumors (3-6).MDM2 negatively regulates p53 function through multiple mechanisms, including direct binding that masks the p53 transactivation domain, impairing nuclear import of the p53 protein, and ubiquitination and proteasomal degradation of the p53 protein (6, 7). Consequently, aberrant MDM2 overexpression and gene amplification contribute to accelerated cancer development and growth (1, 8). The other negative regulator, MDMX, possesses a similar p53-binding activity and also effectively inhibits p53 transcriptional activity. Amplification of MDMX is seen in many tumors, including melanoma, breast, head and neck, hepatocellular, and retinoblastoma, and, interestingly, amplification of MDMX appears to correlate with both p53WT status and an absence of MDM2 amplification (6, 9, 10). MDMX does not have the intrinsic E3 ubiquitin ligase activity of MDM2 and cannot affect p53 stability, but MDM2/MDMX heterodimers can increase ubiquitin ligase activity relative to the MDM2 monomer. Given these functional differences, MDM2 and MDMX are each unable to compensate for the loss of the other, and they regulate nonoverlapping fu...

The prominent symptoms associated with central demyelinating diseases such as multiple sclerosis (MS) are primarily caused by conduction deficits in affected axons. The symptoms may go into remission, but the mechanisms underlying remissions are uncertain. One factor that could be important is the restoration of conduction to affected axons, but it is not known whether demyelinated central axons resemble their peripheral counterparts in being able to conduct in the absence of repair by remyelination. In the present study we have made intraaxonal recordings from central axons affected by a demyelinating lesion, and then the axons have been labeled ionophoretically to permit their subsequent identification. Ultrastructural examination of 23 labeled preparations has established that some segmentally demyelinated central axons can conduct, and that they can do so over continuous lengths of demyelination exceeding several internodes (2500 m). Such segmentally demyelinated central axons were found to conduct with the anticipated reduction in velocity and a refractory period of transmission (RPT) as much as 34 times the value obtained from the nondemyelinated portion of the same axon; the RPT was typically prolonged to 2-5 times the normal value. We conclude that some segmentally demyelinated central axons can conduct, and we propose that the restoration of conduction to such axons is likely to contribute to the remissions commonly observed in diseases such as MS. Key words: demyelination; multiple sclerosis; axon; conduction properties; glia; ionophoresisMultiple sclerosis (MS) is a disease of the C NS in which affected central axons often lose one or more internodes of their myelin sheath; the continuity of the axon through the lesion is frequently maintained, although degeneration becomes more prominent as the disease progresses (McDonald, 1994). The disease is associated with symptoms such as paralysis, blindness, and numbness, which can be explained by conduction block in the relevant pathways. Such symptoms may spontaneously go into remission; however, the mechanisms underlying such remissions are not well understood. It is now clear that some demyelinated lesions are partially repaired by remyelination and that this phenomenon can be quite common and extensive in early lesions (Prineas et al., 1993a). Remyelination is known to restore secure conduction to central (Smith et al., 1979(Smith et al., , 1981Blight and Young, 1989;Felts and Smith, 1992;Honmou et al., 1996) and peripheral (Saida et al., 1980;Smith and Hall, 1980;Sedal et al., 1983;Shrager, 1988) demyelinated axons, and it is reasonable to believe that conduction in remyelinated central axons will contribute to remissions; however, even where remyelination occurs in MS, it may be temporary (Prineas et al., 1993b), and persistently demyelinated lesions are a common feature of the disease. It is known that some of these persistently demyelinated lesions are clinically silent, i.e., they do not produce symptoms (Ghatak et al., 1974;Phadke and Best, 1983; f...

Both CT and MRI are superior to DR for detection of subchondral bone pathology, but underestimate the extent of joint adaptation and pathologic damage. MRI was able to detect cartilage degeneration.

Androgen deprivation, a consequence of hypogonadism, certain cancer treatments, or normal aging in men, leads to loss of muscle mass, increased adiposity, and osteoporosis. In the present study, using a soluble chimeric form of activin receptor type IIB (ActRIIB) we sought to offset the adverse effects of androgen deprivation on muscle, adipose tissue, and bone. Castrated (ORX) or sham-operated (SHAM) mice received either TBS [vehicle-treated (VEH)] or systemic administration of ActRIIB-mFc, a soluble fusion protein comprised of a form of the extracellular domain of ActRIIB fused to a murine IgG2aFc subunit. In vivo body composition imaging demonstrated that ActRIIB-mFc treatment results in increased lean tissue mass of 23% in SHAM mice [19.02 +/- 0.42 g (VEH) versus 23.43 +/- 0.35 g (ActRIIB-mFc), P < 0.00001] and 26% in ORX mice [15.59 +/- 0.26 g (VEH) versus 19.78 +/- 0.26 g (ActRIIB-mFc), P < 0.00001]. Treatment also caused a decrease in adiposity of 30% in SHAM mice [5.03 +/- 0.48 g (VEH) versus 3.53 +/- 0.19 g (ActRIIB-mFc), NS] and 36% in ORX mice [7.12 +/- 0.53 g (VEH) versus 4.57 +/- 0.28 g (ActRIIB-mFc), P < 0.001]. These changes were also accompanied by altered serum levels of leptin, adiponectin, and insulin, as well as by prevention of steatosis (fatty liver) in ActRIIB-mFc-treated ORX mice. Finally, ActRIIB-mFc prevented loss of bone mass in ORX mice as assessed by whole body dual x-ray absorptiometry and micro-computed tomography of proximal tibias. The data demonstrate that treatment with ActRIIB-mFc restored muscle mass, adiposity, and bone quality to normal levels in a mouse model of androgen deprivation, thereby alleviating multiple adverse consequences of such therapy.

Background Increased IL-17A production has been associated with more severe asthma, however the mechanisms whereby IL-17A may contribute to IL-13-driven pathology in asthma remain unclear. Objective We sought to gain mechanistic insight into how IL-17A can influence IL-13-driven responses. Methods The effect of IL-17A on IL-13-induced airway hyperresponsiveness (AHR), gene expression, mucus hypersecretion, and airway inflammation was assessed using in vivo models of IL-13-induced lung pathology and in vitro culture of murine fibroblast cell lines and primary fibroblasts, and human epithelial cell lines or primary human epithelial cells exposed to IL-13, IL-17A, or IL-13 and IL-17A. Results Compared to mice given intratracheal IL-13 alone, those exposed to IL-13 and IL-17A displayed augmented AHR, mucus production, airway inflammation and IL-13-induced gene expression. In vitro, IL-17A enhanced IL-13-induced gene expression in asthma-relevant murine and human cells. In contrast to the exacerbating influence of IL-17A on IL-13-induced responses, co-exposure to IL-13 inhibited IL-17A-driven antimicrobial gene expression in vivo and in vitro. Mechanistically, in both primary human and murine cells, IL-17A-driven elevation of IL-13-induced gene expression was associated with enhanced IL-13-driven STAT6 activation. Conclusions Our data suggest that IL-17A contributes to asthma pathophysiology by increasing the capacity of IL-13 to activate intracellular signaling pathways such as STAT6. These data represent the first mechanistic explanation of how IL-17A may directly contribute to the pathogenesis of IL-13-driven pathology.