Design and Synthesis of Covalent Inhibitors of FabA.

There is an urgent need for the development of new therapeutics with novel modes of action to target Gram-negative bacterial infections, due to resistance to current drugs. Previously, FabA, an enzyme in the bacterial type II fatty acid biosynthesis pathway, was identified as a potential drug target in Pseudomonas aeruginosa, a Gram-negative bacteria of significant clinical concern. A chemical starting point was also identified. There is a cysteine, Cys15, in the active site of FabA, adjacent to where this compound binds. This paper describes the preparation of analogues containing an electrophilic warhead with the aim of covalent inhibition of the target. A wide variety of analogues were successfully prepared. Unfortunately, these analogues did not increase inhibition, which may be due to a loop within the enzyme partially occluding access to the cysteine.

Synthesis of a Series of Diaminoindoles.

A selection of 3,4-diaminoindoles were required for a recent drug discovery project. To this end, a 10-step synthesis was developed from 4-nitroindole. This synthesis was subsequently adapted and used to synthesize 3,5-; 3,6-; and 3,7-diaminoindoles from the corresponding 5-, 6-, or 7-nitroindole. These novel intermediates feature orthogonal protecting groups that allow them to be further diversified. This is the first reported synthesis of these types of compounds.

Characterising covalent warhead reactivity.

Many drugs currently used are covalent inhibitors and irreversibly inhibit their targets. Most of these were discovered through serendipity. Covalent inhibitions can have many advantages from a pharmacokinetic perspective. However, until recently most organisations have shied away from covalent compound design due to fears of non-specific inhibition of off-target proteins leading to toxicity risks. However, there has been a renewed interest in covalent modifiers as potential drugs, as it possible to get highly selective compounds. It is therefore important to know how reactive a warhead is and to be able to select the least reactive warhead possible to avoid toxicity. A robust NMR based assay was developed and used to measure the reactivity of a variety of covalent warheads against serine and cysteine - the two most common targets for covalent drugs. A selection of these warheads also had their reactivity measured against threonine, tyrosine, lysine, histidine and arginine to better understand our ability to target non-traditional residues. The reactivity was also measured at various pHs to assess what effect the environment in the active site would have on these reactions. The reactivity of a covalent modifier was found to be very dependent on the amino acid residue.

Author Correction: Loss of Phd2 cooperates with BRAFV600E to drive melanomagenesis.

The original version of this Article contained an error in the spelling of the author Brett L. Ecker, which was incorrectly given as Brett Ecker. This has now been corrected in both the PDF and HTML versions of the Article.

Loss of Phd2 cooperates with BRAFV600E to drive melanomagenesis.

Prolyl hydroxylase domain protein 2 (PHD2) is a well-known master oxygen sensor. However, the role of PHD2 in tumor initiation remains controversial. We find that during the transition of human nevi to melanoma, the expression of PHD2 protein is significantly decreased and lower expression PHD2 in melanoma is associated with worse clinical outcome. Knockdown of PHD2 leads to elevated Akt phosphorylation in human melanocytes. Mice with conditional melanocyte-specific expression of Phd2lox/lox (Tyr::CreER;Phd2lox/lox) fail to develop pigmented lesions. However, deletion of Phd2 in combination with expression of BRafV600E in melanocytes (Tyr::CreER;Phd2lox/lox;BRafCA) leads to the development of melanoma with 100% penetrance and frequent lymph node metastasis. Analysis of tumor tissues using reverse phase protein arrays demonstrates that Phd2 deletion activates the AKT-mTOR-S6 signaling axis in the recovered tumors. These data indicate that PHD2 is capable of suppressing tumor initiation largely mediated through inhibiting of the Akt-mTOR signaling pathway in the melanocyte lineage.

Hypoxia-activated prodrug enhances therapeutic effect of sunitinib in melanoma.

Angiogenesis is a critical step during tumor progression. Anti-angiogenic therapy has only provided modest benefits in delaying tumor progression despite its early promise in cancer treatment. It has been postulated that anti-angiogenic therapy may promote the emergence of a more aggressive cancer cell phenotype by generating increased tumor hypoxia-a well-recognized promoter of tumor progression. TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug (HAP) which has been shown to selectively target the hypoxic tumor compartment and reduce tumor volume. Here, we show that melanoma cells grown under hypoxic conditions exhibit increased resistance to a wide variety of therapeutic agents in vitro and generate larger and more aggressive tumors in vivo than melanoma cells grown under normoxic conditions. However, hypoxic melanoma cells exhibit a pronounced sensitivity to TH-302 which is further enhanced by the addition of sunitinib. Short term sunitinib treatment fails to prolong the survival of melanoma bearing genetically engineered mice (Tyr::CreER; BRafCA;Ptenlox/lox ) but increases tumor hypoxia. Long term TH-302 alone modestly prolongs the overall survival of melanoma bearing mice. Combination therapy of TH-302 with sunitinib further increases the survival of treated mice. These studies provide a translational rationale for combining hypoxic tumor cell targeted therapies with anti-angiogenics for treatment of melanoma.

Everyday sitting behavior of full-time wheelchair users.

The objective of this study was to describe the in-seat movement and weight-shifting behavior of full-time wheelchair users. We measured everyday sitting behavior for 192 d across 28 individuals who used manual wheelchairs as their primary mobility device. To obtain the measurements, we used eight thin force sensors placed under participants' wheelchair cushions. On a typical day, participants spent an average of 10.6 +/- 3.0 h in their wheelchair and transferred out of the wheelchair 8.4 +/- 4.3 times. Participants only performed pressure reliefs (90% off-loading of the entire buttocks for at least 15 s) 0.4 +/- 0.5 times per hour they were seated in the chair, but they performed weight shifts (WSs) (30%-90% off-loading of at least one side of the buttocks for 15 s) with a frequency of 2.4 +/- 2.2 times per hour. Despite the higher frequency of WSs, they were not performed in a routine manner. Half of the days studied included one segment of upright sitting lasting at least 2 h without a WS. Given these observations, we conclude that seating evaluations should emphasize positioning individuals in a way that facilitates reaching, leaning, and transferring in a safe manner, not only to improve function but also to affect buttocks loading.

Measurement of Load Redistribution Properties of Wheelchair Cushions Using a Compliant Cushion Loading Indenter.

The aim of this project was to develop and validate a compliant cushion loading indenter (CCLI) capable of evaluating wheelchair cushion performance by measuring internal pressures and deflection. The design of the CCLI consists of 3 subsystems: 1) an internal substructure with medial and lateral protuberances to mimic the load-bearing ischial tuberosities and trochanters, 2) an elastomeric shell to mimic soft tissue and 3) instrumentation to measure internal pressures at both protuberances and deflection of the elastomer at 7 locations. It is parametrically designed so can be scaled larger or smaller to represent different body sizes. To assess the repeatability and sensitivity of measurements, the model was loaded onto two wheelchair cushions, 3″ flat foam and Jay3, using two loads, 44kgf and 53kgf, representing the average upper body mass of 70kg and 83kg persons, respectively. The results showed a high precision of pressure and deflection measurement across two different cushions and loads. Under both loads, pressure measurements exhibited a standard error of < 1 mm and <3 mmHg. The standard deviations of deflection values were less than 2.5 mm (0.1 in.). The pressures and absolute deflection differed significantly across load and cushion type indicating sensitivity to change.

Norcantharidin induces melanoma cell apoptosis through activation of TR3 dependent pathway.

Norcantharidin (NCTD) has been reported to induce tumor cell apoptosis. However, the underlying mechanism behinds its antitumor effect remains elusive. We have previously shown that TR3 expression is significantly decreased in metastatic melanomas and involved in melanoma cell apoptosis. In this study, we showed that NCTD inhibited melanoma cell proliferation and induced apoptosis in a dose related manner. NCTD induced translocation of TR3 from nucleus to mitochondria where it co-localized with Bcl-2 in melanoma cells. NCTD also increased cytochome c release from mitochondria to the cytoplasm. These changes were accompanied by increased expression of Bax and cleaved caspase-3 along with decreased expression of Bcl2 and NF-κB2. The effects of NCTD were inhibited by knockdown of TR3 expression using TR3 specific shRNA in melanoma cells. Furthermore, NCTD significantly decreased tumor volume and improved survival of Tyr::CreER; BRAF(Ca/+); Pten(lox/lox) transgenic mice. Our data indicates that NCTD inhibits melanoma growth by inducing tumor cell apoptosis via activation of a TR3 dependent pathway. These results suggest that NCTD is a potential therapeutic agent for melanoma.

Snail1 mediates hypoxia-induced melanoma progression.

Tumor hypoxia is a known adverse prognostic factor, and the hypoxic dermal microenvironment participates in melanomagenesis. High levels of hypoxia inducible factor (HIF) expression in melanoma cells, particularly HIF-2α, is associated with poor prognosis. The mechanism underlying the effect of hypoxia on melanoma progression, however, is not fully understood. We report evidence that the effects of hypoxia on melanoma cells are mediated through activation of Snail1. Hypoxia increased melanoma cell migration and drug resistance, and these changes were accompanied by increased Snail1 and decreased E-cadherin expression. Snail1 expression was regulated by HIF-2α in melanoma. Snail1 overexpression led to more aggressive tumor phenotypes and features associated with stem-like melanoma cells in vitro and increased metastatic capacity in vivo. In addition, we found that knockdown of endogenous Snail1 reduced melanoma proliferation and migratory capacity. Snail1 knockdown also prevented melanoma metastasis in vivo. In summary, hypoxia up-regulates Snail1 expression and leads to increased metastatic capacity and drug resistance in melanoma cells. Our findings support that the effects of hypoxia on melanoma are mediated through Snail1 gene activation and suggest that Snail1 is a potential therapeutic target for the treatment of melanoma.

Investigation of microphones as near-ground sensors for seismic detection of buried landmines.

Commercially available microphones were investigated as near-ground sensors to measure the acoustic pressure and the vertical pressure gradient of evanescent air-acoustic waves associated with audio-frequency seismic waves. Measurements in close proximity to the surface and the use of waveguides were found to improve the microphone signal's quality, the comparison of its seismic sensitivity to its sensitivity to propagating sound (ambient acoustic noise and nonseismic reverberation). Landmine images formed using microphone data collected in a laboratory experimental model clearly locate buried inert landmines but exhibit more clutter than images of the same objects formed with seismic displacement data collected using other techniques.

Special issues and concerns for the high school- and college-aged athletes.

Increasing numbers of high school- and college-aged students are participating in sports. As sport participation and intensity increases, the frequency of associated heat related illnesses and acute and chronic overuse injuries will continue to become more prevalent. The sports medicine physician plays an essential role not only in the practice and treatment of injuries but also in educating athletes about safe and healthy exercise habits that will provide life long benefits.