Macrocyclic Oxindole Peptide Epoxyketones-A Comparative Study of Macrocyclic Inhibitors of the 20S Proteasome.

Peptide macrocycles have recently gained attention as protease inhibitors due to their metabolic stability and specificity. However, the development of peptide macrocycles with improved binding potency has so far been challenging. Here we present macrocyclic peptides derived from the clinically applied proteasome inhibitor carfilzomib with an oxindole group that mimics the natural product TMC-95A. Fluorescence kinetic activity assays reveal a high potency of the oxindole group (IC50 = 0.19 µM) compared with agents lacking this motif. X-ray structures of the ligands with the ß5-subunit of the yeast 20S proteasome illustrate that the installed macrocycle forces strong hydrogen bonding of the oxindole group with ß5-Gly23NH. Thus, the binding of our designed oxindole epoxyketones is entropically and enthalpically favored in contrast to more flexible proteasome inhibitors such as carfilzomib.

Enhancing Alkyne-Based Raman Tags with a Sulfur Linker.

Alkyne-based Raman tags have proven their utility for biological imaging. Although the alkynyl stretching mode is a relatively strong Raman scatterer, the detection sensitivity of alkyne-tagged compounds is ultimately limited by the magnitude of the probe's Raman response. In order to improve the performance of alkyne-based Raman probes, we have designed several tags that benefit from π-π conjugation as well as from additional n-π conjugation with a sulfur linker. We show that the sulfur linker provides additional enhancement and line width narrowing, offering a simple yet effective strategy for improving alkyne-based Raman tags. We validate the utility of various sulfur-linked alkyne tags for cellular imaging through stimulated Raman scattering microscopy.

Fluorogenic Cyclopropenones for Multicomponent, Real-Time Imaging.

Fluorogenic bioorthogonal reactions enable biomolecule visualization in real time. These reactions comprise reporters that "light up" upon reaction with complementary partners. While the spectrum of fluorogenic chemistries is expanding, few transformations are compatible with live cells due to cross-reactivities or insufficient signal turn-on. To address the need for more suitable chemistries for cellular imaging, we developed a fluorogenic reaction featuring cyclopropenone reporters and phosphines. The transformation involves regioselective activation and cyclization of cyclopropenones to form coumarin products. With optimal probes, the reaction provides >1600-fold signal turn-on, one of the highest fluorescence enhancements reported to date. The bioorthogonal motifs were evaluated in vitro and in cells. The reaction was also found to be compatible with other common fluorogenic transformations, enabling multicomponent, real-time imaging. Collectively, these data suggest that the cyclopropenone-phosphine reaction will bolster efforts to track biomolecule targets in their native settings.

Bioorthogonal Reactions of Triarylphosphines and Related Analogues.

Bioorthogonal phosphines were introduced in the context of the Staudinger ligation over 20 years ago. Since that time, phosphine probes have been used in myriad applications to tag azide-functionalized biomolecules. The Staudinger ligation also paved the way for the development of other phosphorus-based chemistries, many of which are widely employed in biological experiments. Several reviews have highlighted early achievements in the design and application of bioorthogonal phosphines. This review summarizes more recent advances in the field. We discuss innovations in classic Staudinger-like transformations that have enabled new biological pursuits. We also highlight relative newcomers to the bioorthogonal stage, including the cyclopropenone-phosphine ligation and the phospha-Michael reaction. The review concludes with chemoselective reactions involving phosphite and phosphonite ligations. For each transformation, we describe the overall mechanism and scope. We also showcase efforts to fine-tune the reagents for specific functions. We further describe recent applications of the chemistries in biological settings. Collectively, these examples underscore the versatility and breadth of bioorthogonal phosphine reagents.

Butenolide Synthesis from Functionalized Cyclopropenones.

A general method to synthesize substituted butenolides from hydroxymethylcyclopropenones is reported. Functionalized cyclopropenones undergo ring-opening reactions with catalytic amounts of phosphine, forming reactive ketene ylides. These intermediates can be trapped by pendant hydroxy groups to afford target butenolide scaffolds. The reaction proceeds efficiently in diverse solvents and with low catalyst loadings. Importantly, the cyclization is tolerant of a broad range of functional groups, yielding a variety of α- and γ-substituted butenolides.

Reliability of preoperative diagnostics and location of lymph node metastases in presumed unilateral prostate cancer.

OBJECTIVE: To investigate the reliability of preoperative diagnostics in predicting the true histopathological stage and grade of prostate cancer, and to examine whether lymph node (LN) metastases in unilateral prostate cancer are located unilaterally and therefore whether it is justified to dissect only the ipsilateral LNs in presumed unilateral disease. PATIENTS AND METHODS: LN metastases in clinically localized prostate cancer are often located near the internal iliac vessels. They will be detected by extended or sentinel pelvic LN dissection (PLND). Both techniques might be time-consuming and require extensive surgical experience. In all, 564 men with impalpable or unilateral palpable prostate cancer and positive biopsy cores only in one prostate lobe had a radical prostatectomy (RP) combined with radio-guided PLND and in some cases an extended PLND. RESULTS: A median of six sentinel LNs (mean, seven) and six non-sentinel LNs (mean, seven) were dissected per patient; 52 of 564 men (9.2%) had positive LNs. Most men with unilateral disease had LN metastases on the same side of the pelvis. Comparing the clinical stage and grade with the tumour stage and grade of the RP specimen, there was a high percentage of upstaging and upgrading even in men with only one positive biopsy core. CONCLUSION: Unilateral prostate cancer preferentially metastasizes to the ipsilateral pelvic LNs. Because there are a few cases of bilateral LN metastases even in unilateral disease, and as it is not possible to reliably predict unilateral disease on the basis of biopsy features, PLND only on the tumour-bearing side has a high risk of understaging, and would possibly leave LN metastases behind.

Sentinel lymph node dissection for prostate cancer: experience with more than 1,000 patients.

PURPOSE: We determined the incidence of positive pelvic lymph nodes in men undergoing radical retropubic prostatectomy and describe the correlation with prostate specific antigen, histological grade and stage. We examined whether tumor cells are localized in the sentinel nodes only or also in other nonsentinel lymph nodes. MATERIALS AND METHODS: A total of 1,055 men with prostate cancer underwent radio guided pelvic lymph node dissection and radical retropubic prostatectomy. In men with prostate specific antigen 20 ng/ml or less and biopsy Gleason score 7 or less only sentinel nodes were removed. In men with prostate specific antigen more than 20 ng/ml or Gleason score greater than 7 extended pelvic lymph node dissection was also performed. RESULTS: Positive lymph nodes were found in 207 men (19.6%). In 63.3% of the men these lymph nodes were detected outside of the region of standard lymphadenectomy. The percent of patients with positive nodes was greater than predicted by currently used nomograms. The higher the preoperative prostate specific antigen, pathological stage and grade, the greater the percent of men with positive sentinel and nonsentinel lymph nodes (p<0.001). CONCLUSIONS: When deciding on pelvic lymph node dissection, sentinel or extended lymphadenectomy should be performed since more than half of patients have positive nodes outside of the region of standard lymphadenectomy. In cases of positive sentinel nodes extended lymph node dissection should be performed since tumor cells are also detectable in nonsentinel lymph nodes.

Limitations of radioguided surgery in high-risk prostate cancer.

OBJECTIVES: To determine how many men with high-risk prostate cancer (prostate-specific antigen [PSA]>20 ng/ml or biopsy Gleason score 8-10) have positive lymph nodes (sentinel lymph nodes [SLNs] and nonsentinel lymph nodes [NSLNs]) and whether these positive nodes are localised in the region of SLN dissection or in other regions, too. METHODS: In 228 men with high-risk prostate cancer radical retropubic prostatectomy combined with radioguided pelvic lymph node dissection and extended lymphadenectomy were performed. Serial sections of the SLNs were analysed immunohistochemically. RESULTS: A median of 7 SLNs (mean, 7) and 11 NSLNs (mean, 11) were dissected per patient. Ninety-six of 228 men (42.1%) had lymph node metastases. Most men had positive lymph nodes along the internal iliac artery alone or in combination with other regions. Twenty-two men had only micrometastatic disease. In 94 of 96 men the SLNs were positive. Twenty-six of 96 men had also positive NSLNs. When SLNs and NSLNs were positive, in more than half the patients the NSLNs were localised outside the region of sentinel lymphadenectomy. CONCLUSIONS: The dissection of SLNs in prostate cancer has a high sensitivity in detecting positive nodes. When SLNs are negative, the other pelvic lymph nodes are also negative in a high percentage of men (sensitivity 97.1%). When the SLNs are positive, patients with high-risk disease also have a high incidence of positive NSLNs. Therefore, when it is aspired to remove all pelvic lymph node metastases sentinel and extended lymphadenectomy should be performed.

Incidence of positive pelvic lymph nodes in patients with prostate cancer, a prostate-specific antigen (PSA) level of < or =10 ng/mL and biopsy Gleason score of < or =6, and their influence on PSA progression-free survival after radical prostatectomy.

OBJECTIVE: To investigate how many men with low-risk prostate cancer had positive lymph nodes detected by radio-guided surgery and whether they had a higher biochemical relapse rate after radical prostatectomy, because in such patients most urologists dispense with operative lymph node staging, as nomograms indicate only a low percentage of lymph node metastases. PATIENTS AND METHODS: The study included 474 men with a prostate-specific antigen (PSA) level of < or = 10 ng/mL, biopsy Gleason score of < or = 6 and positive biopsies in one (group 1, 315 men) or both lobes (group 2, 159 men); follow-up data were available in 357 men. Men with adjuvant radiation or hormone therapy before the occurrence of biochemical relapse were excluded. RESULTS: Positive lymph nodes were detected in 17 men in group 1, and in 18 in group 2. In more than half of the patients (19/35) these nodes were found outside the region of standard lymphadenectomy. Men with node-positive disease had a higher biochemical relapse rate (P < 0.001). When the tumour was organ-confined and well differentiated in node-positive disease (Gleason score < or = 6) the biochemical relapse rate was lower than in men with higher tumour stage and grade. CONCLUSIONS: When dissecting pelvic lymph nodes, extended or sentinel lymphadenectomy should be preferred. Removing the diseased nodes could improve the PSA progression-free survival, especially in well differentiated organ-confined disease.

Dosimetry-guided radioactive iodine treatment in patients with metastatic differentiated thyroid cancer: largest safe dose using a risk-adapted approach.

UNLABELLED: This study is a retrospective analysis of 124 differentiated thyroid cancer patients who underwent dosimetric evaluation using MIRD methodology over a period of 15 y. The objectives of the study were to demonstrate the clinical use of dosimetry-guided radioactive iodine ([RAI] (131)I) treatment and the safe and effective application of a 3-Gy bone marrow (BM) dose in patients with differentiated thyroid cancer. METHODS: Tumor and BM dose estimates were obtained. The administered activity that would deliver a maximum safe dose to the organ at risk (red BM or lungs) was determined as well as the resulting doses to the metastases. The clinical benefit of an individual RAI treatment was predicted on the basis of the dose estimates and the expected therapeutic response. Each patient's response to treatment was assessed clinically and by monitoring the hematologic profile. RESULTS: One hundred twenty-four patients underwent 187 dosimetric evaluations. One hundred four RAI treatments were performed. A complete response at metastatic deposits was attained with absorbed doses of >100 Gy. No permanent BM suppression was observed in patients who received absorbed doses of <3 Gy to BM. The maximum administered dose was 38.5 GBq (1,040 mCi) with the BM dose limitation. CONCLUSION: Dosimetry-guided RAI treatment allows administration of the maximum possible RAI dose to achieve the maximum therapeutic benefit. Estimation of tumor dose rates helps to determine the curative versus the palliative intent of the therapy.