Correlation of Baseline Tumor Burden with Clinical Outcome in Melanoma Patients Treated with Ipilimumab.

INTRODUCTION: Tumor burden is a frequently mentioned parameter; however, a commonly accepted definition is still lacking. METHODS: In this double-center prospective and retrospective study, 76 patients with unresectable stage III or stage IV melanoma treated with ipilimumab were included. We defined the baseline tumor burden (BTB) as the global sum of all metastases' longest diameters before treatment started and correlated the calculated BTB with disease control rate (DCR), progression-free survival (PFS), overall survival (OS), and with the baseline levels of LDH, S100B, and sULPB2. RESULTS: BTB correlated significantly with DCR (p = 0.009), PFS (p = 0.002), OS (p = 0.032), and the occurrence of NRAS mutation (p = 0.006). BTB was also correlated to baseline serum levels of LDH (p = 0.011), S100B (p = 0.027), and SULBP (p < 0.0001). Multivariate analysis revealed that BPB and LDH were independently correlated with PFS and OS. With increasing BTB, disease control was less likely; no patient with a BTB >200 mm achieved disease control. For patients with brain metastasis, no correlation of BTB with DCR (p = 0.251), PFS (p = 0.059), or OS (p = 0.981) was observed. CONCLUSION: Calculated BTB is an independent prognostic factor for patients with metastatic melanoma treated with ipilimumab. Using calculated BTB as a definition of tumor burden may help increase comparability of outcome of therapies in future studies.

Evaluation of the heat sensitivity of Trichophyton rubrum and Trichophyton interdigitale.

BACKGROUND: Onychomycosis affects up to 50% of patients in the older population. OBJECTIVES: This study aimed to explore heat sensitivity of Trichophyton rubrum and Trichophyton interdigitale as pathogens of onychomycosis. MATERIALS & METHODS: The fungi were heated in sterile saline solution up to 100°C for five or 10 minutes with or without additional previous treatment with 1% ciclopirox solution or chitinase and 1,3 -galactidase or for 45 minutes at 40°C or 60°C with washing powder. Subsequently, the fungi were cultured and regrowth was assessed after one week. RESULTS: After heating T. rubrum for five minutes at 60°C, growth was completely inhibited. After heating T. interdigitale for five minutes at 60°C, all of the samples regrew, and at 95°C, none of the samples regrew. No difference between five and 10-minute heating was observed. Previous incubation with 1% ciclopirox solution for 24 hours inhibited the growth of T. rubrum completely. T. interdigitale was still able to regrow to 100% after five minutes at 40°C, to 33% after 60°C, and to 22% after 80°C. Incubation for 45 minutes with washing powder solution at 40°C or 60°C did not lead to significant growth reduction of T. rubrum or interdigitale. Two hours incubation with -1,3-glucanase and chitinase prior to five minutes of heating to 60°C and 80°C reduced the heat resistance of T. interdigitale; growth was inhibited in 56% and 100% of the samples, respectively. CONCLUSION: The heat resistance of T. rubrum and interdigitale should be considered using non-medical thermal treatment.

Droplet Microarray as a Powerful Platform for Seeking New Antibiotics Against Multidrug-Resistant Bacteria.

Multidrug-resistant (MDR) bacteria is a severe threat to public health. Therefore, it is urgent to establish effective screening systems for identifying novel antibacterial compounds. In this study, a highly miniaturized droplet microarray (DMA) based high-throughput screening system is established to screen over 2000 compounds for their antimicrobial properties against carbapenem-resistant Klebsiella pneumoniae and methicillin resistant Staphylococcus aureus (MRSA). The DMA consists of an array of hydrophilic spots divided by superhydrophobic borders. Due to the differences in the surface wettability between the spots and the borders, arrays of hundreds of nanoliter-sized droplets containing bacteria and different drugs can be generated for screening applications. A simple colorimetric viability readout utilizing a conventional photo scanner is developed for fast single-step detection of the inhibitory effect of the compounds on bacterial growth on the whole array. Six hit compounds, including coumarins and structurally simplified estrogen analogs are identified in the primary screening and validated with minimum inhibition concentration assay for their antibacterial effect. This study demonstrates that the DMA-based high-throughput screening system enables the identification of potential antibiotics from novel synthetic compound libraries, offering opportunities for development of new treatments against multidrug-resistant bacteria.

Chalcone-Supported Cardiac Mesoderm Induction in Human Pluripotent Stem Cells for Heart Muscle Engineering.

Human pluripotent stem cells (hPSCs) hold great promise for applications in cell therapy and drug screening in the cardiovascular field. Bone morphogenetic protein 4 (BMP4) is key for early cardiac mesoderm induction in hPSC and subsequent cardiomyocyte derivation. Small-molecular BMP4 mimetics may help to standardize cardiomyocyte derivation from hPSCs. Based on observations that chalcones can stimulate BMP4 signaling pathways, we hypothesized their utility in cardiac mesoderm induction. To test this, we set up a two-tiered screening strategy, (1) for directed differentiation of hPSCs with commercially available chalcones (4'-hydroxychalcone [4'HC] and Isoliquiritigen) and 24 newly synthesized chalcone derivatives, and (2) a functional screen to assess the propensity of the obtained cardiomyocytes to self-organize into contractile engineered human myocardium (EHM). We identified 4'HC, 4-fluoro-4'-methoxychalcone, and 4-fluoro-4'-hydroxychalcone as similarly effective in cardiac mesoderm induction, but only 4'HC as an effective replacement for BMP4 in the derivation of contractile EHM-forming cardiomyocytes.

Aldosterone Glucuronide, an Important Biomarker: Synthesis and Structure Elucidation of Novel Isomers.

Aldosterone 1 is a mineralocorticoid, it has great influence on the blood pressure and its glucuronide is an important marker for the detection of several diseases. Here, we describe the chemical synthesis of different aldosterone-18- and 20-glucuronides. Reaction of trimethylsilyl 2,3,4-tri- acetyl-1-ß-glucuronic acid methyl ester 5 b and aldosterone diacetate 11 in the presence of TMSOTf gave the 18-α-glucuronide 9 a. The 18-ß-glucuronide 15 b and the 20-ß-glucuronide 16 b could be obtained by reaction of methyl 2,3,4-tri-O-isobutyryl-1α-glucuronate trichloroacetimidate 14 and aldosterone 21-acetate 8 in the presence of TMSOTf or BF3 ⋅OEt2 . Finally, reaction of aldosterone 21-acetate 8 and methyl 2,3,4-triacetyl-1α-glucuronate trichloroacetimidate 19 in the presence of TMSOTf gave the corresponding methyl 18-ß-triacetylglucuronate 9 b, which was transformed into the desired aldosterone-18-ß-glucuronide 3 by two enzyma- tic transformations.

Galactose-modified duocarmycin prodrugs as senolytics.

Senescence is a stable growth arrest that impairs the replication of damaged, old or preneoplastic cells, therefore contributing to tissue homeostasis. Senescent cells accumulate during ageing and are associated with cancer, fibrosis and many age-related pathologies. Recent evidence suggests that the selective elimination of senescent cells can be effective on the treatment of many of these senescence-associated diseases. A universal characteristic of senescent cells is that they display elevated activity of the lysosomal ß-galactosidase, and this has been exploited as a marker for senescence (senescence-associated ß-galactosidase activity). Consequently, we hypothesized that galactose-modified cytotoxic prodrugs will be preferentially processed by senescent cells, resulting in their selective killing. Here, we show that different galactose-modified duocarmycin (GMD) derivatives preferentially kill senescent cells. GMD prodrugs induce selective apoptosis of senescent cells in a lysosomal ß-galactosidase (GLB1)-dependent manner. GMD prodrugs can eliminate a broad range of senescent cells in culture, and treatment with a GMD prodrug enhances the elimination of bystander senescent cells that accumulate upon whole-body irradiation treatment of mice. Moreover, taking advantage of a mouse model of adamantinomatous craniopharyngioma (ACP), we show that treatment with a GMD prodrug selectively reduced the number of ß-catenin-positive preneoplastic senescent cells. In summary, the above results make a case for testing the potential of galactose-modified duocarmycin prodrugs to treat senescence-related pathologies.

Bifunctional Duocarmycin Analogues as Inhibitors of Protein Tyrosine Kinases.

Bifunctional duocarmycin analogues are highly cytotoxic compounds that have been shown to be irreversible aldehyde dehydrogenase 1 inhibitors. Interestingly, cells with low aldehyde dehydrogenase 1 expression are also sensitive to bifunctional duocarmycin analogues, suggesting the existence of another target. Through in silico approaches, including principal component analysis, structure-similarity search, and docking calculations, protein tyrosine kinases, and especially the vascular endothelial growth factor receptor 2 (VEGFR-2), were predicted as targets of bifunctional duocarmycin analogues. Biochemical validation was performed in vitro, confirming the in silico results. Structural optimization was performed to mainly target VEGFR-2, but not aldehyde dehydrogenase 1. The optimized bifunctional duocarmycin analogue was synthesized. In vitro assays revealed this bifunctional duocarmycin analogue as a strong inhibitor of VEGFR-2, with low residual aldehyde dehydrogenase 1 activity. Altogether, studies revealed bifunctional duocarmycin analogues as a new class of naturally derived compounds that express a very high cytotoxicity to cancer cells overexpressing aldehyde dehydrogenase 1 as well as VEGFR-2.

Enantioselective Total Synthesis of Chromanone Lactone Homo- and Heterodimers.

A one pot borylation/Suzuki-Miyaura reaction of the 4-bromochromanone lactones 21 and 23, respectively, followed by cleavage of the methyl ether moieties gave the homodimeric chromanone lactones 10 and 11. Reaction of a 1:1 mixture of 21 and 23 under otherwise identical conditions gave a 1:1:2-mixture of the two homodimers 10 and 11 and the heterodimer 12. This is the first example of the preparation of a heterodimeric chromanone lactone. For the enantioselective synthesis of the starting material, phenol 17 was transformed into the chromane 18 using a Wacker-type cyclisation with 99 % ee and 80 % yield.

Enantioselective Total Synthesis of Blennolide†H and Phomopsis-H76†A and Determination of Their Structure.

This work reports on the enantioselective total synthesis of the two dimeric natural chromanone lactones phomopsis-H76 A (5) and blennolide H (6). Both syntheses could be achieved from chromane 11, which was obtained by an enantioselective Wacker-type cyclization with >99 % ee. The dimerization of the corresponding monomers was performed using a palladium-catalyzed Suzuki reaction. Moreover, within this work it was possible to revise the absolute configuration of phomopsis-H76 A and determine the relative as well as absolute configuration of blennolide H.

Enantioselective Total Synthesis of the Fungal Metabolite Blennolide D and the Enantiomers of Blennolide E and F.

An enantioselective total synthesis of blennolide D and the enantiomers of blennolide E and F is described using an enantioselective Wacker-type oxidation followed by the formation of the lactone moiety. For the introduction of the hydroxyl group in the γ-lactone, a TEMPO-mediated α-oxygenation was used which was followed by a benzylic oxidation and deprotection to give the desired compounds. In addition, an unknown diastereomer was synthesized.

Palladium-Catalyzed 4-Fold Domino Reaction for the Synthesis of a Polymeric Double Switch.

A palladium-catalyzed 4-fold domino reaction consisting of two carbopalladation reactions and two C-H activation reactions, followed by the introduction of an acrylate moiety, led to the tetra-substituted helical alkene A2, using the dialkyne A3 as a substrate. The alkene was copolymerized with butyl acrylate by using the reversible addition-fragmentation chain transfer polymerization (RAFT) to give the desired polymeric switch A1.

Enantioselective Total Synthesis and Structure Confirmation of the Natural Dimeric Tetrahydroxanthenone Dicerandrol†C.

The first enantioselective total synthesis of natural dicerandrol C (1 c) as its enantiomer containing a dimeric tetrahydroxanthenone skeleton is described starting from the enantiopure chromane 6 which was obtained through a Wacker-type cyclization with >99 % ee. For the formation of the dimeric skeleton a palladium-catalyzed Suzuki reaction was used. The synthesis allowed the confirmation of the absolute configuration of the dicerandrols.

In vivo imaging of tumour xenografts with an antibody targeting the potassium channel Kv10.1.

The Kv10.1 (Eag1) voltage-gated potassium channel represents a promising molecular target for novel cancer therapies or diagnostic purposes. Physiologically, it is only expressed in the brain, but it was found overexpressed in more than 70 % of tumours of diverse origin. Furthermore, as a plasma membrane protein, it is easily accessible to extracellular interventions. In this study we analysed the feasibility of the anti-Kv10.1 monoclonal antibody mAb62 to target tumour cells in vitro and in vivo and to deliver therapeutics to the tumour. Using time-domain near infrared fluorescence (NIRF) imaging in a subcutaneous MDA-MB-435S tumour model in nude mice, we showed that mAb62-Cy5.5 specifically accumulates at the tumour for at least 1 week in vivo with a maximum intensity at 48 h. Blocking experiments with an excess of unlabelled mAb62 and application of the free Cy5.5 fluorophore demonstrate specific binding to the tumour. Ex vivo NIRF imaging of whole tumours as well as NIRF imaging and microscopy of tumour slices confirmed the accumulation of the mAb62-Cy5.5 in tumours but not in brain tissue. Moreover, mAb62 was conjugated to the prodrug-activating enzyme ß-D-galactosidase (ß-gal; mAb62-ß-gal). The ß-gal activity of the mAb62-ß-gal conjugate was analysed in vitro on Kv10.1-expressing MDA-MB-435S cells in comparison to control AsPC-1 cells. We show that the mAb62-ß-gal conjugate possesses high ß-gal activity when bound to Kv10.1-expressing MDA-MB-435S cells. Moreover, using the ß-gal activatable NIRF probe DDAOG, we detected mAb62-ß-gal activity in vivo over the tumour area. In summary, we could show that the anti-Kv10.1 antibody is a promising tool for the development of novel concepts of targeted cancer therapy.

Design, Synthesis, and Biological Evaluation of Quercetagetin Analogues as JNK1 Inhibitors.

The recent discovery of c-Jun NH2-terminal kinase JNK1 suppression by natural quercetagetin (1) is a promising lead for the development of novel anticancer agents. Using both X-ray structure and docking analyses we predicted that 5'-hydroxy- (2) and 5'-hydroxymethyl-quercetagetin (3) would inhibit JNK1 more actively than the parent compound 1. Notably, our drug design was based on the active enzyme-ligand complex as opposed to the enzyme's relatively open apo structure. In this paper we test our theoretical predictions, aided by docking-model experiments, and report the first synthesis and biological evaluation of quercetagetin analogues 2 and 3. As calculated, both compounds strongly suppress JNK1 activity. The IC50 values were determined to be 3.4 µM and 12.2 µM, respectively, which shows that 2 surpasses the potency of the parent compound 1 (IC50 =4.6 µM). Compound 2 was also shown to suppress matrix metalloproteinase-1 expression with high specificity after UV irradiation.

Enantioselective Total Synthesis of Secalonic Acid E.

The first enantioselective synthesis of a secalonic acid containing a dimeric tetrahydroxanthenone skeleton is described, using a Wacker-type cyclization of a methoxyphenolic compound to form a chiral chroman with a quaternary carbon stereogenic center with >99% ee. Further steps are a Sharpless dihydroxylation and a Dieckmann condensation to give a tetrahydroxanthenone. A late-stage one-pot palladium-catalyzed Suzuki-dimerization reaction leads to the 2,2'-biphenol linkage to complete the enantioselective total synthesis of secalonic acid E in 18 steps with 8% overall yield.

Structural, Biochemical, and Computational Studies Reveal the Mechanism of Selective Aldehyde Dehydrogenase 1A1 Inhibition by Cytotoxic Duocarmycin Analogues.

Analogues of the natural product duocarmycin bearing an indole moiety were shown to bind aldehyde dehydrogenase 1A1 (ALDH1A1) in addition to DNA, while derivatives without the indole solely addressed the ALDH1A1 protein. The molecular mechanism of selective ALDH1A1 inhibition by duocarmycin analogues was unraveled through cocrystallization, mutational studies, and molecular dynamics simulations. The structure of the complex shows the compound embedded in a hydrophobic pocket, where it is stabilized by several crucial π-stacking and van der Waals interactions. This binding mode positions the cyclopropyl electrophile for nucleophilic attack by the noncatalytic residue Cys302, thereby resulting in covalent attachment, steric occlusion of the active site, and inhibition of catalysis. The selectivity of duocarmycin analogues for ALDH1A1 is unique, since only minor alterations in the sequence of closely related protein isoforms restrict compound accessibility.

Four- and Sixfold Tandem-Domino Reactions Leading to Dimeric Tetrasubstituted Alkenes Suitable as Molecular Switches.

A highly efficient palladium-catalyzed fourfold tandem-domino reaction consisting of two carbopalladation and two C-H-activation steps was developed for the synthesis of two types of tetrasubstituted alkenes 3 and 6 with intrinsic helical chirality starting from substrates 1 and 4, respectively. A sixfold tandem-domino reaction was also developed by including a Sonogashira reaction. 20 compounds with different substitution patterns were prepared with yields of up to 97 %. Structure elucidation by X-ray crystallography confirmed helical chirality of the two alkene moieties. Photophysical investigations of some of the compounds showed pronounced switching properties through light-controlled changes of their stereochemical configuration.

Enantioselective total synthesis of the lignan (+)-linoxepin.

An enantioselective total synthesis of the natural (+)-linoxepin (1) was accomplished in eleven steps from bromovanin (24). Key steps are a domino carbopalladation/ Mizoroki-Heck reaction with the formation of a pentacyclic system, an asymmetric hydroboration as well as an oxidative lactonization.

A fast way to fluorescence: a fourfold domino reaction to condensed polycyclic compounds.

A fast and efficient palladium-catalyzed fourfold domino Sonogashira/double-carbopalladation/CH-activation reaction that converts simple aromatic systems into complex polycyclic hydrocarbons has been developed. A number of substituted products has thus been prepared in yields up to 89 %. The structural assignment has been confirmed by using single-crystal X-ray crystallography. The products show intriguing fluorescence activity and thus might serve as chemical sensors or fluorescent imaging dyes.