Single Live Cell Imaging of Multidrug Resistance Using Silver Ultrasmall Nanoparticles as Biosensing Probes in Triple-Negative Breast Cancer Cells.

Silver ultrasmall nanoparticles (Ag UNPs) (size < 5 nm) were used as biosensing probes to analyze the efflux kinetics contributing to multidrug resistance (MDR) in single live triple-negative breast cancer (TNBC) cells by using dark-field optical microscopy to follow their size-dependent localized surface plasmon resonance. TNBC cells lack expression of estrogen (ER-), progesterone (PR-), and human epidermal growth factor 2 (HER2-) receptors and are more likely to acquire resistance to anticancer drugs due to their ability to transport harmful substances outside the cell. The TNBC cells displayed greater nuclear and cytoplasmic efflux, resulting in less toxicity of Ag UNPs in a concentration-independent manner. In contrast, more Ag UNPs and an increase in cytotoxic effects were observed in the receptor-positive breast cancer cells that have receptors for ER+, PR+, and HER2+ and are known to better respond to anticancer therapies. Ag UNPs accumulated in receptor-positive breast cancer cells in a time-and concentration-dependent mode and caused decreased cellular growth, whereas the TNBC cells due to the efflux were able to continue to grow. The TNBC cells demonstrated a marked increase in survival due to their ability to have MDR determined by efflux of Ag UNPs outside the nucleus and the cytoplasm of the cells. Further evaluation of the nuclear efflux kinetics of TNBC cells with Ag UNPs as biosensing probes is critical to gain a better understanding of MDR and potential for enhancement of cancer drug delivery.

Antagonists for Constitutively Active Mutant Estrogen Receptors: Insights into the Roles of Antiestrogen-Core and Side-Chain.

A major risk for patients having estrogen receptor α (ERα)-positive breast cancer is the recurrence of drug-resistant metastases after initial successful treatment with endocrine therapies. Recent studies have implicated a number of activating mutations in the ligand-binding domain of ERα that stabilize the agonist conformation as a prominent mechanism for this acquired resistance. There are several critical gaps in our knowledge regarding the specific pharmacophore requirements of an antagonist that could effectively inhibit all or most of the different mutant ERs. To address this, we screened various chemotypes for blocking mutant ER-mediated transcriptional signaling and identified RU58668 as a model compound that contains structural elements that support potent ligand-induced inhibition of mutant ERs. We designed and synthesized a focused library of novel antagonists and probed how small and large perturbations in different ligand structural regions influenced inhibitory activity on individual mutant ERs in breast cancer cells. Effective inhibition derives from both nonpolar and moderately polar motifs in a multifunctional side chain of the antagonists, with the nature of the ligand core making important contributions by increasing the potency of ligands possessing similar types of side chains. Some of our new antagonists potently blocked the transcriptional activity of the three most common mutant ERs (L536R, Y537S, D538G) and inhibited mutant ER-mediated cell proliferation. Supported by our molecular modeling, these studies provide new insights into the role of specific components, involving both the ligand core and multifunctional side chain, in suppressing wild-type and mutant ER-mediated transcription and breast cancer cell proliferation.

New Class of Selective Estrogen Receptor Degraders (SERDs): Expanding the Toolbox of PROTAC Degrons.

An effective endocrine therapy for breast cancer is to selectively and effectively degrade the estrogen receptor (ER). Up until now, there have been largely only two molecular scaffolds capable of doing this. In this study, we have developed new classes of scaffolds that possess selective estrogen receptor degrader (SERD) and ER antagonistic properties. These novel SERDs potently inhibit MCF-7 breast cancer cell proliferation and the expression of ER target genes, and their efficacy is comparable to Fulvestrant. Unlike Fulvestrant, the modular protein-targeted chimera (PROTAC)-type design of these novel SERDs should allow easy diversification into a library of analogs to further fine-tune their pharmacokinetic properties including oral availability. This work also expands the pool of currently available PROTAC-type scaffolds that could be beneficial for targeted degradation of various other therapeutically important proteins.

Triaryl Pyrazole Toll-Like Receptor Signaling Inhibitors: Structure-Activity Relationships Governing Pan- and Selective Signaling Inhibitors.

The immune system uses members of the toll-like receptor (TLR) family to recognize a variety of pathogen- and host-derived molecules in order to initiate immune responses. Although TLR-mediated, pro-inflammatory immune responses are essential for host defense, prolonged and exaggerated activation can result in inflammation pathology that manifests in a variety of diseases. Therefore, small-molecule inhibitors of the TLR signaling pathway might have promise as anti-inflammatory drugs. We previously identified a class of triaryl pyrazole compounds that inhibit TLR signaling by modulation of the protein-protein interactions essential to the pathway. We have now systematically examined the structural features essential for inhibition of this pathway, revealing characteristics of compounds that inhibited all TLRs tested (pan-TLR signaling inhibitors) as well as compounds that selectively inhibited certain TLRs. These findings reveal interesting classes of compounds that could be optimized for particular inflammatory diseases governed by different TLRs.

Identification of Toll-like receptor signaling inhibitors based on selective activation of hierarchically acting signaling proteins.

Toll-like receptors (TLRs) recognize various pathogen- and host tissue-derived molecules and initiate inflammatory immune responses. Exaggerated or prolonged TLR activation, however, can lead to etiologically diverse diseases, such as bacterial sepsis, metabolic and autoimmune diseases, or stroke. Despite the apparent medical need, no small-molecule drugs against TLR pathways are clinically available. This may be because of the complex signaling mechanisms of TLRs, which are governed by a series of protein-protein interactions initiated by Toll/interleukin-1 receptor homology domains (TIR) found in TLRs and the cytoplasmic adaptor proteins TIRAP and MyD88. Oligomerization of TLRs with MyD88 or TIRAP leads to the recruitment of members of the IRAK family of kinases and the E3 ubiquitin ligase TRAF6. We developed a phenotypic drug screening system based on the inducible homodimerization of either TIRAP, MyD88, or TRAF6, that ranked hits according to their hierarchy of action. From a bioactive compound library, we identified methyl-piperidino-pyrazole (MPP) as a TLR-specific inhibitor. Structure-activity relationship analysis, quantitative proteomics, protein-protein interaction assays, and cellular thermal shift assays suggested that MPP targets the TIR domain of MyD88. Chemical evolution of the original MPP scaffold generated compounds with selectivity for distinct TLRs that interfered with specific TIR interactions. Administration of an MPP analog to mice protected them from TLR4-dependent inflammation. These results validate this phenotypic screening approach and suggest that the MPP scaffold could serve as a starting point for the development of anti-inflammatory drugs.

Exploring the Structural Compliancy versus Specificity of the Estrogen Receptor Using Isomeric Three-Dimensional Ligands.

The estrogen receptors (ERs) bind with high affinity to many structurally diverse ligands by significantly distorting the contours of their ligand-binding pockets. This raises a question: To what degree is ER able to distinguish between structurally related regioisomers and enantiomers? We have explored the structural compliance and specificity of ERα with a set of ligands having a 7-oxa-bicyclo[2.2.1]hept-5-ene sulfonate core and basic side chains typical of selective ER modulators (SERMs). These ligands have two regioisomers, each of which is a racemate of enantiomers. Using orthogonal protecting groups and chiral HPLC, we isolated all 4 isomers and assigned their absolute stereochemistry by X-ray analysis. The 1S,2R,4S isomer has a 80-170-fold higher affinity for ERα than the others, and it profiles as a partial agonist/antagonist in cellular reporter gene assays and in suppressing proliferation of MCF-7 breast cancer cells with subnanomolar potency, far exceeding that of the other isomers. It is the only isomer found bound to ERα by X-ray analysis after crystallization with four-isomer mixtures of closely related analogs. Thus, despite the general compliance of this receptor for binding a large variety of ligand structures, ER demonstrates marked structural specificity and stereospecificity by selecting a single component from a mixture of structurally related isomers to drive ER-regulated cellular activity. Our findings lay the necessary groundwork for seeking unique ER-mediated pharmacological profiles by rational structural perturbations of two different types of side chains in this unprecedented class of ER ligands, which may prove useful in developing more effective endocrine therapies for breast cancer.

Stilbene-chalcone hybrids: design, synthesis, and evaluation as a new class of antimalarial scaffolds that trigger cell death through stage specific apoptosis.

Novel stilbene-chalcone (S-C) hybrids were synthesized via a sequential Claisen-Schmidt-Knoevenagel-Heck approach and evaluated for antiplasmodial activity in in vitro red cell culture using SYBR Green I assay. The most potent hybrid (11) showed IC(50) of 2.2, 1.4, and 6.4 µM against 3D7 (chloroquine sensitive), Indo, and Dd2 (chloroquine resistant) strains of Plasmodium falciparum, respectively. Interestingly, the respective individual stilbene (IC(50) > 100 µM), chalcone (IC(50) = 11.5 µM), or an equimolar mixture of stilbene and chalcone (IC(50) = 32.5 µM) were less potent than 11. Studies done using specific stage enriched cultures and parasite in continuous culture indicate that 11 and 18 spare the schizont but block the progression of the parasite life cycle at the ring or the trophozoite stages. Further, 11 and 18 caused chromatin condensation, DNA fragmentation, and loss of mitochondrial membrane potential in Plasmodium falciparum, thereby suggesting their ability to cause apoptosis in malaria parasite.

Pd-catalyzed orthogonal Knoevenagel/Perkin condensation-decarboxylation-Heck/Suzuki sequences: tandem transformations of benzaldehydes into hydroxy-functionalized antidiabetic stilbene-cinnamoyl hybrids and asymmetric distyrylbenzenes.

Tandem reactions that involve chemoselective Knoevenagel/Perkin condensation-decarboxylation-Heck/Suzuki coupling or Heck-aldol sequences have been achieved. This enabled the first concise and efficient synthesis of several important hydroxy-functionalized compound classes, such as stilbene-cinnamoyl hybrids (potent protein tyrosine phosphatase1B inhibitors), cinnamoyl-cinnamic acid hybrids, asymmetric distyrylbenzenes, and biarylstyrenes. Previously reported synthesis require multiple steps and protection/deprotection manipulations.

Tandem allylic oxidation-condensation/esterification catalyzed by silica gel: an expeditious approach towards antimalarial diaryldienones and enones from natural methoxylated phenylpropenes.

A new one-pot strategy has been developed, wherein abundantly available methoxylated phenylpropenes are directly transformed into corresponding dienones (1,5-diarylpenta-2,4-dien-1-ones) and enones (chalcones and cinnamic esters) via allylic oxidation-condensation or allylic oxidation-esterification sequences. Preliminary antimalarial activity studies of the above synthesized diaryldienones and enones against Plasmodium falciparum (Pf3D7) have shown them to be promising lead candidates for developing newer and economical antimalarial agents. In particular, two enones (12b and 13b) were found to possess comparatively better activity (IC(50) = 4.0 and 3.4 µM, respectively) than licochalcone (IC(50) = 4.1 µM), a well known natural antimalarial compound.

Metal-free activation of H2O2 by synergic effect of ionic liquid and microwave: chemoselective oxidation of benzylic alcohols to carbonyls and unexpected formation of anthraquinone in aqueous condition.

H(2)O(2) mediated oxidation of alcohols in ionic liquid is revisited, wherein, ionic liquids under the influence of microwave irradiation have been found to facilitate activation of H(2)O(2) without any metal catalyst in aqueous condition. The method utilizes a neutral ionic liquid [hmim]Br both as catalyst and solvent for efficient and chemoselective oxidation of benzyl alcohol derivatives on aromatic (ß, γ) alcohols, cyclic and aliphatic analogues, which can be a useful synthetic approach in total synthesis of complex organic compounds/natural products. Moreover, an unexpected oxidation of 9-anthracenyl propanol, a polyaromatic benzyl alcohol, resulting in the formation of 9,10-anthraquinone by the loss of propyl side chain was observed. Plausible mechanism and further exploration of this method on various other related substrates are discussed in detail.

Direct olefination of benzaldehydes into hydroxy functionalized oligo (p-phenylenevinylene)s via Pd-catalyzed heterodomino Knoevenagel-decarboxylation-Heck sequence and its application for fluoride sensing pi-conjugated units.

A new approach for one step olefination of benzaldehydes into hydroxy functionalized OPVs is achieved through the first domino Knoevenagel-decarboxylation-Heck sequence using a single catalyst system. The methodology also led to new oxygen based OPV scaffolds capable of selective and visible fluoride recognition in organic or aqueous medium.

Water-promoted cascade synthesis of alpha-arylaldehydes from arylalkenes using N-halosuccinimides: an avenue for asymmetric oxidation using Cinchona organocatalysis.

The direct oxidation of arylalkenes into alpha-arylaldehydes is achieved for the first time in water without relying on transition metal catalysts, and a novel organocatalytic enantioselective approach is also explored to provide up to 30% ee in initial investigations.

Isolation and purification of acetylshikonin and beta-acetoxyisovalerylshikonin from cell suspension cultures of Arnebia euchroma (Royle) Johnston using rapid preparative HPLC.

Shikonin and its derivatives are important red colored naphthoquinone pigments found in a large number of Arnebia species, including A. euchroma, that are responsible for the various pharmacological activities exhibited by the plant. The precise separation of each naphthoquinone is essential for total quality evaluation and bioactivity analysis of herbal formulations of A. euchroma. Furthermore, the overexploitation of this useful plant has resulted in species becoming endangered. With this in mind, a simple and rapid preparative scale HPLC method with single compound recovery for the isolation and purification of two shikonin derivatives (i. e. acetylshikonin, beta-acetoxyisovalerylshikonin) from cell suspension cultures of A. euchroma is presented. The compounds were separated on a C(18) column within 10 min using acetonitrile/methanol (95:5) as mobile phase in isocratic mode. The isolated compounds were found to be more than 98% pure. The LOD for acetylshikonin and beta-acetoxyisovalerylshikonin was estimated at 0.063 and 0.146 mug/mL, respectively, while the LOQ was found to be 0.209 and 0.487 mug/mL, respectively. The recoveries accomplished for both the shikonin derivatives were in the range of 94.7-96.8%. The repeatability, expressed as %RSD, of acetylshikonin and beta-acetoxyisovalerylshikonin was found to be 1.74 and 1.27, respectively.