Excited State Rotational Freedom Impacts Viscosity Sensitivity in Arylcyanoamide Fluorescent Molecular Rotor Dyes.

The microviscosity of intracellular environments plays an important role in monitoring cellular function. Thus, the capability of detecting changes in viscosity can be utilized for the detection of different disease states. Viscosity-sensitive fluorescent molecular rotors are potentially excellent probes for these applications; however, the predictable relationships between chemical structural features and viscosity sensitivity are poorly understood. Here, we investigate a set of arylcyanoamide-based fluorescent probes and the effect of small aliphatic substituents on their viscosity sensitivity. We found that the location of the substituents and the type of π-network of the fluorophore can significantly affect the viscosity sensitivity of these fluorophores. Computational analysis supported the notion that the excited state rotational energy barrier plays a dominant role in the relative viscosity sensitivity of these fluorophores. These findings provide valuable insight into the design of molecular rotor-based fluorophores for viscosity measurement.

Fluorescent molecular rotors as versatile in situ sensors for protein quantitation.

Accurate protein quantitation is essential for many cellular mechanistic studies. Existing technology relies on extrinsic sample evaluation that requires significant volumes of sample as well as addition of assay-specific reagents and importantly, is a terminal analysis. This study exploits the unique chemical features of a fluorescent molecular rotor that fluctuates between twisted-to-untwisted states, with a subsequent intensity increase in fluorescence depending on environmental conditions (e.g., viscosity). Here we report the development of a rapid, sensitive in situ protein quantitation method using ARCAM-1, a representative fluorescent molecular rotor that can be employed in both non-terminal and terminal assays.

Electrochemical potential enables dormant spores to integrate environmental signals.

The dormant state of bacterial spores is generally thought to be devoid of biological activity. We show that despite continued dormancy, spores can integrate environmental signals over time through a preexisting electrochemical potential. Specifically, we studied thousands of individual Bacillus subtilis spores that remain dormant when exposed to transient nutrient pulses. Guided by a mathematical model of bacterial electrophysiology, we modulated the decision to exit dormancy by genetically and chemically targeting potassium ion flux. We confirmed that short nutrient pulses result in step-like changes in the electrochemical potential of persistent spores. During dormancy, spores thus gradually release their stored electrochemical potential to integrate extracellular information over time. These findings reveal a decision-making mechanism that operates in physiologically inactive cells.

Exploring the Effect of Aliphatic Substituents on Aryl Cyano Amides on Enhancement of Fluorescence upon Binding to Amyloid-ß Aggregates.

The self-assembly of amyloid-ß (Aß) peptides into amyloid aggregates is a pathological hallmark of Alzheimer's Disease. We previously reported a fluorescent Aryl Cyano Amide (ARCAM) probe that exhibits an increase in fluorescence emission upon binding to Aß aggregates in solution and in neuronal tissue. Here, we investigate the effect of introducing small aliphatic substituents on the spectroscopic properties of ARCAM both free in solution and when bound to aggregated Aß. We found that introducing substituents designed to hinder the rotation of bonds between the electron donor and acceptor on these fluorophores can affect the overall brightness of fluorescence emission of the probes in amyloid-free solutions, but the relative fluorescence enhancement of these probes in amyloid-containing solutions is dependent on the location of the substituents on the ARCAM scaffold. We also observed the capability to tune the excitation or emission wavelength of these probes by introducing electron-donating or -withdrawing substituents that putatively affect either the energy required for photoexcitation or the stability of the photoexcited state. These studies reveal new design principles for developing ARCAM-based fluorescent Aß-binding probes with an enhanced fluorescence signal compared to background and tunable spectroscopic properties, which may lead to improved chemical tools for aiding in the diagnosis of amyloid-associated neurodegenerative diseases.

Chiral resolution of a caged xanthone and evaluation across a broad spectrum of breast cancer subtypes.

Racemic resolution of (+/-)-MAD28, a representative caged xanthone, was accomplished using (1S, 4R)-(-)-camphanic chloride as the chiral agent. Selective crystallization of the resulting diastereomers in acetonitrile produced, after hydrolysis, the pure enantiomers. Screening of racemic MAD28 and both enantiomers across a broad spectrum of breast cancer cell lines revealed that they: (a) are equipotent in each of the breast cancer subtypes examined; and (b) exhibit a higher degree of cytotoxicity against breast cancer cell lines of basal-like subtype and triple negative receptor status. The results support the notion that MAD28 and related caged xanthones are promising drug leads against chemoresistant and metastatic cancers.

Marine Spirotetronates: Biosynthetic Edifices That Inspire Drug Discovery.

Spirotetronates are actinomyces-derived polyketides that possess complex structures and exhibit potent and unexplored bioactivities. Due to their anticancer and antimicrobial properties, they have potential as drug hits and deserve further study. In particular, abyssomicin C and tetrocarcin A have shown significant promise against antibiotic-resistant S. aureus and tuberculosis, as well as for the treatment of various lymphomas and solid tumors. Improved synthetic routes to these compounds, particularly the class II spirotetronates, are needed to access sufficient quantities for structure optimization and clinical applications.

Synthesis, structure-activity relationship and in vitro pharmacodynamics of A-ring modified caged xanthones in a preclinical model of inflammatory breast cancer.

Inflammatory breast cancer (IBC) is a highly metastatic, lethal form of breast cancer that lacks targeted therapeutic strategies. Inspired by the promising cytotoxicity of gambogic acid and related caged xanthones in spheroidsMARY-X, an in vitro preclinical IBC model, we constructed a library of synthetic analogs and performed structure-activity relationship studies. The studies revealed that functionalizing the A-ring of the caged xanthone framework can significantly affect potency. Specifically, introduction of hydroxyl or fluorine groups at discrete positions of the A-ring leads to enhanced cytotoxicity at submicromolar concentrations. These compounds induce complete dissolution of spheroidsMARY-X with subsequent apoptosis of both the peripherally- and centrally-located cells, proliferative and quiescent-prone (e.g. hypoxic), respectively. These results highlight the structural flexibility and pharmacological potential of the caged xanthone motif for the design of IBC-targeting therapeutics.

Solvation-Guided Design of Fluorescent Probes for Discrimination of Amyloids.

The deposition of insoluble protein aggregates in the brain is a hallmark of many neurodegenerative diseases. While their exact role in neurodegeneration remains unclear, the presence of these amyloid deposits often precedes clinical symptoms. As a result, recent progress in imaging methods that utilize amyloid-specific small molecule probes have become a promising avenue for antemortem disease diagnosis. Here, we present a series of amino-aryl cyanoacrylate (AACA) fluorophores that show a turn-on fluorescence signal upon binding to amyloids in solution and in tissue. Using a theoretical model for environmental sensitivity of fluorescence together with ab initio computational modeling of the effects of polar environment on electron density distribution and conformational dynamics, we designed, synthesized, and evaluated a set of fluorophores that (1) bind to aggregated forms of Alzheimer's-related ß-amyloid peptides with low micromolar to high nanomolar affinities and (2) have the capability to fluorescently discriminate different amyloids based on differences in amino acid composition within the binding pocket through exploitation of their solvatochromic properties. These studies showcase the rational design of a family of amyloid-binding imaging agents that could be integrated with new optical approaches for the clinical diagnosis of amyloidoses, where accurate identification of the specific neurodegenerative disease could aid in the selection of a proper course for treatment.

Caged Garcinia Xanthones, a Novel Chemical Scaffold with Potent Antimalarial Activity.

Caged Garcinia xanthones (CGXs) constitute a family of natural products that are produced by tropical/subtropical trees of the genus Garcinia CGXs have a unique chemical architecture, defined by the presence of a caged scaffold at the C ring of a xanthone moiety, and exhibit a broad range of biological activities. Here we show that synthetic CGXs exhibit antimalarial activity against Plasmodium falciparum, the causative parasite of human malaria, at the intraerythrocytic stages. Their activity can be substantially improved by attaching a triphenylphosphonium group at the A ring of the caged xanthone. Specifically, CR135 and CR142 were found to be highly effective antimalarial inhibitors, with 50% effective concentrations as low as ∼10 nM. CGXs affect malaria parasites at multiple intraerythrocytic stages, with mature stages (trophozoites and schizonts) being more vulnerable than immature rings. Within hours of CGX treatment, malaria parasites display distinct morphological changes, significant reduction of parasitemia (the percentage of infected red blood cells), and aberrant mitochondrial fragmentation. CGXs do not, however, target the mitochondrial electron transport chain, the target of the drug atovaquone and several preclinical candidates. CGXs are cytotoxic to human HEK293 cells at the low micromolar level, which results in a therapeutic window of around 150-fold for the lead compounds. In summary, we show that CGXs are potent antimalarial compounds with structures distinct from those of previously reported antimalarial inhibitors. Our results highlight the potential to further develop Garcinia natural product derivatives as novel antimalarial agents.

Gambogic acid identifies an isoform-specific druggable pocket in the middle domain of Hsp90ß.

Because of their importance in maintaining protein homeostasis, molecular chaperones, including heat-shock protein 90 (Hsp90), represent attractive drug targets. Although a number of Hsp90 inhibitors are in preclinical/clinical development, none strongly differentiate between constitutively expressed Hsp90ß and stress-induced Hsp90α, the two cytosolic paralogs of this molecular chaperone. Thus, the importance of inhibiting one or the other paralog in different disease states remains unknown. We show that the natural product, gambogic acid (GBA), binds selectively to a site in the middle domain of Hsp90ß, identifying GBA as an Hsp90ß-specific Hsp90 inhibitor. Furthermore, using computational and medicinal chemistry, we identified a GBA analog, referred to as DAP-19, which binds potently and selectively to Hsp90ß. Because of its unprecedented selectivity for Hsp90ß among all Hsp90 paralogs, GBA thus provides a new chemical tool to study the unique biological role of this abundantly expressed molecular chaperone in health and disease.

Ratiometric mechanosensitive fluorescent dyes: Design and applications.

Fluorescent molecules, with their almost instantaneous response to external influences and relatively low-cost measurement instrumentation, have been attractive analytical tools and biosensors for centuries. More recently, advanced chemical synthesis and targeted design have accelerated the development of fluorescent probes. This article focuses on dyes with segmental mobility (known as fluorescent molecular rotors) that act as mechanosensors, which are known for their relationship of emission quantum yield with microviscosity. Fluorescence lifetime is directly related to quantum yield, but steady-state emission intensity is not. To remove confounding factors with steady-state instrumentation, dual-band emission dyes can be used, and molecular rotors have been developed that either have intrinsic dual emission or that have a non-sensitive reference unit to provide a calibration emission band. We report on theory, chemical structure, applications and targeted design of several classes of dual-emission molecular rotors.

Synthetic Strategies Toward the Decalin Motif of Maklamicin and Related Spirotetronates.

Herein we describe a scalable approach to the decalin moiety of maklamicin. Key to the synthesis is an intramolecular Diels-Alder (IMDA) reaction that proceeds via an endo-axial transition state to generate the desired stereochemistry. We explored the diastereoselectivity of the IMDA reaction as a function of both chiral catalysis and acyclic precursor stereochemistry.

Real-Time Monitoring of Alzheimer's-Related Amyloid Aggregation via Probe Enhancement-Fluorescence Correlation Spectroscopy.

This work describes the use of fluorescence correlation spectroscopy (FCS) and a novel amyloid-binding fluorescent probe, ARCAM 1, to monitor the aggregation of the Alzheimer's disease-associated amyloid ß-peptide (Aß). ARCAM 1 exhibits a large increase in fluorescence emission upon binding to Aß assemblies, making it an excellent candidate for probe enhancement FCS (PE-FCS). ARCAM 1 binding does not change Aß aggregation kinetics. It also exhibits greater dynamic range as a probe in reporting aggregate size by FCS in Aß, when compared to thioflavin T (ThT) or an Aß peptide modified with a fluorophore. Using fluorescent burst analysis (via PE-FCS) to follow aggregation of Aß, we detected soluble aggregates at significantly earlier time points compared to typical bulk fluorescence measurements. Autocorrelation analysis revealed the size of these early Aß assemblies. These results indicate that PE-FCS/ARCAM 1 based assays can detect and provide size characterization of small Aß aggregation intermediates during the assembly process, which could enable monitoring and study of such aggregates that transiently accumulate in biofluids of patients with Alzheimer's and other neurodegenerative diseases.

Spontaneously-forming spheroids as an in vitro cancer cell model for anticancer drug screening.

The limited translational value in clinic of analyses performed on 2-D cell cultures has prompted a shift toward the generation of 3-dimensional (3-D) multicellular systems. Here we present a spontaneously-forming in vitro cancer spheroid model, referred to as spheroids(MARY-X), that precisely reflects the pathophysiological features commonly found in tumor tissues and the lymphovascular embolus. In addition, we have developed a rapid, inexpensive means to evaluate response following drug treatment where spheroid dissolution indices from brightfield image analyses are used to construct dose-response curves resulting in relevant IC50 values. Using the spheroids(MARY-X) model, we demonstrate the unique ability of a new class of molecules, containing the caged Garcinia xanthone (CGX) motif, to induce spheroidal dissolution and apoptosis at IC50 values of 0.42 +/-0.02 µM for gambogic acid and 0.66 +/-0.02 µM for MAD28. On the other hand, treatment of spheroids(MARY-X) with various currently approved chemotherapeutics of solid and blood-borne cancer types failed to induce any response as indicated by high dissolution indices and subsequent poor IC50 values, such as 7.8 +/-3.1 µM for paclitaxel. Our studies highlight the significance of the spheroids(MARY-X) model in drug screening and underscore the potential of the CGX motif as a promising anticancer pharmacophore.

Total Synthesis and Structural Revision of Antibiotic CJ-16,264.

The total synthesis and structural revision of antibiotic CJ-16,264 is described. Starting with citronellal, the quest for the target molecule featured a novel bis-transannular Diels-Alder reaction that casted stereoselectively the decalin system and included the synthesis of six isomers before demystification of its true structure.

The Fe-S cluster-containing NEET proteins mitoNEET and NAF-1 as chemotherapeutic targets in breast cancer.

Identification of novel drug targets and chemotherapeutic agents is a high priority in the fight against cancer. Here, we report that MAD-28, a designed cluvenone (CLV) derivative, binds to and destabilizes two members of a unique class of mitochondrial and endoplasmic reticulum (ER) 2Fe-2S proteins, mitoNEET (mNT) and nutrient-deprivation autophagy factor-1 (NAF-1), recently implicated in cancer cell proliferation. Docking analysis of MAD-28 to mNT/NAF-1 revealed that in contrast to CLV, which formed a hydrogen bond network that stabilized the 2Fe-2S clusters of these proteins, MAD-28 broke the coordinative bond between the His ligand and the cluster's Fe of mNT/NAF-1. Analysis of MAD-28 performed with control (Michigan Cancer Foundation; MCF-10A) and malignant (M.D. Anderson-metastatic breast; MDA-MB-231 or MCF-7) human epithelial breast cells revealed that MAD-28 had a high specificity in the selective killing of cancer cells, without any apparent effects on normal breast cells. MAD-28 was found to target the mitochondria of cancer cells and displayed a surprising similarity in its effects to the effects of mNT/NAF-1 shRNA suppression in cancer cells, causing a decrease in respiration and mitochondrial membrane potential, as well as an increase in mitochondrial iron content and glycolysis. As expected, if the NEET proteins are targets of MAD-28, cancer cells with suppressed levels of NAF-1 or mNT were less susceptible to the drug. Taken together, our results suggest that NEET proteins are a novel class of drug targets in the chemotherapeutic treatment of breast cancer, and that MAD-28 can now be used as a template for rational drug design for NEET Fe-S cluster-destabilizing anticancer drugs.

Cyclopenta[b]naphthalene cyanoacrylate dyes: synthesis and evaluation as fluorescent molecular rotors.

We describe the design, synthesis and fluorescent profile of a family of environment-sensitive dyes in which a dimethylamino (donor) group is conjugated to a cyanoacrylate (acceptor) unit via a cyclopenta[b]naphthalene ring system. This assembly satisfies the typical D-π-A motif of a fluorescent molecular rotor and exhibits solvatochromic and viscosity-sensitive fluorescence emission. The central naphthalene ring system of these dyes was synthesized via a novel intramolecular dehydrogenative dehydro-Diels-Alder (IDDDA) reaction that permits incorporation of the donor and acceptor groups in variable positions around the aromatic core. A bathochromic shift of excitation and emission peaks was observed with increasing solvent polarity but the dyes exhibited a complex emission pattern with a second red emission band when dissolved in nonpolar solvents. Consistent with other known molecular rotors, the emission intensity increased with increasing viscosity. Interestingly, closer spatial proximity between the donor and the acceptor groups led to decreased viscosity sensitivity combined with an increased quantum yield. This observation indicates that structural hindrance of intramolecular rotation dominates when the donor and acceptor groups are in close proximity. The examined compounds give insight into how excited state intramolecular rotation can be influenced by both the solvent and the chemical structure.

3,4-diaminobenzoic acid derivatives as inhibitors of the oxytocinase subfamily of M1 aminopeptidases with immune-regulating properties.

Members of the oxytocinase subfamily of M1 aminopeptidases (ERAP1, ERAP2, and IRAP) play important roles in both the adaptive and innate human immune responses. Their enzymatic activity can contribute to the pathogenesis of several major human diseases ranging from viral and parasitic infections to autoimmunity and cancer. We have previously demonstrated that diaminobenzoic acid derivatives show promise as selective inhibitors for this group of aminopeptidases. In this study, we have thoroughly explored a series of 3,4-diaminobenzoic acid derivatives as inhibitors of this class of enzymes, achieving submicromolar inhibitors for ERAP2 (IC50 = 237 nM) and IRAP (IC50 = 105 nM). Cell-based analysis indicated that the lead compounds can be effective in downregulating macrophage activation induced by lipopolysaccharide and interferon-γ as well as cross-presentation by bone marrow-derived dendritic cells. Our results indicate that this class of inhibitors may be useful for the targeted downregulation of immune responses.

Spirotetronate polyketides as leads in drug discovery.

The discovery of chlorothricin (1) defined a new family of microbial metabolites with potent antitumor antibiotic properties collectively referred to as spirotetronate polyketides. These microbial metabolites are structurally distinguished by the presence of a spirotetronate motif embedded within a macrocyclic core. Glycosylation at the periphery of this core contributes to the structural complexity and bioactivity of this motif. The spirotetronate family displays impressive chemical structures, potent bioactivities, and significant pharmacological potential. This review groups the family members based on structural and biosynthetic considerations and summarizes synthetic and biological studies that aim to elucidate their mode of action and explore their pharmacological potential.