The Pseudo-Natural Product Tafbromin Selectively Targets the TAF1 Bromodomain 2.

Phenotypic assays detect small-molecule bioactivity at functionally relevant cellular sites, and inherently cover a variety of targets and mechanisms of action. They can uncover new small molecule-target pairs and may give rise to novel biological insights. By means of an osteoblast differentiation assay which employs a Hedgehog (Hh) signaling agonist as stimulus and which monitors an endogenous marker for osteoblasts, we identified a pyrrolo[3,4-g]quinoline (PQ) pseudo-natural product (PNP) class of osteogenesis inhibitors. The most potent PQ, termed Tafbromin, impairs canonical Hh signaling and modulates osteoblast differentiation through binding to the bromodomain 2 of the TATA-box binding protein-associated factor 1 (TAF1). Tafbromin is the most selective TAF1 bromodomain 2 ligand and promises to be an invaluable tool for the study of biological processes mediated by TAF1(2) bromodomains.

Illuminating Dark Chemical Matter Using the Cell Painting Assay.

Screening for small-molecule modulators of disease-relevant targets and phenotypes is the first step on the way to new drugs. Large compound libraries have been synthesized by academia and, particularly, pharmaceutical companies to meet the need for novel chemical entities that are as diverse as possible. Screening of these compound libraries revealed a portion of small molecules that is inactive in more than 100 different assays and was therefore termed "dark chemical matter" (DCM). Deorphanization of DCM promises to yield very selective compounds as they are expected to have less off-target effects. We employed morphological profiling using the Cell Painting assay to detect bioactive DCM. Within the DCM collection, we identified bioactive compounds and confirmed several modulators of microtubules, DNA synthesis, and pyrimidine biosynthesis. Profiling approaches are, therefore, powerful tools to probe compound collections for bioactivity in an unbiased manner and are particularly suitable for deorphanization of DCM.

A divergent intermediate strategy yields biologically diverse pseudo-natural products.

The efficient exploration of biologically relevant chemical space is essential for the discovery of bioactive compounds. A molecular design principle that possesses both biological relevance and structural diversity may more efficiently lead to compound collections that are enriched in diverse bioactivities. Here the diverse pseudo-natural product (PNP) strategy, which combines the biological relevance of the PNP concept with synthetic diversification strategies from diversity-oriented synthesis, is reported. A diverse PNP collection was synthesized from a common divergent intermediate through developed indole dearomatization methodologies to afford three-dimensional molecular frameworks that could be further diversified via intramolecular coupling and/or carbon monoxide insertion. In total, 154 PNPs were synthesized representing eight different classes. Cheminformatic analyses showed that the PNPs are structurally diverse between classes. Biological investigations revealed the extent of diverse bioactivity enrichment of the collection in which four inhibitors of Hedgehog signalling, DNA synthesis, de novo pyrimidine biosynthesis and tubulin polymerization were identified from four different PNP classes.

A highly enantioselective intramolecular 1,3-dipolar cycloaddition yields novel pseudo-natural product inhibitors of the Hedgehog signalling pathway.

De novo combination of natural product (NP) fragments by means of efficient, complexity- and stereogenic character-generating transformations to yield pseudo-natural products (PNPs) may explore novel biologically relevant chemical space. Pyrrolidine- and tetrahydroquinoline fragments rarely occur in combination in nature, such that PNPs that embody both fragments might represent novel NP-inspired chemical matter endowed with bioactivity. We describe the synthesis of pyrrolo[3,2-c]quinolines by means of a highly enantioselective intramolecular exo-1,3-dipolar cycloaddition catalysed by the AgOAc/(S)-DMBiphep complex. The cycloadditions proceeded in excellent yields (up to 98%) and with very high enantioselectivity (up to 99% ee). Investigation of the resulting PNP collection in cell-based assays monitoring different biological programmes led to the discovery of a structurally novel and potent inhibitor of the Hedgehog signalling pathway that targets the Smoothened protein.

Unprecedented Combination of Polyketide Natural Product Fragments Identifies the New Hedgehog Signaling Pathway Inhibitor Grismonone.

Pseudo-natural products (pseudo-NPs) are de novo combinations of natural product (NP) fragments that define novel bioactive chemotypes. For their discovery, new design principles are being sought. Previously, pseudo-NPs were synthesized by the combination of fragments originating from biosynthetically unrelated NPs to guarantee structural novelty and novel bioactivity. We report the combination of fragments from biosynthetically related NPs in novel arrangements to yield a novel chemotype with activity not shared by the guiding fragments. We describe the synthesis of the polyketide pseudo-NP grismonone and identify it as a structurally novel and potent inhibitor of Hedgehog signaling. The insight that the de novo combination of fragments derived from biosynthetically related NPs may also yield new biologically relevant compound classes with unexpected bioactivity may be considered a chemical extension or diversion of existing biosynthetic pathways and greatly expands the opportunities for exploration of biologically relevant chemical space by means of the pseudo-NP principle.

Spatial targeting of Bcl-2 on endoplasmic reticulum and mitochondria in cancer cells by lipid nanoparticles.

The presence of the same proteins at different sub-cellular locations with completely different functions adds to the complexity of signalling pathways in cancer. Subsequently, it becomes indispensable to understand the diverse critical roles of these proteins based on their spatial distribution for the development of improved cancer therapeutics. To address this, in this work, we report the development of endoplasmic reticulum (ER) and mitochondria targeted nanoscale particles to spatially impair anti-apoptotic Bcl-2 protein in these organelles in HeLa cervical cancer cells. Confocal microscopy and gel electrophoresis confirmed that these nanoparticles selectively home into ER and mitochondria and inhibited Bcl-2 localized there. Interestingly, Bcl-2 inhibition in ER induced ER stress leading to autophagy, whereas inhibition of Bcl-2 in mitochondria leads to mitochondrial damage and programmed cell death (apoptosis) in HeLa cells. These nanoscale platforms can be further explored as chemical biology tools to decipher the location-function relationship of proteins towards next generation cancer therapeutics.

Mitochondrial Impairment by Cyanine-Based Small Molecules Induces Apoptosis in Cancer Cells.

Mitochondrion, the powerhouse of the cells, has emerged as one of the unorthodox targets in anticancer therapy due to its involvement in several cellular functions. However, the development of small molecules for selective mitochondrial damage in cancer cells remained limited and less explored. To address this, in our work, we have synthesized a natural product inspired cyanine-based 3-methoxy pyrrole small molecule library by a concise strategy. This strategy involves Vilsmeier and Pd(0) catalyzed Suzuki cross-coupling reactions as key steps. The screening of the library members in HeLa cervical cancer cells revealed two new molecules that localized into subcellular mitochondria and damaged them. These small molecules perturbed antiapoptotic (Bcl-2/Bcl-xl) and pro-apoptotic (Bax) proteins to produce reactive oxygen species (ROS). Molecular docking studies showed that both molecules bind more tightly with the BH3 domain of Bcl-2 proteins compared to obatoclax (a pan-Bcl-2 inhibitor). These novel small molecules arrested the cell cycle in the G0/G1 phase, cleaved caspase-3/9, and finally prompted late apoptosis. This small molecule-mediated mitochondrial damage induced remarkably high cervical cancer cell death. These unique small molecules can be further explored as chemical biology tools and next-generation organelle-targeted anticancer therapy.

Hydrazide-Hydrazone Small Molecules as AIEgens: Illuminating Mitochondria in Cancer Cells.

Aggregation-induced-emission luminogens (AIEgens) have gained considerable attention as interesting tools for several biomedical applications, especially for bioimaging due to their brightness and photostability. Numerous AIEgens have been developed for lighting up the subcellular organelles to understand their forms and functions not only healthy but also unhealthy states, such as in cancer cells. However, there is lack of easily synthesizable, biocompatible small molecules for illuminating mitochondria (powerhouses) inside cells. To address this issue, an easy and short synthesis of new biocompatible hydrazide-hydrazone-based small molecules with remarkable aggregation-induced emission (AIE) properties is described. These small-molecule AIEgens showed hitherto unobserved AIE properties due to dual intramolecular H-bonding confirmed by theoretical calculation, pH- and temperature-dependent fluorescence and X-ray crystallographic studies. Confocal microscopy showed that these AIEgens were internalized into the HeLa cervical cancer cells without showing any cytotoxicity. One of the AIEgens was tagged with a triphenylphosphine (TPP) moiety, which successfully localized in the mitochondria of HeLa cells in a selective way compared to L929 noncancerous fibroblast cells. These unique hydrazide-hydrazone-based biocompatible AIEgens can serve as powerful tools to illuminate multiple subcellular organelles to elucidate their forms and functions in cancer cells for next-generation biomedical applications.

Impairing Powerhouse in Colon Cancer Cells by Hydrazide-Hydrazone-Based Small Molecule.

Mitochondrion has emerged as one of the unconventional targets in next-generation cancer therapy. Hence, small molecules targeting mitochondria in cancer cells have immense potential in the next-generation anticancer therapeutics. In this report, we have synthesized a library of hydrazide-hydrazone-based small molecules and identified a novel compound that induces mitochondrial outer membrane permeabilization by inhibiting antiapoptotic B-cell CLL/lymphoma 2 (Bcl-2) family proteins followed by sequestration of proapoptotic cytochrome c. The new small molecule triggered programmed cell death (early and late apoptosis) through cell cycle arrest in the G2/M phase and caspase-9/3 cleavage in HCT-116 colon cancer cells, confirmed by an array of fluorescence confocal microscopy, cell sorting, and immunoblotting analysis. Furthermore, cell viability studies have verified that the small molecule rendered toxicity to a panel of colon cancer cells (HCT-116, DLD-1, and SW-620), keeping healthy L929 fibroblast cells unharmed. The novel small molecule has the potential to form a new understudied class of mitochondria targeting anticancer agent.

Iron-Catalyzed Batch/Continuous Flow C-H Functionalization Module for the Synthesis of Anticancer Peroxides.

Iron-catalyzed dehydrogenative cross-coupling of carbonyl compounds with aliphatic peroxide was developed under mild conditions. A library of linear alkylated and arylated peroxides are synthesized in good to excellent yield. This method is highly selective and general for a range of biologically important derivatives of 2-oxindole, barbituric acid, and 4-hydroxy coumarin with a good functional group tolerance and without the cleavage of the peroxide bond. This peroxidation reaction is upscalable to grams and also synthesizable in continuous flow with increased safety in short duration. Mechanistic investigation reveals Fe-(II) undergoes redox type process to generate the radical intermediates, which subsequently recombine selectively to form the stable peroxides. The potential of peroxides is evaluated by cell viability assay and found to exhibit the good anticancer activity with minimum IC50= 5.3 µM.

Biological Activity of Coumarin Derivatives as Anti-Leishmanial Agents.

Cutaneous leishmaniasis affects nearly 0.7 to 1.3 million people annually. Treatment of this disease is difficult due to lack of appropriate medication and the growing problem of drug resistance. Natural compounds such as coumarins serve as complementary therapeutic agents in addition to the current treatment modalities. In this study, we have performed an in-silico screening of the coumarin derivatives and their anti-leishmanial properties has been explored both in-vitro and in-vivo. One of the compounds (compound 2) exhibited leishmanicidal activity and to further study its properties, nanoliposomal formulation of the compound was developed. Treatment of cutaneous lesions in BALB/c mice with compound 2 showed significantly reduced lesion size as compared to the untreated mice (p<0.05) suggesting that compound 2 may possess anti-leishmanial properties.

Synthesis, characterization and in vitro evaluation of novel vitamin D3 nanoparticles as a versatile platform for drug delivery in cancer therapy.

Development of novel nanotechnology based platforms can impact cancer therapeutics in a paradigm shifting manner. The major concerns in drug delivery in cancer therapy are the biocompatibility, biodegradability, non-toxic nature, easy and short synthesis and versatility of the nanovectors. Herein we report the engineering of versatile nanoparticles from biocompatible, biodegradable and non-toxic lipid soluble vitamin D3. We have conjugated different clinically used cytotoxic drugs (paclitaxel and doxorubicin) as well as PI3 kinase inhibitor (PI103) with vitamin D3 using a succinic acid linker. Sub-200 nm, monodispersed nanoparticles with high drug loading were engineered from the vitamin D3-succinic acid-drug conjugates. These nanoparticles released the active drugs at pH 5.5 in a slow and sustained manner over 100 h. Furthermore, these nanoparticles were taken up by HeLa cells into the low pH lysosomal compartments through an endocytosis mechanism in 6 h. Finally, these drug loaded vitamin D3 nanoparticles induced HeLa cervical cancer cell death in a dose dependent manner at 48 h to show their potential in cancer therapeutics.