a-fluorescence-based-protocol-for-preliminary-screening-protein

A Fluorescence-based Protocol for Preliminary Screening of Protein Synthesis Inhibitors from Natural Sources

Natural products represent a rich source of therapeutic compounds with applications across multiple diseases, particularly cancer. By leveraging these ...

Louisiana State University

The abietane diterpenoids ferruginol (), tanshinone IIA (), and carnosol () are well-known for their interesting pharmacological properties, including antitumor, similar to other natural and semisynthetic abietanes. In this study, a pair of semisynthetic C18-functionalized analogues of  and  were prepared from the commercially available (+)-dehydroabietylamine or readily obtained methyl dehydroabietate. Semisynthetic ferruginol () and some selected analogues, together with the synthesized analogues, were tested in vitro for the inhibition of proliferation in four breast cancer cell lines, SUM149, MDA-MB231, T47D, and MCF07. As a result, several tested abietane analogues decreased cell proliferation and enhanced cell death, with IC in the range 1.3-18.7 μM. This work demonstrates the antitumor activities of two tested compounds, making these molecules interesting for the development of new anticancer agents.

Discovery of Novel Bioactive Tanshinones and Carnosol Analogues against Breast Cancer.

Combining antiviral drugs with different mechanisms of action can help prevent the development of resistance by attacking the infectious agent through multiple pathways. Additionally, by using faster and more economical screening methods, effective synergistic drug candidates can be rapidly identified, facilitating faster paths to clinical testing. In this work, a rapid method was standardized to identify possible synergisms from drug combinations. We analyzed the possible reduction in the antiviral effective concentration of drugs already approved by the FDA, such as ivermectin (IVM), ribavirin (RIBA), and acyclovir (ACV) against Zika virus (ZIKV), Chikungunya virus (CHIKV), and herpes virus type 2 (HHV-2). Essential oils (EOs) were also included in the study since they have been reported for more than a couple of decades to have broad-spectrum antiviral activity. We also continued studying the antiviral properties of one of our patented molecules with broad-spectrum antiviral activity, the ferruginol analog 18-(phthalimid-2-yl)ferruginol (phthFGL), which presented an IC of 25.6 μM for the three types of virus. In general, the combination of IVM, phthFGL, and oregano EO showed the greatest synergism potential against CHIKV, ZIKV, and HHV-2. For instance, this combination achieved reductions in the IC value of each component up to ~8-, ~27-, and ~12-fold for CHIKV, respectively. The ternary combination of RIBA, phthFGL, and oregano EO was slightly more efficient than the binary combination RIBA/phthFGL but much less efficient than IVM, phthFGL, and oregano EO, which indicates that IVM could contribute more to the differentiation of cell targets (for example via the inhibition of the host heterodimeric importin IMP α/β1 complex) than ribavirin. Statistical analysis showed significant differences among the combination groups tested, especially in the HHV-2 and CHIKV models, with  = 0.0098. Additionally, phthFGL showed a good pharmacokinetic profile that should encourage future optimization studies.

Synergistic In Vitro Antiviral Effect of Combinations of Ivermectin, Essential Oils, and 18-(Phthalimid-2-yl)ferruginol against Arboviruses and Herpesvirus.

Monoterpenes and meroterpenes are two large classes of isoprene-based molecules produced by terrestrial plants and unicellular organisms as diverse secondary metabolites. The global rising incidence of cancer has led to a renewed interest in natural products. These monoterpenes and meroterpenes represent a novel source of molecular scaffolds that can serve as medicinal chemistry platforms for the development of potential preclinical leads. Furthermore, some of these natural products are either abundant, or their synthetic strategies are scalable as it will be indicated here, facilitating their derivatization to expand their scope in drug discovery. This review is a collection of representative updates (from 2016-2023) in biologically active monoterpene and meroterpenoid natural products and focuses on the recent findings of the pharmacological potential of these bioactive compounds as well as the newly developed synthetic strategies employed to access them. Particular emphasis will be placed on the anticancer and antioxidant potential of these compounds in order to raise knowledge for further investigations into the development of potential anti-cancer therapeutics. The mounting experimental evidence from various research groups across the globe regarding the use of these natural products at pre-clinical levels, renders them a fast-track research area worth of attention.

Recent Advances in Chemistry and Antioxidant/Anticancer Biology of Monoterpene and Meroterpenoid Natural Product.

Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor type 2 expression. It is known for its high malignancy, invasiveness, and propensity for metastasis, resulting in a poor prognosis due to the absence of beneficial therapeutic targets. Natural products derived from mushrooms have gained significant attention in neoplastic therapy due to their potential medicinal properties. The therapeutic potential of  in breast cancer has been highlighted by our group, suggesting its use as an adjuvant treatment. The present study aims to assess the potential antineoplastic capacity of two Caribbean native  species found in Puerto Rico,  () and  (). Antiproliferative studies were conducted via cell viability assays after cultivation, harvesting, and fractionation of both species. The obtained results indicate that most of the fractions show some cytotoxicity against all cell lines, but 33% of the fractions (F1, F2, F7, F12) display selectivity towards cancer cell models. We demonstrate for the first time that native  species can generate metabolites with anti-TNBC properties. Future avenues will focus on structure elucidation of the most active fractions of these  extracts.

Selective Antineoplastic Potential of Fractionated Caribbean Native  Species Extracts on Triple-Negative Breast Cancer Cells.

 is a medicinal mushroom that produces various pharmacological compounds, including triterpenoids. A major bioactive component of  is ergosterol peroxide (EP), which is attributed to its anticancer effects. The current study focuses on the  ADME (absorption, distribution, metabolism, and elimination),  efficacy and toxicity of EP, and the synthesis of new EP derivatives to improve aqueous solubility. It was found that EP is metabolically stable in liver microsomes and plasma.  studies showed that EP inhibits tumor growth in murine cancer models, and it is well tolerated by mice. The maximum tolerated dose was investigated in mice at escalating doses with a defined maximum amount of 500 mg/kg, which indicated no signs of toxicity, confirmed by plasma chemistry and analysis of harvested tissues. Complementary organ toxicity assays including cardio and hepatotoxicity assays of EP demonstrated no inhibitory effects. Next, a focused library of EP derivatives was developed to investigate the iterative addition of heteroatoms to improve the aqueous solubility properties of EP. Significant solubility improvement was observed by the introduction of hydrogen bonding promoting groups, particularly the sulfate group. Superior aqueous solubility properties are directly correlated with the biological activity of the compound against triple-negative breast cancer cellular (TNBC) models. The EP derivatives maintain ample therapeutic index at the tested concentrations, indicating they engage with the same biological target(s) as the parental compound (EP). The combined studies indicate that EP and its derivatives are selective TNBC cell death inducers, while sparing noncancerous tissue.

Early Preclinical Studies of Ergosterol Peroxide and Biological Evaluation of Its Derivatives.

Ergosterol peroxide (EP) triggers apoptosis pathways by inducing reactive oxygen species (ROS) in TNBC cell lines. Excess ROS production is associated with major damage to mitochondria. We hypothesized that EP may act through ROS-induced mitochondrial dysfunction. Therefore, we performed a series of assays that assessed mitochondrial membrane potential (MMP), cellular respiration, and glycolysis in TNBC models. Cardiomyocytes derived from human-induced pluripotent stem cells were chosen as a non-cancerous model because of their high mitochondrial content. Two in vivo TNBC models were used to quantify the effect of EP on tumor volume and metastases. EP reduced MMP and disrupted mitochondrial functions exclusively in TNBC cells. In vivo EP was effective in reducing tumor volume without affecting liver function. There was also a significant decrease in metastasis to the lung, liver, and cancer stem cells following treatment. These results suggest EP is a promising therapy for TNBC.

Ergosterol Peroxide Disrupts Triple-Negative Breast Cancer Mitochondrial Function and Inhibits Tumor Growth and Metastasis.

The synthesis of organic molecules through regioselective and enantioselective processes represents a significant challenge in organic chemistry, but constructing molecules with all-carbon quaternary centers poses even greater complexity. The importance of synthetic organic chemistry in daily life cannot be overstated, as organic molecules are fundamental to human existence - from the nutrients that sustain us to the pharmaceutical products that maintain our health. Fundamental knowledge on chemical reactivity, particularly that of carbon-carbon bond formation in sterically hindered substrates, is of great interest in developing and studying new chemical matter. Increases in precision medicine have also highlighted the importance of molecular selectivity towards biological targets and the need to access complex molecular scaffolds with unique stereochemistry features such as quaternary carbon centers. The scarcity of molecules containing all-carbon quaternary centers has constrained our ability to fully explore their properties. This report examines recent methodological advances since 2019 in their synthesis, focusing on three key synthetic approaches: single electron transfer (SET) mediated reactions, reactions mediated by chemical phenomena associated with charge separation (electrochemistry), Lewis acid and transition metal mediated cyclization/rearrangement reactions. These innovative synthetic strategies open new pathways for creating complex chemical structures, advancing our ability to develop both novel therapeutic candidates and new materials. The enhanced efficiency of these approaches enables the exploration of increasingly sophisticated molecular architectures, expanding opportunities for discovering compounds with unique biological activities and material properties.

Recent synthetic methodology advancements towards all-carbon quaternary center formation and applications.

Ferruginol, tanshinones and carnosol are considered privileged natural products due to their demonstrated diverse biological activities with relevance to cancer research. Globally, cancer continues to be a major contributor to mortality rates, making these compounds potentially valuable molecular scaffolds for further development as potential anticancer agents. In this work, a focused library of ferruginol, tanshinone IIA, and carnosol analogues was studied to examine their effectiveness against various solid tumor models. The compounds were efficiently synthesized from either methyl 12-hydroxy-dehydroabietate or 12-hydroxydehydroabietylamine in 1-3 step processes with good chemical yields. The compounds that were synthesized underwent a methodical evaluation using multiple biological tests (including viability assays, clonogenic assays, and mitochondrial membrane polarization measurements) to determine their ability to inhibit in vitro the growth of three breast cancer cell linages. It was determined that while most compounds exhibited biological activity, compounds  and  demonstrated significant efficacy against triple negative breast cancer cells. These compounds continue to show promising biological activity, suggesting that additional studies to understand their mechanisms of action would be valuable.

Article	Year
Discovery of Novel Bioactive Tanshinones and Carnosol Analogues against Breast Cancer. Cancers\| PubMed ID: 36831660	2023
Synergistic In Vitro Antiviral Effect of Combinations of Ivermectin, Essential Oils, and 18-(Phthalimid-2-yl)ferruginol against Arboviruses and Herpesvirus. Pharmaceuticals (Basel, Switzerland)\| PubMed ID: 38004467	2023
Recent Advances in Chemistry and Antioxidant/Anticancer Biology of Monoterpene and Meroterpenoid Natural Product. Molecules (Basel, Switzerland)\| PubMed ID: 38202861	2024
Selective Antineoplastic Potential of Fractionated Caribbean Native Species Extracts on Triple-Negative Breast Cancer Cells. Pharmaceuticals (Basel, Switzerland)\| PubMed ID: 39065715	2024
Early Preclinical Studies of Ergosterol Peroxide and Biological Evaluation of Its Derivatives. ACS omega\| PubMed ID: 39246459	2024
Ergosterol Peroxide Disrupts Triple-Negative Breast Cancer Mitochondrial Function and Inhibits Tumor Growth and Metastasis. International journal of molecular sciences\| PubMed ID: 40429733	2025
Recent synthetic methodology advancements towards all-carbon quaternary center formation and applications. Tetrahedron\| PubMed ID: 40454198	2025
In Vitro Effect of Ferruginol, Tanshinone, and Carnosol Analogues on the Proliferation of Three Breast Cancer Cell Lines. Molecules (Basel, Switzerland)\| PubMed ID: 40572493	2025

Fatima Rivas

Biography

JoVE Journal Publications

Other Publications