Skeletal Transformations of Terpenoid Forskolin Employing an Oxidative Rearrangement Strategy.

The skeletal transformations of diterpenoid forskolin were achieved by employing an oxidative rearrangement strategy. A library of 36 forskolin analogues with structural diversity was effectively generated. Computational analysis shows that 12 CTD compounds with unique scaffolds and ring systems were produced during the course of this work.

Unveiling the Antiviral Efficacy of Forskolin: A Multifaceted In Vitro and In Silico Approach.

Coleus forskohlii (Willd.) Briq. is a medicinal herb of the Lamiaceae family. It is native to India and widely present in the tropical and sub-tropical regions of Egypt, China, Ethiopia, and Pakistan. The roots of C. forskohlii are edible, rich with pharmaceutically bioactive compounds, and traditionally reported to treat a variety of diseases, including inflammation, respiratory disorders, obesity, and viral ailments. Notably, the emergence of viral diseases is expected to quickly spread; consequently, these data impose a need for various approaches to develop broad active therapeutics for utilization in the management of future viral infectious outbreaks. In this study, the naturally occurring labdane diterpenoid derivative, Forskolin, was obtained from Coleus forskohlii. Additionally, we evaluated the antiviral potential of Forskolin towards three viruses, namely the herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), hepatitis A virus (HAV), and coxsackievirus B4 (COX-B4). We observed that Forskolin displayed antiviral activity against HAV, COX-B4, HSV-1, and HSV-2 with IC50 values of 62.9, 73.1, 99.0, and 106.0 µg/mL, respectively. Furthermore, we explored the Forskolin's potential antiviral target using PharmMapper, a pharmacophore-based virtual screening platform. Forskolin's modeled structure was analyzed to identify potential protein targets linked to its antiviral activity, with results ranked based on Fit scores. Cathepsin L (PDB ID: 3BC3) emerged as a top-scoring hit, prompting further exploration through molecular docking and MD simulations. Our analysis revealed that Forskolin's binding mode within Cathepsin L's active site, characterized by stable hydrogen bonding and hydrophobic interactions, mirrors that of a co-crystallized inhibitor. These findings, supported by consistent RMSD profiles and similar binding free energies, suggest Forskolin's potential in inhibiting Cathepsin L, highlighting its promise as an antiviral agent.

Study on Chemotypic Variability of Coleus forskohlii Briq., Samples Collected from Different Phytogeographical Locations of India and Evaluation of Its Inhibitory Potential.

Coleus forskohlii Briq. is an important medicinal herb, endowed with a wide range of medicinal properties against the variety of ailments. Seven germplasm of C. forskohlii collected from different phyto-geographical locations and identification of elite chemotype was performed with the help of high performance thin layer chromatography. Data of soil analysis correlated with the bioactive compounds and inhibitory potential of the species. Quantification of forskolin and its isomer (iso-forskolin) content were done in all the collected samples of C. forskohlii, which revealed a wide range of variations, varying from 1.15-0.004% and 0.0091 to 0.1077% per dry weights basic, respectively. Variation in the bioactive content may be due to the soil nature and environmental factors. Soil analysis of collected samples demonstrated that there is significant variation in available NPK and micronutrient content and may be reasoned for existing chemotypic variability. In vitro biological activity (antioxidant and antidiabetic) analyses were performed, which reveals that germplasms have a high amount of forskolin and iso-forskolin, both show more activity. The aim of this study was to elucidate the effect of elicitors and precursors on the production of bioactive compounds and identification of best elite germplasm among the populations, to provide basic lead to the industry for commercial exploitability including its location-specific commercial cultivation.

Transdifferentiation of goat ear fibroblasts into lactating mammary epithelial cells induced by small molecule compounds.

Mammary epithelial cells are the only cells in the mammary glands that are capable of lactation and they are ideal for studying cellular and molecular biology mechanisms during growth, development and lactation of the mammary glands. The limiting factors in most of the currently available mammary epithelial cells are low cell viability, transgenerational efficiency and lactation function that renders them unsuitable for subsequent studies on mammary gland's cellular and lactation mechanisms and utilizing them as bioreactors. Hence, new methods are required to obtain mammary epithelial cells with high transgenerational efficiency and lactation function. In this study, transdifferentiation of goat ear fibroblasts (GEFs) into goat mammary epithelial cells (CiMECs) was induced in only eight days by five small molecule compounds, including 500 µg/mL VPA, 10 µM Tranylcypromine, 10 µM Forskolin, 1 µM TTNPB, 10 µM RepSox. Morphological observation, marker genes comparison, specific antigen expression and comparison of gene expression levels by transcriptome sequencing between the two types of cells that led to the primary deduction that CiMECs have similar biological properties to goat mammary epithelial cells (GMECs) and comparatively more lactation capacity. Therefore, we establish a novel reprogramming route to convert fibroblasts into CiMECs under fully chemically conditions. This study is expected to provide an in vitro platform for understanding cellular mechanisms such as mammary epithelial cells' fate determination and developmental differentiation, and also to find a new way to obtain a large number of functional mammary epithelial cells in vitro.

HtrA4 is up-regulated during trophoblast syncytialization and BeWo cells fail to syncytialize without HtrA4.

The outer layer of the human placenta comprises syncytiotrophoblast, which forms through fusion of cytotrophoblasts (syncytialization), and plays a critical role in maternal-fetal communication including nutrient/oxygen transportation and hormone secretion. Impairment in syncytialization inevitably affects pregnancy outcomes. High temperature requirement factor A 4 (HtrA4) is a placental-specific protease, expressed by various trophoblasts including syncytiotrophoblast, and significantly elevated in preeclampsia at disease presentation. However, it is unknown whether HtrA4 is important for syncytialization. Here we first examined HtrA4 expression in primary human cytotrophoblasts during syncytialization which occurs spontaneously in culture, and in BeWo cells which syncytialize upon forskolin stimulation. The success of syncytialization in each model was confirmed by significant up-regulation/secretion of ß-hCG, and the concurrent down-regulation of E-cadherin. In both models, HtrA4 mRNA and protein increased concomitantly with syncytialization. Furthermore, the secreted levels of ß-hCG and HtrA4 correlated significantly and positively in both models. We next knocked out HtrA4 in BeWo by CRISPR/Cas9. Upon forskolin treatment, control BeWo profoundly up-regulated ß-hCG and syncytin-1, down-regulated E-cadherin, and at the same time increased the formation of multinucleated cells, whereas BeWo cells without HtrA4 did not alter any of these parameters. Our data thus suggest that HtrA4 plays an essential role in syncytialization.

The isolation, purification and complete characterization of the diterpene forskolin from nutritional supplements.

Forskolin (1) is a diterpene found in the Coleus forskohlii plant that has been examined for its medical properties resulting from adenylyl cyclase activation. This article describes a straightforward purification method of 1 from commercially available weight loss capsules. In addition, there has been some ambiguity with respect to the use of the name 'forskolin' to describe 1 and related diterpenes, which this report serves to eliminate. Herein we detail the complete spectroscopic characterization of purified 1 as well as its single crystal X-ray structure.

Progress in heterologous biosynthesis of forskolin.

Forskolin, a class of labdane-type diterpenoid, has significant medicinal value in anticancer, antiasthmatic, antihypertensive, and heart-strengthening treatments. The main source of natural forskolin is its extraction from the cork tissue of the root of Coleus forskohlii. However, conventional modes of extraction pose several challenges. In recent years, the construction of microbial cell factories to produce medicinal natural products via synthetic biological methods has effectively solved the current problems and is a research hotspot in this field. This review summarizes the recent progress in the heterologous synthesis of forskolin via synthetic biological technology, analyzes the current challenges, and proposes corresponding strategies.

Total barley maiya alkaloids inhibit prolactin secretion by acting on dopamine D2 receptor and protein kinase A targets.

ETHNOPHARMACOLOGICAL RELEVANCE: Barley maiya from gramineous plants (Hordeum vulgare L.) is obtained from ripe fruits through germination and drying. It is often used to treat diseases associated with high prolactin levels. OBJECTIVE: To investigate the anti-hyperprolactinemia (anti-HPRL) mechanisms of total barley maiya alkaloids (TBMA) and hordenine. METHODS: This experiment included 9 groups: Normal group, TBMA group, hordenine group, TBMA + haloperidol group, TBMA + forskolin group, TBMA + 8-bromo-cAMP group, hordenine + haloperidol group, hordenine + forskolin group, and hordenine + 8-bromo-cAMP group. The prolactin (PRL) concentration in the supernatant and the total cAMP concentration in the cells were detected by ELISA. The expression levels of PRL, dopamine D2 receptor (DRD2) and cAMP/PKA/CREB protein were measured by Western Blot. RESULTS: In the TBMA group and the hordenine group, the PRL level in MMQ cells was significantly decreased, but in GH3 cells there was no change. DRD2 expression level was markedly increased, cAMP concentration was decreased, and the activity of PKA and CREB declined in MMQ cells. Compared with the TBMA group, there was a significant decrease of DRD2 expression level, a remarkable increase of PRL secretion and an increase of cAMP/PKA/CREB expression in MMQ cells within the TBMA + haloperidol group. Compared with the forskolin group, there was no significant change in PRL secretion and cAMP/PKA/CREB expression level in MMQ cells within the TBMA + forskolin group. There was a decrease in PRL secretion and cAMP/PKA/CREB expression level in MMQ cells within the TBMA + 8-bromo-cAMP group compared with the 8-bromo-cAMP group. Compared with the hordenine group, DRD2 expression level was significantly decreased, PRL secretion was markedly increased, and cAMP/PKA/CREB expression level was increased in MMQ cells within the hordenine + haloperidol group. There was no significant change in PRL secretion and cAMP/PKA/CREB expression level in MMQ cells within the hordenine + forskolin group compared with the forskolin group and within the hordenine + 8-bromo-cAMP group compared with the 8-bromo-cAMP group. CONCLUSION: TBMA and hordenine can both play an anti-HPRL role via DRD2, and TBMA can also act on PKA targets to exert its anti-HPRL effect. TBMA and hordenine may be potential treatment strategies for HPRL.

Reprogramming Cancer Stem-like Cells with Nanoforskolin Enhances the Efficacy of Paclitaxel in Targeting Breast Cancer.

Cancer stem-like cells (CSCs) have emerged as an important target for breast cancer therapy owing to their self-renewability, proliferation, and elevated chemoresistance properties. Here, we present a strategy of eliminating CSCs by differentiation therapy where "forced differentiation" reprograms CSCs so that they lose their intrinsic properties and become susceptible for conventional chemotherapeutic drugs. In this study, we report that a conventional chemotherapeutic paclitaxel enhances the stemness of CSCs, while a phytochemical forskolin being essentially nontoxic to CSCs possesses the intrinsic ability to reprogram them. To achieve simultaneous targeting of CSCs and bulk tumor cells, we used a co-delivery system where liquid crystal nanoparticles (LCN) were co-encapsulated with both paclitaxel and forskolin. LCN showed higher uptake, retention, and penetration potential in CSCs overcoming their high drug efflux property. Moreover, LCN improved the pharmacokinetic parameters of forskolin, which otherwise had very low retention and bioavailability. Forskolin-loaded LCN forced CSCs to exit from their mesenchymal state, which reduced their stemness and chemosensitized them while inhibiting E-cadherin-mediated survival and tumor-initiating potential as well as reversing paclitaxel-induced stemness. We further showed that upon administration of paclitaxel and forskolin co-loaded LCN to an orthotropic xenograft mouse model, the nanomedicine showed enhanced passive tumor targeting capability with very potent antitumor activity that eradicated small solid tumor in a single dose and showed no sign of tumor relapse or systemic toxicity over a long period. Overall, these findings give a proof of concept that co-delivery of forskolin and paclitaxel in a single nanoformulation can achieve overall tumor targeting where forskolin can efficiently reprogram/differentiate CSCs and paclitaxel can induce cytotoxicity in both differentiated CSCs and bulk tumor cells simultaneously. Hence, this study can provide a nanoformulation that can offer an efficient strategy for cancer therapy.

Small molecular compounds efficiently convert human fibroblasts directly into neurons.

No effective treatment is currently available for neurodegenerative diseases, and existing pharmacotherapy is inconsistent with severe side effects. Cell replacement therapy is promising for neurodegenerative disease treatment, and the induction of neurons is an unmet need for such therapy. The present study investigated the potential of a combined medium composed of conditioned medium and eight small molecular compounds in reprogramming human foreskin fibroblasts (HFFs) into neurons. HFFs were cultured from foreskin and then induced by small molecules to generate neurons. The results demonstrated that the conditioned medium containing forskolin, RepSox, SP600125, CHIR99021, Go6983, Y­27632, IXS9 and I­BET151 effectively induced human fibroblasts to change into neurons in vitro. Following a 30­day induction, the cells exhibited neuronal properties as determined by morphological and phenotypical alterations. The induced cells exhibited expression of neuronal markers, including class III ß­tubulin, microtubule­associated protein 2, vesicular glutamate transporter 1 and γ­aminobutyric acid, accompanied by increased expression of neuronal transcription factors, including neuronal differentiation 1 and achaete­scute family bHLH transcription factor 1, and decreased expression levels of fibroblast­specific genes. Furthermore, these cells also exhibited electrophysiological properties of neurons. Notably, the course of cell morphological alterations demonstrated the differentiation of fibroblasts into neurons. The present study provided a novel combination of existing small molecular compounds that efficiently reprogramed human fibroblasts into neurons.

Evaluation of Priming Efficiency of Forskolin in Tissue-Specific Human Mesenchymal Stem Cells into Dopaminergic Neurons: An In Vitro Comparative Study.

BACKGROUND: Human mesenchymal stem cells (hMSC) can be derived from various tissue sources and differentiated into dopaminergic (DAergic) neurons using various types of inducers. There are several strategies that have been reported to generate functional dopaminergic neuronal cells from hMSCs in the most efficient manner possible. However, this area is still under extensive research. In this study, we aim to compare hMSCs derived from bone marrow (BM), adipose tissue (AD) and dental pulp (DP) to generate functional dopaminergic neurons, using FGF2 and forskolin. Post-differentiation, multiple factors were used to characterize the cells at morphological, morphometric, ultra-structural, mRNA and protein levels for various markers (Nestin, NF, MAP2, Tuj1, TH, DAT, PitX3, Ngn2, Kv4.2, SCN5A). Cells' functionality was studied by calcium ion imaging, along with the amount of dopamine secreted by the cells in the culture medium. RESULTS: Data analysis revealed that forskolin has comparable effect on BM- and AD-derived MSC (28.43% and 29.46% DAergic neurons, respectively), whereas DP-MSC (42.78 ± 1.248% DAergic neurons) show better outcome in terms of efficient generation of DAergic neuronal cells, expression of neuronal associated markers, dopamine release and calcium ion efflux. Ultra-structural studies by SEM and TEM also revealed a substantial change in both cellular morphology and composition of cellular organelles. It was observed that AD-MSCs showed the best neuronal features, at morphological, gene, and protein levels upon induction with the above-mentioned induction cocktail. CONCLUSION: It may be concluded that a combination of FGF2 and forskolin yields functionally active dopaminergic neuronal cells in vitro, with highest percentage of the same from AD-MSCs, as compared to that in BM-MSCs and DP-MSCs. The outcomes and comparative evaluation provide a substantial platform for further studies on molecular pathways involved in the process of DAergic neurogenesis in individual cases.

A developed serum-free medium and an optimized chemical cocktail for direct conversion of human dermal fibroblasts into brown adipocytes.

Brown adipocytes coordinate systemic energy metabolism associated with the pathogenesis of obesity and related metabolic diseases including type 2 diabetes. We have previously reported chemical compound-induced brown adipocytes (ciBAs) converted from human dermal fibroblasts without using transgenes. In this study, to reveal a precise molecular mechanism underlying the direct conversion and human adipocyte browning, we developed serum-free brown adipogenic medium (SFBAM) with an optimized chemical cocktail consisting of Rosiglitazone, Forskolin, and BMP7. During the direct conversion, treatment with BMP7 enhanced Ucp1 expression rather than the conversion efficiency in the absence of BMP signalling inhibitors. Moreover, treatment with a TGF-ß signalling pathway inhibitor was no longer required in the serum-free medium, likely because the TGF-ß pathway was already suppressed. SFBAM and the chemical cocktail efficiently converted human dermal fibroblasts into ciBAs within four weeks. The ciBAs exhibited increased mitochondrial levels, elevated oxygen consumption rate, and a response to ß-adrenergic receptor agonists. Thus the ciBAs converted by the serum-free medium and the chemical cocktail provide a novel model of human brown (beige) adipocytes applicable for basic research, drug screening, and clinical applications.

Enhanced bioluminescent sensor for longitudinal detection of CREB activation in living cells.

Cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is associated with memory formation and controls cell survival and proliferation via regulation of downstream gene expression in tumorigenesis. As a transcription factor, CREB binds to cAMP response elements. Phosphorylation of CREB triggers transcriptional activation of CREB downstream genes following the interaction of the kinase-inducible domain (KID) of CREB with the KID interaction domain (KIX) of CREB-binding protein. Nevertheless, because of the lack of single-cell analytical techniques, little is known about spatiotemporal regulation of CREB phosphorylation. To analyze CREB activation in single living cells, we developed genetically encoded bioluminescent sensors using luciferase-fragment complementation: the sensors are designed based on KID-KIX interaction with a single-molecule format. The luminescence intensity of the sensor, designated as CREX (a sensor of CREB activation based on KID(CREB)-KIX interaction), increased by phosphorylation of CREB. Moreover, the luminescence intensity of CREX was sufficient to detect CREB activation in live-cell bioluminescence imaging for single-cell analysis because of the higher sensitivity. CREX sensor is expected to contribute to elucidation of the spatiotemporal regulation of CREB phosphorylation by applying single-cell analysis.

Rapid Screening of Forskolin-Type Diterpenoids of Blumea aromatica DC Using Ultra-High-Performance Liquid Chromatography Tandem Quadrupole Time-of-Flight Mass Spectrometry Based on the Mass Defect Filtering Approach.

The discovery of new active compounds of natural products tends to be increasingly more challenging due to chemical complexity and unpredictable matrices. Forskolin is an active natural labdane-type diterpenoid ingredient widely used worldwide for the treatment of glaucoma, heart failure, hypertension, diabetes, and asthma, and is expected to be a promising anticancer, anti-inflammation, and anti-HIV agent. In recent years, demand for forskolin in the medicine market has increased dramatically. However, natural forskolin originates exclusively from traditional Indian herb medicine Coleus forskohlii (Willd.) Briq. In a previous study, we isolated a series of diterpenoids including an 8,13-epoxy-14ene labdane carbon skeleton from Blumea aromatica DC. In order to identify alternative plant resources, a novel and effective strategy was proposed for the screening of potential forskolin-type diterpenoids (FSKD) compounds obtained from B. aromatica, using the mass defect filtering (MDF) strategy via ultra-high-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) approach. Within a narrow, well-defined mass defect range, the strategy developed could significantly improve the detection efficiency of selected FSKD compounds by filtering out certain major or moderate interference compounds. Additionally, the MS/MS cleavage behavior and the characteristic diagnostic ions of the FSKD compounds were proposed to be used in aiding structural identification of the filtration compounds. As a result, a total of 38 FSKD of B. aromatica were filtered out and tentatively identified. To the best of our knowledge, it was the first time that these forskolin-type diterpenoids were identified in B. aromatica, which significantly expands our understanding of the chemical constituents of Blumea species, and allows B. aromatica to be used as a potential alternative plant resource that contains these forskolin-type active compounds. The strategy proposed was proven efficient and reliable for the discovery of novel compounds of herbal extracts.

A Radical-Polar Crossover Annulation To Access Terpenoid Motifs.

A new catalytic radical-polar crossover annulation between two unsaturated carbonyl compounds is described. The annulation proceeds under exceptionally mild conditions and provides direct and expedient access to complex terpenoid motifs. Application of this chemistry allows for synthesis of forskolin, a densely functionalized terpenoid, in 14 steps from commercially available material.

Engineering of CYP76AH15 can improve activity and specificity towards forskolin biosynthesis in yeast.

BACKGROUND: Forskolin is a high-value diterpenoid produced exclusively by the Lamiaceae plant Coleus forskohlii. Today forskolin is used pharmaceutically for its adenyl-cyclase activating properties. The limited availability of pure  forskolin is currently hindering its full utilization, thus a new environmentally friendly, scalable and sustainable strategy is needed for forskolin production. Recently, the entire biosynthetic pathway leading to forskolin was elucidated. The key steps of the pathway are catalyzed by cytochrome P450 enzymes (CYPs), which have been shown to be the limiting steps of the pathway. Here we study whether protein engineering of the substrate recognition sites (SRSs) of CYPs can improve their efficiency towards forskolin biosynthesis in yeast. RESULTS: As a proof of concept, we engineered the enzyme responsible for the first putative oxygenation step of the forskolin pathway: the conversion of 13R-manoyl oxide to 11-oxo-13R-manoyl oxide, catalyzed by the CYP76AH15. Four CYP76AH15 variants-engineered in the SRS regions-yielded at least a twofold increase of 11-oxo-13R-manoyl oxide when expressed in yeast cells grown in microtiter plates. The highest titers (5.6-fold increase) were observed with the variant A99I, mutated in the SRS1 region. Double or triple CYP76AH15 mutant variants resulted in additional enzymes with optimized performances. Moreover, in planta CYP76AH15 can synthesize ferruginol from miltiradiene. In this work, we showed that the mutants affecting 11-oxo-13R-manoyl oxide synthesis, do not affect ferruginol production, and vice versa. The best performing variant, A99I, was utilized to reconstruct the forskolin biosynthetic pathway in yeast cells. Although these strains showed increased 11-oxo-manoyl oxide production and higher accumulation of other pathway intermediates compared to the native CYP76AH15, lower production of forskolin was observed. CONCLUSIONS: As demonstrated for CYP76AH15, site-directed mutagenesis of SRS regions of plant CYPs may be an efficient and targeted approach to increase the performance of these enzymes. Although in this work we have managed to achieve higher efficiency and specificity of the first CYP of the pathway, further work is necessary in order to increase the overall production of forskolin in yeast cells.

Natural products as modulators of the cyclic-AMP pathway: evaluation and synthesis of lead compounds.

It is now well recognized that the normal cellular response in mammalian cells is critically regulated by the cyclic-AMP (cAMP) pathway through the appropriate balance of adenylyl cyclase (AC) and phosphodiesterase-4 (PDE4) activities. Dysfunctions in the cAMP pathway have major implications in various diseases like CNS disorders, inflammation and cardiac syndromes and, hence, the modulation of cAMP signalling through appropriate intervention of AC/PDE4 activities has emerged as a promising new drug discovery strategy of current interest. In this context, synthetic small molecules have had limited success so far and therefore parallel efforts on natural product leads have been actively pursued. The early promise of using the diterpene forskolin and its semi-synthetic analogs as AC activators has given way to new leads in the last decade from novel natural products like the marine sesterterpenoids alotaketals and ansellones and the 9,9'-diarylfluorenone cored selaginpulvilins, etc. and their synthesis has drawn much attention. This review captures these contemporary developments, particularly total synthesis campaigns and structure-guided analog design in the context of AC and PDE-4 modulating attributes and the scope for future possibilities.

The Chemical Sensitivity and Electrical Activity of Individual Olfactory Sensory Neurons to a Range of Sex Pheromones and Food Odors in the Goldfish.

Although it is well established that the olfactory epithelium of teleost fish detects at least 6 classes of biologically relevant odorants using 5 types of olfactory sensory neurons (OSNs), little is understood about the specificity of individual OSNs and thus how they encode identity of natural odors. In this study, we used in vivo extracellular single-unit recording to examine the odor responsiveness and physiological characteristics of 109 individual OSNs in mature male goldfish to a broad range of biological odorants including feeding stimuli (amino acids, polyamines, nucleotides), sex pheromones (sex steroids, prostaglandins [PGs]), and a putative social cue (bile acids). Sixty-one OSNs were chemosensitive, with over half of these (36) responding to amino acids, 7 to polyamines, 7 to nucleotides, 5 to bile acids, 9 to PGs, and 7 to sex steroids. Approximately a quarter of the amino acid-sensitive units also responded to polyamines or nucleotides. Three of 6 amino acid-sensitive units responded to more than 1 amino acid compound, and 5 sex pheromone-sensitive units detected just 1 sex pheromone. While pheromone-sensitive OSNs also responded to the adenylyl cyclase activator, forskolin, amino acid-sensitive OSNs responded to either forskolin or a phospholipase C activator, imipramine. Most OSNs responded to odorants and activators with excitation. Our results suggest that pheromone information is encoded by OSNs specifically tuned to single sex pheromones and employ adenylyl cyclase, suggestive of a labeled-line organization, while food information is encoded by a combination of OSNs that use both adenylyl cyclase and phospholipase C and are often less specifically tuned.

Study of adenylyl cyclase-GαS interactions and identification of novel AC ligands.

Adenylyl cyclases (ACs) are membrane bound enzymes that catalyze the production of cAMP from ATP in response to the activation by G-protein Gαs. Different isoforms of ACs are ubiquitously expressed in different tissues involved in regulatory mechanisms in response to specific stimulants. There are 9 AC isoforms present in humans, with AC5 and AC6 proposed to play a vital role in cardiac functions. The activity of AC6 is sensitive to nitric oxide, such that nitrosylation of the protein might regulate its function. However, the information on structural determinants of nitrosylation in ACs and how they interact with Gαs is limited. Here we used homology modeling to build a molecular model of human AC6 bound to Gαs. Based on this 3D model, we predict the nitrosylation amenable cysteines, and identify potential novel ligands of AC6 using virtual ligand screening. Our model suggests Cys1004 in AC6 (subunit C2) and Cys174 in Gαs present at the AC-Gαs interface as the possible residues that might undergo reversible nitrosylation. Docking analysis predicted novel ligands of AC6 that include forskolin-based compounds and its derivatives. Further work involving site-directed mutagenesis of the predicted residues will allow manipulation of AC activity using novel ligands, and crucial insights on the role of nitrosylation of these proteins in pathophysiological conditions.

GDC-0879, a BRAFV600E Inhibitor, Protects Kidney Podocytes from Death.

Progressive kidney diseases affect approximately 500 million people worldwide. Podocytes are terminally differentiated cells of the kidney filter, the loss of which leads to disease progression and kidney failure. To date, there are no therapies to promote podocyte survival. Drug repurposing may therefore help accelerate the development of cures in an area of tremendous unmet need. In a newly developed high-throughput screening assay of podocyte viability, we identified the BRAFV600E inhibitor GDC-0879 and the adenylate cyclase agonist forskolin as podocyte-survival-promoting compounds. GDC-0879 protects podocytes from injury through paradoxical activation of the MEK/ERK pathway. Forskolin promotes podocyte survival by attenuating protein biosynthesis. Importantly, GDC-0879 and forskolin are shown to promote podocyte survival against an array of cellular stressors. This work reveals new therapeutic targets for much needed podocyte-protective therapies and provides insights into the use of GDC-0879-like molecules for the treatment of progressive kidney diseases.