Isolation and design of diterpenoids from Plectranthus species

Cancer is still a serious threat to public health and according to WHO in 2018, thre are 1 in 6 deaths due to cancer. Muilti-drug resistant remains a major challenge to cancer therapy and there for the need to develop new reversal MDR agents. Natural products from medicinal plants represent a major resource of novel therapeutic substances for combating serious diseases including cancer. Cytotoxicity screenings have identified Plectranthus plants as potential sources of cytotoxic compounds. Most importantly, abietane type diterpenoids known for their cytotoxicity. In this work, sixteen Plectranthus spp. were screened for their bioactivity. Their acetone extracts were prepared using the ultrasound-assisted extraction method (10 % (w/v) and screened for their antimicrobial, antioxidant, general toxicity. The most bioactive extracts were selected and tested against three different cancer cell lines. P. hadiensis and P. mutabilis were the most bioactive and their phytochemical studies was done to identify the compounds that may be responsible for their bioactivity. The HPLC analysis of P. hadiensis leaves showed that the known abietane diterpene, 7α-acetoxy-6β-hydroxyroyleanone (Roy) was the major compound in this extract. Roy was isolated and tested against the aggressive type of triple-negative breast cancer (MDA-MB-231S) cell lines. P. hadiensis extract and Roy reduced the viability of MDA-MB-231S cancer cell lines, showing an IC50 value of 25.6 μg/mL and 5.5 μM (2.15 μg/mL) respectively, suggesting that this lead molecule could be responsible for the bioactivity of this extract. Due to the poor aqueous solubility of royleanones, the need to decrease the site effects and enhance a target drug delivery; Roy, its hemisynthetic derivative 7α-acetoxy-6β-benzoyloxyroyleanone (12BzRoy) and 6,7-dehydroroyleanone (DHR) isolated from the essential oil of P. madagascariensis were used as lead molecules for the synthesis of self-assembled nanoparticles. Squalene (Sq), Oleic acid (OA) and 1-bromodecane were used was linkers. Roy-OA, DHR-sq, and 12BzRoy-sq conjugates were successfully synthesized and their nanoassemblies characterized based on size, Pdl, zeta potential, and morphology. The release profile of Roy was determined from Roy-OA NPs at physiological pH 7.4. The biological activity of DHR.sq and, Roy-OA NPs were evaluated, and both were found to have less bioactivity when compared with Roy and DHR respectively. These results suggested that these nanoassemblies act as prodrugs for the release of cytotoxic lead molecules. The second bioactive extract, P. mutabilis was subjected to a bio-guided fractionation of its acetone extract. From this extract, one new nor-abietane diterpene, (+)-(5S,10R)-10,11,12-trihydroxy-6,7-dioxo-20-nor-abieta-8,11,13-triene (1) alongside three known abietane-type diterpenoids Coleon-U-quinone (2), 8α,9α-epoxycoleon-U-quinone (3), and Coleon U (4) were isolated. The structure of the compounds was elucidated using different spectroscopic techniques (1D and 2D-NMR, LCMS, IR) and HRMS. From the ESI+ MS/MS fragmentation patterns analysis, an additional acetoxy derivative of an abietane diterpenoid, compound (5) was tentatively identified. Compounds 1-4 were quantified in the extract using HPLC-DAD. The isolated compounds were found to be unstable. Coleon U for instant was found to rapidly interchanges to its quinone derivative coleon U quinone. For this reason, a biosynthetic relation between the compounds was done. The results suggests that both the quinone (2) and the epoxyquinone (3) are formed directly from Coleon U (4). Cytotoxicity of the isolated compounds was done and their interaction with P-gp studied using the model systems human non-small cell lung carcinoma cells (NCI-H460), its MDR variant with P-gp expression (NCI-H460/R), and human embryonic pulmonary fibroblasts (MRC-5). In conclusion, this work provides important information about the biological activity of several Plectranthus spp. In a review, and published works, we identified the importance of using Artemia salina as a screening model in natural product chemistry. The phytochemical study of P. mutabilis was done for the first time in this study and we identified compounds with a unique way of interacting with P-gp. In addition, we proposed two paths for the possible formation of epoxide 3 from 4. We equally successively prepared self-assembly nanoparticles of royleanones using oleic and squalene linkers these by improving the the aqueous solubility of 7α-acetoxy-6β-hydroxyroyleanone, 7α-acetoxy-6β-benzoyloxyroyleanone and 6,7-dehydroroyleanone

Genetic heterogeneity and actionable mutations in HER2-positive primary breast cancers and their brain metastases

Brain metastases constitute a challenge in the management of patients with HER2- positive breast cancer treated with anti-HER2 systemic therapies. Here we sought to define the repertoire of mutations private to or enriched for in HER2-positive brain metastases. Massively parallel sequencing targeting all exons of 254 genes frequently mutated in breast cancers and/or related to DNA repair was used to characterize the spatial and temporal heterogeneity of HER2-positive breast cancers and their brain metastases in six patients. Data were analyzed with state-of-the-art bioinformatics algorithms and selected mutations were validated with orthogonal methods. Spatial and temporal inter-lesion genetic heterogeneity was observed in the HER2-positive brain metastases from an index patient subjected to a rapid autopsy. Genetic alterations restricted to the brain metastases included mutations in cancer genes FGFR2, PIK3CA and ATR, homozygous deletion in CDKN2A and amplification in KRAS. Shifts in clonal composition and the acquisition of additional mutations in the progression from primary HER2-positive breast cancer to brain metastases following anti-HER2 therapy were investigated in additional five patients. Likely pathogenic mutations private to or enriched in the brain lesions affected cancer and clinically actionable genes, including ATR, BRAF, FGFR2, MAP2K4, PIK3CA, RAF1 and TP53. Changes in clonal composition and the acquisition of additional mutations in brain metastases may affect potentially actionable genes in HER2-positive breast cancers. Our observations have potential clinical implications, given that treatment decisions for patients with brain metastatic disease are still mainly based on biomarkers assessed in the primary tumor

Study of mechanisms of action involved in the Biocontrol of a strain of Aureobasidium pullulans (De Bary) Arnaud against Penicillium expansum Link on postharvest apples

L’agent de lutte biologique Aureobasidium pullulans souche Ach1-1 a présenté une grandepotentialité dans le contrôle de Penicillium expansum, l’agent causal de la pourriture bleue des pommes enconservation. Les mécanismes d’action qui sont les plus impliqués dans son activité antagoniste ont étéanalysés, au cours de ce travail. D’après des essais de protection réalisés sur pommes blessées, il apparaîtque l’efficacité de cette souche n’est pas liée essentiellement à la sécrétion des métabolites toxiques dans lemilieu ou à l’induction de la résistance de fruit. Par contre, le mécanisme de la compétition pour la nutritionsemble jouer un rôle important. Dans les essais in vitro, la souche antagoniste Ach1-1 a eu un importanteffet inhibiteur de la germination des conidies de P. expansum dans des milieux de jus de pomme à desfaibles concentrations. Mais cet effet était réversible et les conidies inhibées étaient capables de germer unefois remises dans des conditions favorables en éléments nutritifs. Sur pommes blessées, l’effet protecteurde la souche Ach1-1 vis-à-vis de P. expansum a été significativement affaibli par l’ajout dans les blessuresde concentrations élevées des principaux composants des pommes en sucres, en vitamines etparticulièrement en acides aminés. Il apparaît que l’antagoniste exerce une activité fongistatique plus quefongicide vis-à-vis de P. expansum et agit par une compétition efficace pour les éléments nutritifs desblessures des pommes sans affecter la viabilité des conidies du pathogène. Une application exogène desacides aminés des pommes avec des concentrations croissantes dans les blessures a progressivement réduitl’activité antagoniste de la souche Ach1-1 sans altérer son développement dans les blessures, montrant quela compétition pour les acides aminés joue un rôle important dans la suppression de P. expansum. Cerésultat a été appuyé par l’analyse biochimique de la cinétique de l’épuisement des acides aminés dans lesblessures des pommes qui a montré que ces composés et particulièrement la sérine, la glycine et l’acideglutamique sont mieux métabolisés par la souche antagoniste que par le pathogène. L’ajout en excès de cestrois acides aminés en groupe ou individuellement dans les blessures des pommes a fortement réduitl’efficacité de la souche Ach1-1 vis-à-vis de P. expansum. De plus, la présence de la sérine et la glycineavec des concentrations élevées dans des milieux synthétiques ne présentant aucune source azotée, a réduitl’effet inhibiteur de la germination des conidies de P. expansum par la souche Ach1-1. Ainsi, ces acidesaminés semble être parmi les éléments les plus limitants dans le mécanisme de la compétition./The biocontrol agent Aureobasidium pullulans strain Ach1-1 was very effective againstPenicillium expansum, the causal agent of blue mold on stored apple. Modes of action that could beinvolved in its biocontrol activity were analysed in this work. According to some biocontrol trials onwounded apples, it appears that neither the production of metabolites nor the induction of fruit resistancewere the principal modes of action of this strain. However the mechanism of nutrient competition appearsto play an important role. In in vitro assays, the strain Ach1-1 had an important inhibitory effect of conidialgermination of P. expansum in apple juice at low concentrations. However this inhibitory effect wassuppressed when inhibited conidia were placed in favourable nutrients conditions. On wounded apples theprotective activity of strain Ach1-1 against P. expansum was significantly reduced by adding, in thewounds, high concentrations of major apple compounds of sugar, vitamins and most particularly aminoacids. It appears that the antagonist exerts a fungistatic rather than fungicidal activi ty on P. expansum as itcan deplete limiting nutrient available at the infection site and inhibit conidia germination without affectingtheir viability. Moreover, an exogenous application of increasing apple amino acids concentrations inwounds had progressively reduced the antagonist activity of strain Ach1-1 without altering its developmentin wounds, suggesting that competition for apple amino acids by strain Ach1-1 plays an important role insuppressing P. expansum. This finding was strengthened by a time-course analysis of wounds amino acidsduring apple incubation in witch the strain Ach1-1 was able to assimilate apple amino acids better than P.expansum, most particularly Serine, Glycin and Glutamic acid. Exogenous additions of these three aminoacids at high concentrations on apple wounds as a mixture or individually, strongly lowered the Biocontrolactivity of strain Ach1-1. Moreover, the existence of amino acids serine and glycin at high concentration insynthetic media, without any nitrogen source, was able to reduce the inhibitory effect of conidialgermination of P. expansum by the strain Ach1-1.Therefore these amino acids could be among the mostlimited nutrients in the mechanism of competition

Hybrid Modeling of Cancer Drug Resistance Mechanisms

Cancer is a multi-scale disease and its overwhelming complexity depends upon the multiple interwind events occurring at both molecular and cellular levels, making it very difficult for therapeutic advancements in cancer research. The resistance to cancer drugs is a significant challenge faced by scientists nowadays. The roots of the problem reside not only at the molecular level, due to multiple type of mutations in a single tumor, but also at the cellular level of drug interactions with the tumor. Tumor heterogeneity is the term used by oncologists for the involvement of multiple mutations in the development of a tumor at the sub-cellular level. The mechanisms for tumor heterogeneity are rigorously being explored as a reason for drug resistance in cancer patients. It is important to observe cell interactions not only at intra-tumoral level, but it is also essential to study the drug and tumor cell interactions at cellular level to have a complete picture of the mechanisms underlying drug resistance. The multi-scale nature of cancer drug resistance problem require modeling approaches that can capture all the multiple sub-cellular and cellular interaction factors with respect to dierent scales for time and space. Hybrid modeling offers a way to integrate both discrete and continuous dynamics to overcome this challenge. This research work is focused on the development of hybrid models to understand the drug resistance behaviors in colorectal and lung cancers. The common thing about the two types of cancer is that they both have dierent mutations at epidermal growth factor receptors (EGFRs) and they are normally treated with anti-EGFR drugs, to which they develop resistances with the passage of time. The acquiring of resistance is the sign of relapse in both kind of tumors. The most challenging task in colorectal cancer research nowadays is to understand the development of acquired resistance to anti-EGFR drugs. The key reason for this problem is the KRAS mutations appearance after the treatment with monoclonal antibodies (moAb). A hybrid model is proposed for the analysis of KRAS mutations behavior in colorectal cancer with respect to moAb treatments. The colorectal tumor hybrid model is represented as a single state automata, which shows tumor progression and evolution by means of mathematical equations for tumor sub-populations, immune system components and drugs for the treatment. The drug introduction is managed as a discrete step in this model. To evaluate the drug performance on a tumor, equations for two types of tumors cells are developed, i.e KRAS mutated and KRAS wild-type. Both tumor cell populations were treated with a combination of moAb and chemotherapy drugs. It is observed that even a minimal initial concentration of KRAS mutated cells before the treatment has the ability to make the tumor refractory to the treatment. Moreover, a small population of KRAS mutated cells has a strong influence on a large number of wild-type cells by making them resistant to chemotherapy. Patient's immune responses are specifically taken into considerations and it is found that, in case of KRAS mutations, the immune strength does not affect medication efficacy. Finally, cetuximab (moAb) and irinotecan (chemotherapy) drugs are analyzed as first-line treatment of colorectal cancer with few KRAS mutated cells. Results show that this combined treatment could be only effective for patients with high immune strengths and it should not be recommended as first-line therapy for patients with moderate immune strengths or weak immune systems because of a potential risk of relapse, with KRAS mutant cells acquired resistance involved with them. Lung cancer is more complicated then colorectal cancer because of acquiring of multiple resistances to anti-EGFR drugs. The appearance of EGFR T790M and KRAS mutations makes tumor resistant to a geftinib and AZD9291 drugs, respectively. The hybrid model for lung cancer consists of two non-resistant and resistant states of tumor. The non-resistant state is treated with geftinib drug until resistance to this drug makes tumor regrowth leading towards the resistant state. The resistant state is treated with AZD9291 drug for recovery. In this model the complete resistant state due to KRAS mutations is ignored because of the unavailability of parameter information and patient data. Each tumor state is evaluated by mathematical differential equations for tumor growth and progression. The tumor model consists of four tumor sub-population equations depending upon the type of mutations. The drug administration in this model is also managed as a discrete step for exact scheduling and dosages. The parameter values for the model are obtained by experiments performed in the laboratory. The experimental data is only available for the tumor progression along with the geftinib drug. The model is then fine tuned for obtaining the exact tumor growth patterns as observed in clinic, only for the geftinib drug. The growth rate for EGFR T790M tumor sub-population is changed to obtain the same tumor progression patterns as observed in real patients. The growth rate of mutations largely depends upon the immune system strength and by manipulating the growth rates for different tumor populations, it is possible to capture the factor of immune strength of the patient. The fine tuned model is then used to analyze the effect of AZD9291 drug on geftinib resistant state of the tumor. It is observed that AZD9291 could be the best candidate for the treatment of the EGFR T790M tumor sub-population. Hybrid modeling helps to understand the tumor drug resistance along with tumor progression due to multiple mutations, in a more realistic way and it also provides a way for personalized therapy by managing the drug administration in a strict pattern that avoid the growth of resistant sub-populations as well as target other populations at the same time. The only key to avoid relapse in cancer is the personalized therapy and the proposed hybrid models promises to do that