Growth Inhibition Profiles Research Articles

Mathematical Modeling of Inhibitory Microbial Lethality Synergistic: Secondary Phytocompounds from Purple Toronjil, Temperature, and Harvest Stress Effects on Escherichia coli

This research investigated the inhibition of *Escherichia coli* ATCC 25922 (E. coli) bacterial growth in situ, specifically on the stems and aerial parts of *Agastache mexicana* subsp. mexicana (Amm) or “purple toronjil” and on food-grade paper, both contained within Kraft paper bags with a plastic window. The qualitative phytochemical profile of an aqueous extract of Amm revealed the presence of various compounds including alkaloids, coumarins, tannins, flavonoids, saponins, triterpenes, and sterols. The results indicate that these secondary metabolites exhibit a synergistic bactericidal effect, especially when combined with temperature and starvation stress. This was quantified using a decay equation referred to as the bacterial growth inhibition profile of E. coli (BGIPEc). Calculations, which included first derivatives, gradients based on substrate effects and temperature as well as the area under the curve of BGIPEc, demonstrated that higher temperatures led to the greater inhibition of colony forming units (CFUs), further enhanced by the presence of secondary metabolites. Additionally, a shorter half-life corresponded to a faster change rate and a lower area under the curve, indicating a reduced survival rate over time. At lower temperatures, E. coli exhibited a survival effect, which was corroborated by the preceding calculations.

Comparative antimicrobial activity of four different endodontic sealers.

Dental cements are widely used in the clinical routine, specifically for root canal sealing. Within this context, it is expected that these materials present antimicrobial activity, since it would help in the prevention of apical and periapical infections. The present study aimed to comparatively verify the antimicrobial activity of four dental cements against microorganisms that are routinely isolated from endodontic infections. Reference strains of Enterococcus faecalis, Candida albicans and Escherichia coli were submitted to the agar diffusion test and to modified direct contact test using four different sealers: an eugenol zinc oxide compound, an epoxy resin associated to calcium hydroxide and bismuth, a mineral trioxide aggregate (MTA) and a bioceramics. Different E. coli, C. albicans and E. faecalis growth inhibition profiles were observed in the agar diffusion assay. In the direct contact test, the bioceramics presented a higher microbicide activity on all microorganisms tested herein. Dental cements have different antimicrobial activities, being that the bioceramics present the most consistent antimicrobial activity, and that the direct contact test presented more uniform results than the agar diffusion test. Thisstudyrevealstheantimicrobial activitiesof different cements and allow dentiststodecidewhichmaterialtoemployintheirdailypractice.

Abstract 4895: Introduction of a platform for preclinical profiling of drug conjugates: a case study with sacituzumab govitecan

Abstract Drug-conjugates are an emerging class of anticancer agents combining the cytotoxic activity of highly potent chemotherapeutic agents with the target specificity of an antibody, a small molecule or a peptide ligand directed against a cancer-associated protein. With nearly half a century of development, profound advancements have been made in the development of these therapeutics. The most advanced are antibody-drug conjugates (ADCs) with currently >100 in development and 11 approved by the FDA. To facilitate preclinical evaluation of novel drug conjugates, Reaction Biology and 4HF Biotec have developed a dedicated platform allowing specific in vitro and in silico analyses for this class of therapeutics. The platform is intended to provide information on the potency of the drug conjugate over the corresponding stand-alone cytotoxin, the cancer entities to be treated, the target engagement and the determinant(s) of cancer cell sensitivity. Here, we present our platform to refine antitumor potential of Sacituzumab govitecan (ScG), a recently approved TROP-2 targeted ADC used for the treatment of triple-negative breast cancer. First, we used the ProLiFiler™ (Reaction Biology), a cell proliferation and survival assay with 140 human cancer cell lines, to establish the antitumor profile of ScG, its payload SN-38 and three other cytotoxins (camptothecin, MMAE and mertansine) to validate the results of our assay. The IC50 obtained for the cytotoxins were next validated using the MoAFinder (4HF Biotec), a tool derived from the NIH COMPARE algorithm that ranks drugs based on the similarity of their growth inhibitory profiles. The analysis confirmed that SN-38 and camptothecin antitumor profiles, as established with the ProLiFiler, correlated best with other topoisomerase-1 inhibitors from our database of more than 1,000 compounds. Mertansine and MMAE correlated best with tubulin inhibitors and were more potent than camptothecin and SN-38. Overall, the response to these cytotoxins was stronger in cell lines from hyperproliferative cancers such as hematological malignancies than in those from solid cancers. Next, by using OMICS data (internal and publicly available), qPCR and flow cytometry connected to drug sensitivity profiles, we will present a differential biomarker screen of ScG vs. SN-38 to identify molecular determinants of sensitivity toward the ADC and its cytotoxin. We will address the expression level of TROP2 and its predictivity for tumor cell sensitivity, screen for predictors of SN-38 response and investigate possible off-target effects in cell lines sensitive to ScG but lacking TROP2 expression. Overall, the study demonstrates the potential of our platform to investigate drug conjugates. The comparative profiling of ScG and its payload allows to identify candidate cancer entities for ScG treatment and to improve patient enrollment into clinical trials using biomarkers. Citation Format: Vincent Vuaroqueaux, Nadine Obier, Anne-Lise Peille, Daniel Feger, Katharina Schaich, Thomas Metz, Sebastian Dempe, Heinz-Herbert Fiebig, Jan Erik Ehlert. Introduction of a platform for preclinical profiling of drug conjugates: a case study with sacituzumab govitecan. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4895.

Biological management of coffee wilt disease (Fusarium xylarioides) using antagonistic Trichoderma isolates.

Coffee wilt disease (CWD) is a serious threat to the food security of small-scale farmers in Ethiopia, causing significant reductions in coffee yield. Currently, there are no effective control measures available against the causative agent of CWD, Fusarium xylarioides. The main objective of this study was therefore to develop, formulate, and evaluate a range of biofungicides against F. xylarioides, derived from Trichoderma species and tested under in vitro, greenhouse, and field conditions. In total, 175 Trichoderma isolates were screened as microbial biocontrol agents against F. xylarioides. The efficacy of two biofungicide formulations, wettable powder and water dispensable granules, were tested on the susceptible Geisha coffee variety in three different agro-ecological zones in southwestern Ethiopia over three years. The greenhouse experiments were set up using a complete block design, while in the field a randomized complete block design was used, with twice yearly applications of biofungicide. The test pathogen spore suspension was applied to the coffee seedlings by soil drenching, and the subsequent incidence and severity of CWD evaluated annually. The mycelial growth inhibition profiles of the Trichoderma isolates against F. xylarioides ranged from 44.5% to 84.8%. In vitro experiments revealed that T. asperelloides AU71, T. asperellum AU131 and T. longibrachiatum AU158 reduced the mycelial growth of F. xylarioides by over 80%. The greenhouse study indicated that wettable powder (WP) of T. asperellum AU131 had the highest biocontrol efficacy (84.3%), followed by T. longibrachiatum AU158 (77.9%) and T. asperelloides AU71 (71.2%); they also had a significant positive impact on plant growth. The pathogen-treated control plants had a disease severity index of 100% across all the field experiments, and of 76.7% in the greenhouse experiments. In comparison to untreated controls, the annual and cumulative disease incidence over the three years of the study period varied from 46.2 to 90%, 51.6 to 84.5%, and 58.2 to 91%, at the Teppi, Gera and Jimma field experimental locations. Overall, the greenhouse and field experiments and in vitro assays support the biocontrol potential of Trichoderma isolates, and T. asperellum AU131 and T. longibrachiatum AU158 in particular are recommended for the management of CWD under field conditions.

New in vitro findings about halogenated boroxine cytotoxicity and deregulation of cell death-related genes in GR-M melanoma cells.

Anti-proliferative effects of halogenated boroxine - K2(B3O3F4OH) (HB) - have been confirmed in multiple cancer cell lines, including melanoma, but the exact mechanism of action is still unknown. This study aimed to determine its cytotoxic effects on human Caucasian melanoma (GR-M) cell growth in vitro as well as on the expression of cell death-related genes BCL-2, BECN1, DRAM1, and SQSTM1. GR-M and peripheral blood mononuclear (PBM) cells were treated with different HB concentrations and their growth inhibition and relative gene expression profiles were determined using the Alamar blue assay and real-time PCR. HB significantly inhibited cell growth of both GR-M and PBM cells but was even more effective in GR-M melanoma cells, as significant inhibition occurred at a lower HB concentration of 0.2 mg/mL. GR-M BCL-2 expression was significantly downregulated (P=0.001) at HB concentration of 0.4 mg/mL, which suggests that HB is a potent tumour growth inhibitor. At the same time, it upregulated BCL-2 expression in normal (PBM) cells, probably by activating protective mechanisms against induced cytotoxicity. In addition, all but the lowest HB concentrations significantly upregulated SQSTM1 (P=0.001) in GR-M cells. Upregulated BECN1 expression suggests early activation of autophagy at the lowest HB concentration in SQSTM1 cells and at all HB concentrations in PBM cells. Our findings clearly show HB-associated cell death and, along with previous cytotoxicity studies, reveal its promising anti-tumour potential.

Terminators or Guardians? Design, Synthesis, and Cytotoxicity Profiling of Chalcone-Sulfonamide Hybrids.

With a "less is more" philosophy, a series of 15 chalcone-sulfonamide hybrids were designed anticipating synergistic anticancer activity. The aromatic sulfonamide moiety was included as a known direct inhibitor of carbonic anhydrase IX activity through its zinc chelating property. The chalcone moiety was incorporated as an electrophilic stressor to indirectly inhibit carbonic anhydrase IX cellular activity. Screening by the Developmental Therapeutics Program of the National Cancer Institute, NCI-60, revealed that 12 derivatives were potent inhibitors of cancer cell growth in multiple cell lines and were promoted to the five-dose screen. The cancer cell growth inhibition profile indicated sub- to two-digit micromolar potency (GI50 down to 0.3 μM and LC50 as low as 4 μM) against colorectal carcinoma cells, in particular. Unexpectedly, most compounds demonstrated low to moderate potency as direct inhibitors of carbonic anhydrase catalytic activity in vitro, with 4d being the most potent, having an average Ki value of 4 μM. Compound 4j showed ca. six-fold selectivity to carbonic anhydrase IX over the other tested isoforms in vitro. Cytotoxicity of both 4d and 4j in live HCT116, U251, and LOX IMVI cells under hypoxic conditions confirmed their targeting of carbonic anhydrase activity. Elevation of oxidative cellular stress was stipulated from the increase in Nrf2 and ROS levels in 4j-treated colorectal carcinoma, HCT116, cells compared to the control. Compound 4j arrested the cell cycle of HCT116 cells at the G1/S phase. In addition, both 4d and 4j showed up to 50-fold cancer cell selectivity compared to the non-cancerous HEK293T cells. Accordingly, this study presents 4d and 4j being new, synthetically accessible, simplistically designed derivatives as potential candidates to be further developed as anticancer therapeutics.

Chemosensitivity-Gene Expression Correlations and Functional Enrichment Analysis Provide Insight into the Mechanism of Action of a Platinum-Acridine Anticancer Agent.

NCI-60 growth inhibition and gene expression profiles were analyzed using Pearson correlation and functional enrichment computational tools to demonstrate critical mechanistic differences between a nucleolus-targeting platinum-acridine anticancer agent (PA) and other DNA-directed chemotherapies. The results support prior experimental data and are consistent with DNA being a major target of the hybrid agent based on the negative correlations observed between its potency and expression levels of genes implicated in DNA double-strand break (DSB) repair. Gene ontology terms related to RNA processing, including ribosome biogenesis, are also negatively enriched, suggesting a mechanism by which these processes render cancer cells more resistant to the highly cytotoxic agent. The opposite trend is observed for oxaliplatin and other DNA-targeted drugs. Significant functional interactions exist between genes/gene products involved in ribosome biogenesis and DSB repair, including the ribosomal protein (RPL5)-MDM2-p53 surveillance pathway, as a response to the nucleolar stress produced by PAs.

Cancer Conformational Landscape Shapes Tumorigenesis.

During tumorigenesis, DNA mutations in protein coding sequences can alter amino acid sequences which can change the structures of proteins. While the 3D structure of mutated proteins has been studied with atomic resolution, the precise impact of somatic mutations on the 3D proteome during malignant transformation remains unknown because methods to reveal in vivo protein structures in high throughput are limited. Here, we measured the accessibility of the lysine ε-amine for chemical modification across proteomes using covalent protein painting (CPP) to indirectly determine alterations in the 3D proteome. CPP is a novel, high-throughput quantitative mass spectrometric method that surveyed a total of 8052 lysine sites across the 60 cell lines of the well-studied anticancer cell line panel (NCI60). Overall, 5.2 structural alterations differentiated any cancer cell line from the other 59. Structural aberrations in 98 effector proteins correlated with the selected presence of 90 commonly mutated proteins in the NCI60 cell line panel, suggesting that different tumor genotypes reshape a limited set of effector proteins. We searched our dataset for druggable conformational aberrations and identified 49 changes in the cancer conformational landscape that correlated with the growth inhibition profiles of 300 drug candidates out of 50,000 small molecules. We found that alterations in heat shock proteins are key predictors of anticancer drug efficacy, which implies that the proteostasis network may have a general but hitherto unrecognized role in maintaining malignancy. Individual lysine sites may serve as biomarkers to guide drug selection or may be directly targeted for anticancer drug development.

Identification and characterization of bacteriocin biosynthetic gene clusters found in multiple bacteriocins producing Lactiplantibacillus plantarum PUK6

The multiple bacteriocins produced by Lactiplantibacillus plantarum PUK6 isolated from misozuke-tofu (tofu pickled in miso) were identified as plantaricins A, EF, and NC8. The pln locus (21,847 bp) containing the three plantaricin structural genes and another newly found putative bacteriocin structural genes (orf1 and orf2) were determined, and a biosynthesis mechanism was proposed. Reverse transcription-PCR analysis revealed that orf1 and orf2, the putative two-peptide bacteriocin structural genes, were expressed after 8h (logarithmic growth phase) and 20h (stationary growth phase) of cultivation of the PUK6 strain. Additionally, the growth inhibition profile obtained using the chemically synthesized mature peptides of Orf1 and Orf2 (referred to as mOrf1 and mOrf2) showed that the equimolar mixture of mOrf1 and mOrf2 exhibited bactericidal effect against the indicator strain Latilactobacillus sakei subsp. sakei JCM 1157T. Furthermore, fluorescence microscopic analysis revealed disruption of the cell membranes. These findings indicate that orf1 and orf2 are structural genes encoding class IIb bacteriocins consisting of two peptides. Therefore, the novel bacteriocin encoded by plnPUK6α (orf1) and plnPUK6β (orf2) genes was named plantaricin PUK6. Since the PUK6 strain produces multiple bacteriocins, when used as a starter culture, it could contribute to improving the shelf life of fermented foods and preventing the appearance of bacteriocin-resistant bacteria.

Abstract LBA009: Orally available ENPP1 inhibitor, TXN10128, restores STING activation in tumor microenvironment and confers anti-tumor responses in combination with immune checkpoint blockade

Abstract Background Recently, there are growing needs of immune modulators that can convert cold tumors into hot tumors, which can be utilized for combination treatment with existing immune related therapies. An orally available small molecule that is capable of activating innate immune response can be an ideal candidate to meet those needs. Upon binding to 2’3’-cGAMP, STING activates TBK1-IRF3 signaling cascade in cancer cells as well as host cells and promotes innate immune responses against cancer cells, leading to T cell mediated anti-tumor immunity by facilitating T cell priming and infiltration. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), a transmembrane protein highly expressed in subset of cancer cells, has been known to hydrolyze 2’3’-cGAMP and negatively regulates the STING activation. Inhibition of ENPP1 can prevent 2’3’-cGAMP degradation in tumor microenvironment (TME) and restore STING activation, leading to innate immune responses. It has been shown that ENPP1 inhibitor can enhance anti-tumor immunity by restoring 2’3’-cGAMP level in TME and can prevent tumor growth when co-treated with immune checkpoint blockade (ICB) as well as with radiation therapy. Methods The activity of ENPP1 inhibitors were measured in enzymatic assay using 2’3’-cGAMP as substrate and in cellular STING activation assay using IRF3-responsive reporter. Also, the effects on innate immune response were estimated in LIN (Lymphocyte INfiltration) assay measuring lymphocyte infiltration and TMED (TME in Dish) assay measuring tumor spheroid growth and immune cell activation. In vivo efficacy of TXN10128 (PO dosing) was evaluated in MC38 syngeneic model in combination with and anti-PD-L1 (IV dosing). Tumor growth was monitored and immune cells in tumor were analyzed. Results TXN10128 inhibited ENPP1 activity with single digit nanomolar potency in enzyme assay and further induced STING activation in cellular assay. In 3D spheroid co-culture condition, TXN10128 enhances lymphocyte infiltration and inhibits the spheroid growth. TXN10128 has a drug-like properties desirable for oral administration in terms of physicochemical properties and pharmacokinetics parameters. Systemic exposure of TXN10128 by PO dosing resulted in synergistic tumor growth inhibition with anti-PD-L1 antibody and improved tumor-infiltrating lymphocytes (TIL) profile as expected in MC38 syngeneic model. Additional in vivo studies are on-going to expand applicable cancer types to TXN10128 treatment. Conclusions TXN10128 is a potent and selective ENPP1 inhibitor that can exert immune response in 3D co-culture condition, which is consistent with tumor growth inhibition and favorable TIL profile in animal model. Together with promising drug-likeness, these studies demonstrate that TXN10128 is a suitable candidate for clinical investigation as a combination partner with existing immunotherapies. Citation Format: Sungjoon Kim, Imran Ali, Ahran Yu, Sun woo Lee, Sung young Park, Jung hwan Choi, Yong-yea Park, Chan sun Park. Orally available ENPP1 inhibitor, TXN10128, restores STING activation in tumor microenvironment and confers anti-tumor responses in combination with immune checkpoint blockade [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA009.

Investigation of the Mechanisms of Cytotoxic Activity of 1,3-Disubstituted Thiourea Derivatives.

Substituted thiourea derivatives possess confirmed cytotoxic activity towards cancer but also normal cells. To develop new selective antitumor agents, a series of 3-(trifluoromethyl)phenylthiourea analogs were synthesized, and their cytotoxicity was evaluated in vitro against the cell line panel. Compounds 1–5, 8, and 9 were highly cytotoxic against human colon (SW480, SW620) and prostate (PC3) cancer cells, and leukemia K-562 cell lines (IC50 ≤ 10 µM), with favorable selectivity over normal HaCaT cells. The derivatives exerted better growth inhibitory profiles towards selected tumor cells than the reference cisplatin. Compounds incorporating 3,4-dichloro- (2) and 4-CF3-phenyl (8) substituents displayed the highest activity (IC50 from 1.5 to 8.9 µM). The mechanisms of cytotoxic action of the most effective thioureas 1–3, 8, and 9 were studied, including the trypan blue exclusion test of cell viability, interleukin-6, and apoptosis assessments. Compounds reduced all cancerous cell numbers (especially SW480 and SW620) by 20–93%. Derivatives 2 and 8 diminished the viability of SW620 cells by 45–58%. Thioureas 1, 2, and 8 exerted strong pro-apoptotic activity. Compound 2 induced late apoptosis in both colon cancer cell lines (95–99%) and in K-562 cells (73%). All derivatives acted as inhibitors of IL-6 levels in both SW480 and SW620 cells, decreasing its secretion by 23–63%.

Nanomolar potency of imidazo[2,1-b]thiazole analogs as indoleamine 2,3-dioxygenase inhibitors.

Novel series of imidazo[2,1-b]thiazole analogs were designed, synthesized, and biologically evaluated as indoleamine 2,3-dioxygenase (IDO1) inhibitors. Imidazo[2,1-b]thiazoles 6, 7, and 8 showed inhibitory profiles against IDO1 at IC50 values of 68.48, 82.39, and 48.48 nM, respectively, compared with IDO5L at IC50 67.40 nM. Benzo[d]imidazo[2,1-b]thiazoles 17, 20, and 22 showed promising IDO1 inhibition at IC50 values of 53.58, 53.16, and 57.95 nM, respectively. Compound 7 showed a growth-inhibitory profile at GI of 39.33% against the MCF7 breast cancer cell line, while 8 proved lethal to ACHN renal cancer cells. Cells treated with compounds 17 and 22 showed a typical apoptosis pattern of DNA fragments that reflected the G0/G1, S, and G2/M phases of the cell cycle, together with a pre-G1 phase corresponding to apoptotic cells, which indicates that cell growth arrest occurred at the S phase. Molecular modeling simulations validated the potential of benzo[d]imidazo[2,1-b]thiazole analogs to chelate iron(III) within the IDO1 binding pocket and, hence, to have a better binding affinity via hydrophobic-hydrophobic interactions.

An assessment of the growth inhibition profiles of Hamamelis virginiana L. extracts against Streptococcus and Staphylococcus spp.

Staphylococcal and streptococcal species trigger a wide variety of infections involving epithelial tissues. Virginian witch hazel (WH; Hamamelis virginiana L.; family: Hamamelidaceae) is a plant that has been used traditionally by Native Americans to treat a variety of skin conditions. Extracts from the leaves were examined for their inhibitory effects on these bacterial species. Solvents of different polarity (water, methanol, ethyl acetate, hexane and chloroform) were used to prepare extracts from WH leaves, and the aqueous resuspensions were screened for antibacterial activities using disc diffusion and liquid dilution assays. Extract phytochemical profiles and toxicities were also examined, and combinations of extracts with conventional antibiotics were tested against each bacterial strain. The methanolic and aqueous extracts inhibited the growth of S. oralis, S. pyogenes, S. epidermidis and S. aureus, but not S. mutans. The extracts were especially active against staphylococcal species, with MIC values between 200 and 500 μg/ml. Combinations of active extracts with conventional antibiotics failed to yield beneficial interactions, except for two cases where additive interactions were observed (aqueous WH extract combined with chloramphenicol against S. oralis, and methanolic WH extract combined with ciprofloxacin against S. aureus). Phytochemical assays indicated an abundance of tannins, triterpenoids and phenolics in the water and methanol extracts, with trace amounts of these components in the ethyl acetate extract. Phytochemicals were not detected in hexane and chloroform extracts. Thus, phytochemical abundance in extracts was concordant with antibacterial activities. All extracts were found to be non-toxic in Artemia nauplii assays. These findings indicate the potential for WH leaf extracts for clinical use in treating staphylococcal and streptococcal infections, while substantiating their traditional Native American uses.

Coibamide A kills cancer cells through inhibiting autophagy

Natural products are useful tools for biological mechanism research and drug discovery. Due to the excellent tumor cell growth inhibitory profile and sub-nanomolar potency, Coibamide A (CA), an N-methyl-stabilized depsipeptide isolated from marine cyanobacterium, has been considered as a promising lead compound for cancer treatment. However, the molecular anti-cancer mechanism of the action of CA remains unclear. Here, we showed that CA treatment induced caspase-independent cell death in breast cancer cells. CA treatment also led to severe lysosome defects, which was ascribed to the impaired glycosylation of lysosome membrane protein LAMP1 and LAMP2. As a consequence, the autophagosome-lysosome fusion was blocked upon CA treatment. In addition, we presented evidence that this autophagy defect partially contributed to the CA treatment-induced tumor cell death. Together, our work uncovers a novel mechanism underlying the anti-cancer action of CA, which will promote its further application for cancer therapy.

Oscillatoxin E and Its C7 Epimer Show Distinct Growth Inhibition Profiles against Several Cancer Cell Lines

Oscillatoxin E and Its C7 Epimer Show Distinct Growth Inhibition Profiles against Several Cancer Cell Lines

COMPARE Analysis, a Bioinformatic Approach to Accelerate Drug Repurposing against Covid-19 and Other Emerging Epidemics.

A novel bioinformatic approach for drug repurposing against emerging viral epidemics like Covid-19 is described. It exploits the COMPARE algorithm, a public program from the National Cancer Institute (NCI) to sort drugs according to their patterns of growth inhibitory profiles from a diverse panel of human cancer cell lines. The data repository of the NCI includes the growth inhibitory patterns of more than 55,000 molecules. When candidate drug molecules with ostensible anti-SARS-CoV-2 activities were used as seeds (e.g., hydroxychloroquine, ritonavir, and dexamethasone) in COMPARE, the analysis uncovered several molecules with fingerprints similar to the seeded drugs. Interestingly, despite the fact that the uncovered drugs were from various pharmacological classes (antiarrhythmic, nucleosides, antipsychotic, alkaloids, antibiotics, and vitamins), they were all reportedly known from published literature to exert antiviral activities via different modes, confirming that COMPARE analysis is efficient for predicting antiviral activities of drugs from various pharmacological classes. Noticeably, several of the uncovered drugs can be readily tested, like didanosine, methotrexate, vitamin A, nicotinamide, valproic acid, uridine, and flucloxacillin. Unlike pure in silico methods, this approach is biologically more relevant and able to pharmacologically correlate compounds regardless of their chemical structures. This is an untapped resource, reliable and readily exploitable for drug repurposing against current and future viral outbreaks.

In Search of Outliers. Mining for Protein Kinase Inhibitors Based on Their Anti-Proliferative NCI-60 Cell Lines Profile.

Protein kinases play a pivotal role in signal transduction, protein synthesis, cell growth and proliferation. Their deregulation represents the basis of pathogenesis for numerous diseases such as cancer and pathologies with cardiovascular, nervous and inflammatory components. Protein kinases are an important target in the pharmaceutical industry, with 48 protein kinase inhibitors (PKI) already approved on the market as treatments for different afflictions including several types of cancer. The present work focuses on facilitating the identification of new PKIs with antitumoral potential through the use of data-mining and basic statistics. The National Cancer Institute (NCI) granted access to the results of numerous previously tested compounds on 60 tumoral cell lines (NCI-60 panel). Our approach involved analyzing the NCI database to identify compounds that presented similar growth inhibition (GI) profiles to that of existing PKIs, but different from approved oncologic drugs with other mechanisms of action, using descriptive statistics and statistical outliers. Starting from 34,000 compounds present in the database, we filtered 400 which displayed selective inhibition on certain cancer cell lines similar to that of several already-approved PKIs.

Novel [(N-alkyl-3-indolylmethylene)hydrazono]oxindoles arrest cell cycle and induce cell apoptosis by inhibiting CDK2 and Bcl-2: synthesis, biological evaluation and in silico studies

As a continuation for our previous work, a novel set of N-alkylindole-isatin conjugates (7, 8a–c, 9 and 10a–e) is here designed and synthesised with the prime aim to develop more efficient isatin-based antitumor candidates. Utilising the SAR outputs from the previous study, our design here is based on appending four alkyl groups with different length (ethyl and n-propyl), bulkiness (iso-propyl) and unsaturation (allyl) on N-1 of indole motif, with subsequent conjugation with different N-unsubstituted isatin moieties to furnish the target conjugates. As planned, the adopted strategy achieved a substantial improvement in the growth inhibitory profile for the target conjugates in comparison to the reported lead VI. The best results were obtained with N-propylindole –5-methylisatin hybrid 8a which displayed broad spectrum anti-proliferative action with efficient sub-panel GI50 (MG-MID) range from 1.33 to 4.23 µM, and promising full-panel GI50 (MG-MID) equals 3.10 µM, at the NCI five-dose assay. Also, hybrid 8a was able to provoke cell cycle disturbance and apoptosis in breast T-47D cells as evidenced by the DNA flow cytometry and Annexin V-FITC/PI assays. Furthermore, hybrid 8a exhibited good inhibitory action against cell cycle regulator CDK2 protein kinase and the anti-apoptotic Bcl-2 protein (IC50= 0.85 ± 0.03 and 0.46 ± 0.02 µM, respectively). Interestingly, molecular docking for hybrid 8a in CDK2 and Bcl-2 active sites unveiled that N-propyl group is involved in significant hydrophobic interactions. Taken together, the results suggested conjugate 8a as a promising lead for further development and optimisation as an efficient antitumor drug.

Abstract 2091: Lysosomal targeting of doxorubicin induces different membrane permeabilization and cytotoxicity in two breast cancer cell lines

Abstract Background: Lysosomal targeting has been investigated for the treatment of cancer but details of the resulting cytotoxicity remain unclear. The induced lysosomal membrane permeabilization (LMP) has been shown to induce apoptosis and cytotoxicity. The purpose of this study is to elucidate details of doxorubicin (DOX) induced LMP in breast cancer cells and identify correlations with cytotoxicity. Methods: A gelatin – doxorubicin conjugate (GDOX) to target lysosomes was synthesized by EDC chemistry in formamide and purified by precipitation and desalting. GDox and free Dox uptake studies at 10 µM or its equivalent were conducted in MCF7 and MDA-MB-231 (triple negative breast cancer-TNBC) cells in growth media. Intracellular localization was followed by fluorescent markers. LMP was shown by cytosolic release of 10 kD Dextran-Alexa Fluor 488 previously loaded into lysosomes. Intra-lysosomal release of Dox from GDOX was examined by rupture of lysosomes and nuclear accumulation of released Dox followed by fluorescent microscopy and ImageJ. Released Dox accumulation in the nucleus without lysosome rupture was analyzed by UHPLC. Viability, growth inhibition profiles, and IC50 values were determined by the MTT procedure and GraphPad Prism 7. Results: GDox localized substantially to the lysosomes in both MCF7 and TNBC cells while free DOX localized to the nucleus, as expected. GDOX induced LMP by 24 hrs in 100% of the TNBC cells but only 20% of the MCF7 cells even up to 48 hrs. Intra-lysosomal DOX release in the MCF7 cells was notable only by 24 hrs and was estimated by fluorescent intensity (300,000 integrated density units per cell) at one-half the amount of free DOX accumulation in the nucleus. Nuclear accumulation of released DOX escaping intact lysosomes in MCF7 cells by 24 hrs was one-half that at 48 hrs which reached 0.13 µg Dox per 106 cells by 48 hrs representing only one-tenth that achieved by free DOX. MCF7 cell viability in GDOX was reduced to 62±3% of controls by 24 hr but reached 19±4% by 48 hrs compared to 11±1% for free Dox. The IC50 values of GDOX after 48 hr were 1.3±0.7 µM and 4.5±2 µM in TNBC and MCF7 cells, respectively. Conclusions: Taken together these results indicate that LMP is not induced by pooling of released DOX in MCF7 lysosomes nor does LMP contribute to cytotoxicity in these cells. The lysosomal accumulation of GDOX in MDA cells, in contrast to MCF7 cells, leads to extensive LMP which may contribute to the greater cytotoxicity in these cells compared to the MCF7 cells. The small amount of released DOX accumulating in the MCF7 nuclei may induce a nuclear pathway of cytotoxicity in these cells. Acknowledgements: Financial support is appreciated from the Agnes Varis Trust for Women’s Leadership and Health, King Abdul-Aziz University, and NIH R15 CA135421. Citation Format: Mohammed Alvi, Rachel Nicoletto, Bayan A. Eshmawi, Clyde M. Ofner. Lysosomal targeting of doxorubicin induces different membrane permeabilization and cytotoxicity in two breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2091.

Stereoisomer-Specific Induction of G2/M Phase Arrest and Apoptosis by 9-(E,Z)-Hydroxyoctadecadienoic Acid in Mouse Lymphoma Cells.

Hydroxyoctadecadienoic acids (HODEs) are generated by oxidation of linoleic acid in vivo and thought to mediate various pathophysiological responses. In this study, we examined the effects of HODEs on EL4 mouse lymphoma cell growth and found that 9-(E,Z)-HODE inhibited EL4 cell growth in a dose-dependent manner, whereas no such growth inhibition was observed with other isomers (9-(E,E)-, 13-(Z,E)-, or 13-(E,E)-HODE), suggesting that the growth-inhibitory effect of HODEs was stereospecific. Analysis by flow cytometry (FACS) with annexin V and propidium iodide (PI) staining showed that 9-(E,Z)-HODE induced apoptosis with G2/M phase arrest. We next examined the growth inhibition profile of 9-(E,Z)-HODE against a panel of 39 human cancer cell lines (JFCR39). The fingerprint of growth inhibition by 9-(E,Z)-HODE exhibited a high degree of similarity to that by MLN4924, an inhibitor of NEDD8-activating enzyme. The intracellular NEDD8 (ubiquitin-like protein) expression in EL4 cells was decreased by the treatment with 9-(E,Z)-HODE as assessed by immunoblotting and flow cytometry. In conclusion, 9-(E,Z)-HODE specifically induced G2/M phase arrest and apoptosis, and the decrease of NEDD8 expression might be involved in this effect.