Phenotypic Screening Research Articles

Effects of exogenous additives on growth and high-value bioproducts accumulation of microalgae

With the rapid development of synthetic biology, genetic engineering, and molecular manipulation methods in recent years, microalgae, as representatives of microbial cell factories, have been widely used as hosts in the production of high-value bioproducts, such as oils, pigments, proteins, and biofuels, demonstrating promising prospects of application in biochemical energy, food and drugs, and environmental protection. Despite these advancements, the low production efficiency of microalgae limits their industrial application. In addition to strain improvement and culture condition optimization, the regulation by exogenous chemical additives serves as a promising optimization strategy. This method relies on straightforward phenotypic screening and circumvents the necessity for intricate understanding of molecular targets in the metabolic and catabolic pathways involved in the synthesis of bioproducts. It enables rapid yield increasing of high-value bioproducts from microalgae and obtaining the required phenotypes. Although studies have reported the use of alternatives means such as exogenous additives to improve the growth of microalgae and the yield of high-value bioproducts, the classification and summarization of the types, applications, targeted strains, and molecular mechanisms of these additives are not comprehensive. Here, we review the studies using chemical inducers or enhancers to improve cell growth and high-value bioproduct accumulation in microalgae in recent years. This paper focuses on the types of exogenous additives, the effects of exogenous additives and their combinations on microalgae growth and high-value bioproduct accumulation, and the molecular mechanisms of related effects. We aim to provide information for researchers to use methods of synthetic biology to develop suitable cell chassis and harness microalgae for industrial production.

A multifunctional sensor for cell traction force, matrix remodeling and biomechanical assays in self-assembled 3D tissues in vitro.

Cell-matrix interactions, mediated by cellular force and matrix remodeling, result in dynamic reciprocity that drives numerous biological processes and disease progression. Currently, there is no available method for directly quantifying cell traction force and matrix remodeling in three-dimensional matrices as a function of time. To address this long-standing need, we developed a high-resolution microfabricated device that enables longitudinal measurement of cell force, matrix stiffness and the application of mechanical stimulation (tension or compression) to cells. Here a specimen comprising of cells and matrix self-assembles and self-integrates with the sensor. With primary fibroblasts, cancer cells and neurons we have demonstrated the feasibility of the sensor by measuring single or multiple cell force with a resolution of 1 nN and changes in tissue stiffness due to matrix remodeling by the cells. The sensor can also potentially be translated into a high-throughput system for clinical assays such as patient-specific drug and phenotypic screening. We present the detailed protocol for manufacturing the sensors, preparing experimental setup, developing assays with different tissues and for imaging and analyzing the data. Apart from microfabrication of the molds in a cleanroom (one time operation), this protocol does not require any specialized skillset and can be completed within 4-5 h.

Assessing phenotypic effect of integrase strand-transfer inhibitor (INSTI)-based resistance substitutions associated with failures on cabotegravir.

International guidelines recommend integrase strand-transfer inhibitor (INSTI)-based regimens as initial and switch therapy in people with HIV. As novel INSTIs become available, understanding how emergence of resistance at virological failures and seroconversions affects subsequent treatment options is needed. For the latest approved INSTI, cabotegravir, resistance patterns comprising Q148K/R, N155H, R263K, G118R, E138A/K and G140A/S (alone or in combination) have been documented in virological failures and seroconversions. Here, the effect of these substitutions on antiviral activity of commercially approved INSTIs, bictegravir and elvitegravir, was assessed. Antiviral testing was performed using person-derived clinical isolates (n = 52) with viral profiles similar to cabotegravir INSTI resistance patterns; susceptibility to cabotegravir, bictegravir and elvitegravir was measured using a phenotypic assay. Substitution patterns from isolates included triple [Q148K/H/R + E138A/K + G140A/C/S (n = 16)], double [Q148R + E138K (n = 3); Q148H/R + G140A/S (n = 24)] and single [N155H (n = 6); Q148R (n = 3)] resistance-associated mutations (RAMs). IC50 fold changes (FCs) for triple RAMs were the highest, at 47.0, 7.59 and >144 for cabotegravir, bictegravir and elvitegravir, respectively. For cabotegravir, bictegravir and elvitegravir, respectively, mean IC50 FCs were 9.5, 2.5 and >144 for double RAMs; and 3.3, 1.4 and >65 for single RAMs. When considering clinical/biological assay cut-offs, 54% (28/52) of isolates were susceptible to bictegravir, 40% (21/52) were partially susceptible and 6% (3/52) were resistant; for elvitegravir, 100% of isolates were resistant. Cabotegravir cut-offs were not available at the time of reporting. Overall, clinical isolates with RAM patterns similar to clinically observed cabotegravir INSTI resistance showed meaningful increases in IC50 FCs, suggesting that cabotegravir-associated resistance may negatively affect efficacy of bictegravir- and elvitegravir-based regimens.

A novel approach for target deconvolution from phenotype-based screening using knowledge graph

Deconvoluting drug targets is crucial in modern drug development, yet both traditional and artificial intelligence (AI)-driven methods face challenges in terms of completeness, accuracy, and efficiency. Identifying drug targets, especially within complex systems such as the p53 pathway, remains a formidable task. The regulation of this pathway by myriad stress signals and regulatory elements adds layers of complexity to the discovery of effective p53 pathway activators. Recent insights into p53 activation have led to two main screening strategies for p53 activators. The target-based approach focuses on p53 and its regulators (MDM2, MDMX, USP7, Sirt proteins), but requires separate systems for each target and may miss multi-target compounds. Phenotype-based screening can reveal new targets but involves a lengthy process to elucidate mechanisms and targets, hindering drug development. Knowledge graphs have emerged as powerful tools that offer strengths in link prediction and knowledge inference to address these issues. In this study, we constructed a protein-protein interaction knowledge graph (PPIKG) and pioneered an integrated drug target deconvolution system that combines AI with molecular docking techniques. Analysis based on the PPIKG narrowed down candidate proteins from 1088 to 35, significantly saving time and cost. Subsequent molecular docking led us to pinpoint USP7 as a direct target for the p53 pathway activator UNBS5162. Leveraging knowledge graphs and a multidisciplinary approach allows us to streamline the laborious and expensive process of reverse targeting drug discovery through phenotype screening. Our findings have the potential to revolutionize drug screening and open new avenues in pharmacological research, increasing the speed and efficiency of pursuing novel therapeutics. The code is available at https://github.com/Xiong-Jing/PPIKG.

DNA Repair Capacity and Clinicopathological Characteristics in Puerto Rican Hispanic/Latino Patients with Metastatic Castration-Resistant Prostate Cancer.

Prostate cancer (PCa) accounts for 22% of the new cases diagnosed in Hispanic/Latino (H/L) men in the US. PCa has the highest incidence (38.3%) and mortality (16.4%) among all types of cancer diagnosed in Puerto Rico. We previously showed that PCa patients (n = 41) have a significant reduction of 59% in their levels of DNA repair capacity (DRC) when compared to controls (n = 14). This study aimed to evaluate DRC levels through the nucleotide excision repair (NER) pathway for the first time in 16 Puerto Rican H/L men with metastatic castration-resistant PCa (mCRPCa) while establishing comparisons with controls and PCa patients with indolent and aggressive disease. Blood samples and clinicopathological data from PCa cases (n = 71) and controls (n = 25) were evaluated. PCa cases were stratified into mCRPCa (n = 16), aggressive (n = 31), and indolent (n = 24). DRC levels through NER were measured in lymphocytes with the CometChip assay. The stratification by Gleason score (GS) was GS6 (n = 7), GS7 (n = 23), GS ≥ 8 (n = 20), and mCRPCa patients (n = 16). Significant statistical differences were found when comparing the DRC values of the controls with any other of the four PCa patient groups. mCRPCa patients had the lowest mean DRC level of all four patient groups studied. The mean DRC level of mCRPCa patients was 6.65%, and compared to the controls, this represented a statistically significant reduction of 62% (p < 0.0001). Further analysis was performed to evaluate the contributions of age, anthropometric measurements, and prostate-specific antigen (PSA) levels to the DRC. Kaplan-Meier curves of mCRPCa revealed that survival probability decreased by approximately 50% by 30 months. This pilot study uses a blood-based phenotypic assay to present the first report of mCRPCa in Puerto Rican men and at a global level of DRC levels of mCRPCa patients. This study evaluated DRC levels through the NER pathway for the first time in 16 Puerto Rican H/L men with mCRPCa. Significant differences in DRC values were found between the controls and the three PCa patient groups. Kaplan-Meier curves revealed that survival probability decreased by approximately 50% by 30 months, and only 20% of the cohort was alive at 50 months, confirming the lethality of mCRPCa in this H/L population. This pilot study represents the first report of metastatic PCa in Puerto Rican men at a global level of DRC levels of mCRPCa patients using a blood-based phenotypic assay.

Bacteriophage Treatment Induces Phenotype Switching and Alters Antibiotic Resistance of ESBL Escherichia coli.

Background/Objectives: Bacteriophage therapy represents a promising strategy to combat multidrug-resistant pathogens, such as Escherichia coli. In this study, we explored the effects of a bacteriophage infection on an Extended Spectrum Beta-Lactamase (ESBL) positive E. coli isolate. Methods: We used next generation sequencing, proteomics and phenotypic screens to investigate the effect of bacteriophage infections on E. coli metabolism and resistance phenotypes. Results: The bacteriophage infection led to notable alterations in colony morphology, indicating profound changes in bacterial metabolism. Proteomic analysis revealed significant shifts in protein expression, with 65 proteins upregulated and 246 downregulated post-infection. The downregulated proteins were involved in various metabolic pathways, including nucleic acid, protein and lipid metabolism, and iron acquisition. Bacteriophage treatment also led to increased bacterial membrane permeability. Altogether, these alterations in bacterial metabolism and membrane permeability may lead to a general reduction in antibiotic resistance. Indeed, the bacteriophage-infected E. coli exhibited increased sensitivity to various classes of antibiotics, including beta-lactams, fluoroquinolones, trimethoprim-sulfamethoxazole, and aminoglycosides. Conclusions: Our findings highlight the potential of bacteriophage therapy as an adjunct to existing antibiotics, enhancing their efficacy against resistant strains.

Phenotypic and Complete Reference Whole Genome Sequence Analyses of Two Paenibacillus spp. Isolates from a Gray Wolf (Canis lupus) Gastrointestinal Tract.

Inflammatory bowel disease (IBD) is increasing among mammals around the world, and domestic dogs are no exception. There is no approved cure for canine IBD with limited treatment options. Novel probiotic bacteria discovery from free-ranging animals for the treatment of IBD in domestic pets can likely yield promising probiotic candidates. Consequently, the overall aim was to isolate bacteria from free-ranging animals that could potentially be utilized as novel probiotics. Two bacteria identified as unique Paenibacillus spp. strains by small ribosomal RNA (16S) gene sequencing were isolated from the gastrointestinal tract of a North American Gray Wolf (Canis lupus). The bacteria were typed as Gram-variable, and both were catalase/oxidase positive as well as sensitive to commonly used antibiotics. The bacteria digested complex carbohydrates and lipids by standard assays. The isolated bacteria also inhibited the growth of Staphylococcus aureus and Micrococcus luteus. The whole genome sequence (WGS) length of bacterial isolate ClWae17B was 6,939,193 bp, while ClWae19 was 7,032,512 bp, both similar in size to other Paenibacillus spp. The genomes of both bacteria encoded enzymes involved with the metabolism of complex starches and lipids, such as lyases and pectinases, along with encoding antimicrobials such as lanthipeptides, lasso peptides, and cyclic-lactone-autoinducers. No pernicious virulence genes were identified in the WGS of either bacterial isolate. Phylogenetically, the most closely related bacteria based on 16S gene sequences and WGS were P. taichungensis for ClWae17B and P. amylolyticus for ClWae19. WGS analyses and phenotypic assays supported the hypothesis that the isolates described constitute two novel candidate probiotic bacteria for potential use in dogs.

Typing of feces-derived Candida albicans strains using a novel seven-locus microsatellite panel reveals associations with yeast phenotype in individuals with inflammatory bowel disease.

Inflammatory diseases of the human gastrointestinal tract are affected by the microbes that reside in the mucosal surfaces. Patients with inflammatory bowel diseases (IBD) have altered bacterial and fungal intestinal compositions, including higher levels of fecal Candida yeasts. Ongoing research indicates that genetic and phenotypic diversity of Candida albicans may be linked with disease severity. Here, we set out to investigate feces-derived C. albicans strains from individuals with IBD and healthy volunteers through microsatellite-based genotyping and phenotypic assays. A seven-locus microsatellite panel was applied, of which six loci were newly developed. It appears that there is no specific lineage of C. albicans that is associated with IBD, but rather that the three study populations (Crohn's disease, ulcerative colitis, healthy volunteers) do have distinguishable distributions of genotypes. In addition, phenotypic characterization by means of enzyme release assays revealed trends between genotypes, virulence-related enzyme activity and clinical biomarkers. We thus show that microsatellite typing can describe genetic diversity of feces-derived C. albicans strains, and that phenotypic diversity of these strains may indeed correlate with fungal genotype or disease. This study opens further possibilities to investigate fecal fungi in relation to severity of inflammation in IBD or in other (intestinal) diseases.

Prevalence and Molecular Characterization of Carbapenemase-Producing Multidrug-Resistant Bacteria in Diabetic Foot Ulcer Infections.

Background: Diabetic foot ulcers (DFUs) represent severe complications in diabetic patients, often leading to chronic infections and potentially resulting in nontraumatic lower-limb amputations. The increasing incidence of multidrug-resistant (MDR) bacteria in DFUs complicates treatment strategies and worsens patient prognosis. Among these pathogens, carbapenemase-producing pathogens have emerged as particularly concerning owing to their resistance to β-lactam antibiotics, including carbapenems. Methods: This study evaluated the prevalence of MDR bacteria, specifically carbapenemase-producing pathogens, in DFU infections. A total of 200 clinical isolates from DFU patients were analyzed via phenotypic assays, including the modified Hodge test (MHT) and the Carba NP test, alongside molecular techniques to detect carbapenemase-encoding genes (blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48). Results: Among the isolates, 51.7% were confirmed to be carbapenemase producers. The key identified pathogens included Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli. The most commonly detected carbapenemase genes were blaKPC (27.6%) and blaNDM (24.1%). Carbapenemase-producing isolates presented high resistance to β-lactam antibiotics, whereas non-carbapenemase-producing isolates presented resistance through mechanisms such as porin loss and efflux pumps. Conclusions: The findings of this study highlight the significant burden of MDR infections, particularly carbapenemase-producing organisms, in DFUs. MDR infections were strongly associated with critical clinical parameters, including pyrexia (p = 0.017), recent antibiotic use (p = 0.003), and the severity of infections. Notably, the need for minor amputations was much higher in MDR cases (p < 0.001), as was the need for major amputations (p < 0.001). MDR infections were also strongly associated with polymicrobial infections (p < 0.001). Furthermore, Wagner ulcer grade ≥II was more common in MDR cases (p = 0.002). These results emphasize the urgent need for enhanced microbiological surveillance and the development of tailored antimicrobial strategies to combat MDR pathogens effectively. Given the high prevalence of carbapenem resistance, there is an immediate need to explore novel therapeutic options to improve clinical outcomes for diabetic patients with DFUs.

High-throughput tracking enables systematic phenotyping and drug repurposing in C. elegans disease models.

There are thousands of Mendelian diseases with more being discovered weekly and the majority have no approved treatments. To address this need, we require scalable approaches that are relatively inexpensive compared to traditional drug development. In the absence of a validated drug target, phenotypic screening in model organisms provides a route for identifying candidate treatments. Success requires a screenable phenotype. However, the right phenotype and assay may not be obvious for pleiotropic neuromuscular disorders. Here, we show that high-throughput imaging and quantitative phenotyping can be conducted systematically on a panel of C. elegans disease model strains. We used CRISPR genome-editing to create 25 worm models of human Mendelian diseases and phenotyped them using a single standardised assay. All but two strains were significantly different from wild-type controls in at least one feature. The observed phenotypes were diverse, but mutations of genes predicted to have related functions led to similar behavioural differences in worms. As a proof-of-concept, we performed a drug repurposing screen of an FDA-approved compound library, and identified two compounds that rescued the behavioural phenotype of a model of UNC80 deficiency. Our results show that a single assay to measure multiple phenotypes can be applied systematically to diverse Mendelian disease models. The relatively short time and low cost associated with creating and phenotyping multiple strains suggest that high-throughput worm tracking could provide a scalable approach to drug repurposing commensurate with the number of Mendelian diseases.

Meeting report about self-organization in biology: Freiburg Spemann-Mangold Centennial Symposium.

From September 16 to 19, 2024, an international symposium to celebrate the centennial of the discovery of the gastrula organizer by Hans Spemann and Hilde Mangold, was held at the University of Freiburg, Germany, where they studied embryology. There were 41 plenary lectures, 11 short talks, and 182 poster presentations, with more than 300 participants from 23 countries. The symposium covered research topics broadly related to developmental, cell, genome, and evolutionary biology, mainly focused on early animal development. In addition to in vivo studies on topics such as gastrulation, embryonic patterning, cell polarity, and morphogenesis, recent studies using gastruloids and organoids, which recapitulate embryogenesis and organogenesis in in vitro cell culture, were also presented at this symposium, entitled Self-Organization in Biology. Most of the reported studies used vertebrate models such as mice, frogs, and zebrafish; however, evolutionary studies involving invertebrate and plant models were also presented. Presentations employing traditional methods such as cell transplantation and phenotype screening, and state-of-the-art technologies such as single-cell omics, high-resolution imaging, and computational analysis showed that experimental embryology has a long history, to which studies of the organizer have contributed significantly. Here we discuss memorable aspects of the symposium in the hope that this report will encourage young scientists to actively participate in face-to-face international conferences.

LTX-315 is a novel broad-spectrum antimicrobial peptide against clinical multidrug-resistant bacteria.

Infections stemming from multidrug-resistant bacteria present a substantial threat to public health today. Discovering or synthesizing novel compounds is crucial to alleviate this pressing situation. The main purpose of this study is to verify the antibacterial activity of LTX-315 and explore its primary action mode. Through antibacterial phenotype assay screening, we obtained a potent compound named LTX-315 from diverse drug libraries, 10,926 compounds in total. Then, the bactericidal effect and its action mode were explored through biochemical and chemistry methods such as atime-killing curve, scanning electronic microscopy, isothermal titration calorimetry analysis, and nuclear magnetic resonance. Finally, the efficacy in vivo of LTX-315 against drug-resistant bacteria was proved through amice infection model. In this study, LTX-315, an oncolytic peptide, was discovered to effectively eliminate gram-positive and gram-negative pathogens, even for those multidrug-resistant strains. Through strong electrostatic interactions, LTX-315 can bind to the membrane component phosphatidylglycerol (PG) with extremely high affinity (nanomolar level). Strikingly, in contrast to the typical electrostatic interactions of antibacterial peptides, the indole group of LTX-315, situated near the alkyl chain, exhibits significantly enhanced recognition and interaction with PG due to the hydrophobic effect of the alkyl chain. Furthermore, it exerts various impacts on cell membranes, including damaging integrity, increasing permeability, and decreasing membrane fluidity. Additionally, microscopy revealed significant cell disintegration. The influence, in turn, disrupts several physiological activities inside cells, such as increasing the reactive oxygen species level, ultimately leading to cell death. Finally, the efficacy of LTX-315 in vivo against multidrug-resistant and hypervirulent Klebsiella pneumoniae was demonstrated. The unique mechanism of LTX-315 involves high-affinity binding to PG and subsequent membrane disruption, providing a novel approach against multidrug-resistant bacteria compared to conventional antibiotics. As a potential candidate, it shows promise in effectively treating bacterial infections, particularly those caused by drug-resistant bacteria, thereby addressing the escalating challenge of antibiotic resistance worldwide.

MALDI biotyper - Phenotypic identification test rapid assay (MBT-PITRA): A new method to detect KPC and NDM in Enterobacterales.

Carbapenemase-producing Enterobacterales are a growing concern in public health. In order to rapidly determine the antimicrobial profile, the MALDI Biotyper - antibiotic susceptibility test rapid assay (MBT-ASTRA) was developed, based on the relative growth of bacteria in the presence of antibiotics. In this study, we added carbapenemase enzymatic inhibitors to the MBT-ASTRA and developed an adapted method named MALDI Biotyper - Phenotypic Identification Test Rapid Assay (MBT-PITRA) in order to perform a rapid and cost-effective phenotypic test to detect Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-beta-lactamase (NDM), including co-producers, in Enterobacterales. Fifty-nine clinical isolates of carbapenemase-producing Enterobacterales and 28 non-carbapenemase-producing isolates collected from 2013 to 2023 were assessed using this approach. The MBT-PITRA method involved incubating bacterial isolates with different solutions of meropenem plus enzymatic inhibitors, phenylboronic acid and/or ethylenediaminetetraacetic acid, followed by analysis using MALDI-TOF MS. Carbapenemase production was inferred based on relative growth (RG) values calculated from peak intensities in the presence and absence of enzymatic inhibitors. KPC-producing isolates presented 90% (18/20) concordance, while isolates positive for NDM (16/16), KPC plus NDM (14/14) and negative (28/28) for carbapenemases were 100% correctly identified. MBT-PITRA demonstrated to be a promising method for identification of carbapenemases. IMPORTANCE: The MBT-PITRA appears to be a promising method for the rapid detection of carbapenemases in Enterobacterales.

Penfluridol targets septin7 to suppress endometrial cancer by septin7-Orai/IP3R-Ca2+-PIK3CA pathway.

Phenotypic screening of existing drugs is a good strategy to discover new drugs. Herein, 33 psychotherapeutic drugs in our drug library were screened by phenotypic screening and penfluridol (PFD) was found to exhibit excellent anti-endometrial cancer (EC) activity both invitro and invivo. Furthermore, the molecular target of PFD was identified as septin7, a tumor suppressor in EC. In septin7-deficient EC cells and xenograft mouse models, PFD exhibited weaker anti-cancer properties, indicating that septin7 was essential for the tumor inhibitory activity. Notably, PFD could induce cell apoptosis by regulating the septin7-Orai/IP3R-Ca2+-PIK3CA pathway. In addition, PFD attenuates the interaction of septin7-tubulin, thereby inhibiting microtubule polymerization. In summary, this study revealed a target and mechanistic insights into EC therapeutic strategies and identified a potential candidate agent for the treatment of EC.

Antimalarial Drug Discovery from Natural and Synthetic Sources.

Malaria remains a significant global health threat despite extensive efforts aimed at its eradication. Numerous challenges persist in eliminating the disease, chief among them being the parasite's ability to mutate, resulting in drug resistance. The discovery of antimalarial drugs has relied on both phenotypic and target-based approaches. While phenotypic screening has identified promising candidates, target-based methods offer a more precise approach by leveraging chemically validated targets and computational tools. Analysis of Plasmodium spp. protein structures reveal druggable targets, offering opportunities for in silico screening. Combining compounds from natural and synthetic sources in a target-based approach accelerates the discovery of new antimalarial agents. This review explores previous breakthroughs in antimalarial drug discovery from natural products and synthetic origins, emphasizing their specific target proteins within Plasmodium species.

Chemical biology research in RIKEN NPDepo aimed at agricultural applications.

This review outlines research on chemical biology using mainly microbial metabolites for agricultural applications. We established the RIKEN Natural Products Depository (NPDepo), housing many microbial metabolites, to support academic researchers who focus on drug discovery. We studied methods to stimulate secondary metabolism in microorganisms to collect various microbial products. The switch of secondary metabolism in microorganisms changes depending on the culture conditions. We discovered compounds that activate biosynthetic gene clusters in actinomycetes and filamentous fungi. Using these compounds, we succeeded in inducing the production of active compounds. Two approaches for screening bioactive compounds are described. One is phenotypic screening to explore antifungal compounds assisted by artificial intelligence (AI). AI can distinguish the morphological changes induced by antifungal compounds in filamentous fungi. The other is the chemical array method for detecting interactions between compounds and target proteins. Our chemical biology approach yielded many new compounds as fungicide candidates.

Rational design strategies for innovative small-molecule scaffolds inspired by three pivotal protein secondary structures.

We developed a design strategy focusing on pivotal secondary structural motifs-α-helix, β-strand, and β-turn-critical for PPI recognition, using a common core skeleton. The resulting peptide-inspired pyrimidodiazepine scaffolds were further subjected to comprehensive phenotypic screening to evaluate their efficacy. Our strategy offers a transformative approach to developing small-molecule PPI modulators with broad therapeutic potential.

Ligand-centred phenotype-driven development of potent kinase inhibitors against oesophageal cancer.

Oesophageal cancer (OC) is one of the leading causes of cancer-related deaths worldwide. Due in part to its high heterogeneity, OC prognosis remains poor despite the introduction of targeted and immunotherapy drugs. Although numerous kinases play a significant role in the oncogenesis and progression of OC, targeting kinases have shown so far limited therapeutic success. Based on our understanding of the pharmacological properties of the pyrazolo[3,4-d]pyrimidine scaffold and the complex biology of OC, we implemented a ligand-centred strategy combined with phenotypic screening to develop novel antiproliferative inhibitors against OC. This approach is specifically designed to accelerate the discovery of lead compounds in cancers of high molecular heterogeneity such as OC. In an iterative process driven by structure-antiproliferative activity relationships (SAARs), we synthesised and tested 54 novel pyrazolo[3,4-d]pyrimidine derivatives against OC cell lines. The lead compound 2D7 (a.k.a. eCCA352) induces pan-OC activity and cell cycle arrest in the submicromolar range and was determined to inhibit Aurora kinase A, providing a new starting point to develop anticancer targeted agents against OC.

A Phase III Randomized Trial of Integrated Genomics and Avatar Models for Personalized Treatment of Pancreatic Cancer: The AVATAR Trial.

Pancreatic ductal adenocarcinoma (PDAC) has limited treatment options. We compared the efficacy of comprehensive precision medicine against that of the conventional treatment in PDAC. We report a phase III trial of advanced PDAC in which patients were randomized (1:2) to a conventional treatment treated at physician's discretion (arm A) or to precision medicine (arm B). Subjects randomized to arm B underwent a tumor biopsy for whole-exome sequencing and to generate avatar mouse models and patient-derived organoids for phenotypic drug screening, with final treatment recommended by the molecular tumor board. The primary objective was median overall survival (OS). A total of 137 patients were enrolled with 125 randomized, 44 to arm A and 81 to arm B. Whole-exome sequencing was performed in 80.3% (65/81) patients of arm B, with potentially actionable mutations detected in 21.5% (14/65). Experimental models were generated in 16/81 patients (19.8%). Second-line treatment was administered to 39 patients in the experimental arm, but only four (10.2%) received personalized treatment, whereas 35 could not receive matched therapy because of rapid clinical deterioration, delays in obtaining study results, or the absence of actionable targets. The median OS was 8.7 and 8.6 months (P = 0.849) and the median progression-free survival was 3.8 and 4.3 months (P = 0.563) for the conventional and experimental arms, respectively. Notably, the four patients who received personalized treatment had a median OS of 19.3 months. Personalized medicine was challenging to implement in most patients with PDAC, limiting the interpretation of intention-to-treat analysis. Survival was improved in the subset of patients who did receive matched therapy.

Establishment of a Mutant Library for Infection Cushion Development and Identification of a Key Regulatory Gene in Botrytis cinerea.

Botrytis cinerea, the grey mould fungus affecting over 1400 plant species, employs infection cushion (IC), a branched and claw-like structure formed by mycelia, as a critical strategy to breach host surface barriers. However, the molecular mechanisms underlying IC formation remain largely unexplored. In this study, we utilized a forward genetics approach to establish a large T-DNA tagged population of B. cinerea, which contained 14,000 transformants. Through phenotype screening, we identified 161 mutants with defects in IC development. Detailed analyses revealed that these mutants exhibited various degrees of impairment in IC formation, ranging from complete failure to form ICs to a reduction in the number and maturity of ICs. Further genetic analysis of one of the mutants led to the identification of EXO70, a gene encoding a component of the exocyst complex, as a key regulatory factor in IC development. Mutants with deletion of EXO70 failed to form ICs, confirming its crucial role in the process. The mutant library reported here provides a rich resource for further large-scale identification of genes involved in IC development. Our findings provide valuable insights into the genetic and molecular basis of IC formation and offer new targets for controlling B. cinerea pathogenicity.