Robust Potency Research Articles

Targeting bacterial efflux pump effectively enhances the efficacy of Ru-based antibacterial agents against Gram-negative pathogen

The rise of antibiotic resistance has posed a great threat to human's life, thus develop novel antibacterial agents is urgently needed. It worthies to noted that Ru-based antibacterial agents often showed robust potency against Gram-positive pathogens, disrupted bacterial membrane and avoided bacterial resistance, making they promising antibiotic candidates. However, they are generally less active when applied to negative pathogens. To address this problem, a Ru-based metalloantibiotic (RuN) modified with a nitrothiophene moiety, which can target bacterial efflux pump, was designed and evaluated in this work. A series of assays demonstrated that RuN not only fully retained the advantages of Ru-based agents, such as destroyed bacterial membrane and induced reactive oxygen species production, but also can targeted bacterial efflux pumps. Of course, these properties make it effective in killing both Gram-positive and negative pathogens, its MIC values against Staphylococcus aureus and Escherichia coli lies at 3.125 and 6.25 μg/mL, respectively. Importantly, RuN also showed low toxicity and has robust anti-infective potency in two animal infection models. Together, our results paved an alternative way to enhance the anti-infective efficacy of Ru-based agents against resistant negative bacteria.

Comprehensive study of the physicochemical properties of three-component deep eutectic solvents and their implications for microbial and anticancerous activity

Sustainable chemistry centers on substituting perilous solvents and materials with eco-conscious alternatives. Deep eutectic solvents (DES) hold substantial potential in this arena. This inquiry includes the formulation of three-component eutectic solvents and an exhaustive scrutiny of their physical and chemical attributes. These encompass solubility, boiling point, pH, density, viscosity, surface tension, refractive index, contact angle, conductivity, Fourier-transform infrared spectroscopy, polarized optical microscopy, thermogravimetric analysis, and differential scanning calorimetry. Furthermore, a biological exploration featured two bacterial strains and two fungal strains. The entire spectrum of ten three-component DES was administered to these microorganisms to discern plausible impacts. In addition, the biomedical promise of these DES was unveiled through anticancer assays employing MCF-7 and HeLa cell lines. The outcomes were favorable, underscoring robust anticancer potency, thereby hinting at future oncological utility. These interdisciplinary endeavors envelop the progression of sustainable solvent innovation, meticulous physicochemical scrutiny, microbial analysis, and anticancer appraisal. This study propels inventive resolutions with ecological and biomedical reverberations by amalgamating these distinct yet interconnected facets.

Chemical Proteomics Approaches for Screening Small Molecule Inhibitors Covalently Binding to SARS-Cov-2.

Although various strategies have been used to prevent and treat SARS-CoV-2, the spread and evolution of SARS-CoV-2 is still progressing rapidly. The emerging variants Omicron and its sublineage have a greater ability to spread and escape nearly all current monoclonal antibodies treatments, highlighting an urgent need to develop therapeutics targeting current and emerging Omicron variants or recombinants with breadth and potency. Here, some small molecule drugs are rapidly identified that could covalently binding to receptor binding domain (RBD) protein of Omicron through the combined application of artificial intelligence (AI) and activity-based protein profiling (ABPP) technology. The surface plasmon resonance (SPR) and pseudo-virus neutralization experiments further reveal that an FDA-approved drug gallic acid has robust neutralization potency against Omicron pseudo-virus with the IC50 values of 23.56 × 10-6 m. Taken together, a platform combining AI intelligence, biochemical, SPR, molecular docking, and pseudo-virus-based screening for rapid identification and evaluation of potential anti-SARS-CoV-2 small molecule drugs is established and the effectiveness of the platform is validated.

Anti-tumor activity of camptothecin analog conjugate of a RSPO4-based peptibody targeting LGR4/5/6 in preclinical models of colorectal cancer.

Antibody-drug conjugates (ADCs) have emerged as a major modality of targeted cancer therapy, yet no ADC has been approved for colorectal cancer (CRC). LGR4/5/6 (leucine-rich repeat containing, G protein-coupled receptor 4, 5, 6) are three related receptors that are expressed at high levels together or alternately in nearly all cases of CRC. ADCs targeting LGR5 have been shown to have robust anti-tumor potency, but not all CRC cells express LGR5 and LGR5-positive tumor cells may lose LGR5 expression due to cancer cell plasticity. R-spondin 4 (RSPO4) is a natural protein ligand of LGR4/5/6 with high affinity for all three receptors. We fused a mutant form of RSPO4 that retains high affinity binding to LGR4/5/6 to IgG1 Fc to create a peptibody designated R462. Conjugation of R462 with a camptothecin analog (CPT2) at eight drugs per peptibody led to the synthesis of R462-CPT2 that showed highly potent cytotoxic activity in vitro in CRC cell lines expressing any of LG4/5/6. In cell line xenograft and PDX models of CRC, R462-CPT2 demonstrated robust anti-tumor effect. Importantly, R462-CPT2 showed no major adverse effect at therapeutically effective dose levels. These results strongly support the use of RSPO ligand drug-conjugates that target LGR4/5/6 simultaneously for the treatment of CRC.

Triple broadly neutralizing antibody (bnAb) combination therapy: a cure for pediatric HIV?

Abstract Background In 2022, UNAIDS estimated out of the 1.7 million children living with HIV, only 57.1% had access to antiretroviral therapy (ART). Though ART has significantly decreased HIV-associated morbidity and mortality, it cannot eliminate the latent viral reservoir. Consequently, most people living with HIV demonstrate viral rebound when ART is interrupted. There is therefore a critical need for alternative therapeutic to achieve long term ART free viral control. Broadly neutralizing antibodies (bnAb) have shown promising results in preclinical and clinical studies, but their potential efficacy is limited by the emergence of escape variants, especially with monotherapy. We previously tested the neutralizing and non-neutralizing functions of different bnAb combinations and identified a triple bnAb combination that showed robust neutralization potency and breadth, as well as non-neutralizing functions. In this project, we investigated the impact of combination bnAb therapy on viral rebound in SHIV infected infant rhesus macaques (RMs). Methods Ten infant rhesus macaques were orally challenged with SHIV.C.CH505 at 4 weeks of age. Oral challenge is a well-established method that is used to mimic breastmilk transmission of HIV, which contributes to almost 50% of pediatric HIV infections every year. A triple bnAb combination of simianized 3BNC117 (CD4 binding site), PGDM1400 (V2 glycan dependent), and PGT151 (interface) was subcutaneously administered twice at 40 mg/kg of each antibody. The initial infusion was administered at ART initiation (8 weeks post-infection) while the second infusion with administered at the time of analytical treatment interruption (ATI) (49 weeks post-infection). RMs were monitored weekly from the beginning of infection until 12 weeks post-ATI. Enzyme-linked Immunosorbent Assay (ELISA) was used to monitor plasma concentrations of each bNab and Env-specific IgG binding responses. Results Env-specific antibody binding levels against HIV envelope proteins gp120 and gp41 peaked in plasma 8 weeks after infection. The antibody levels then slightly decreased during ART and increased again after viral rebound following ATI. BnAb plasma concentrations peaked 1-2 weeks after each bnAb infusion, then slowly declined. After the initial infusion, all RMs had undetectable levels of 3BNC117 and PGT151 by 8 weeks post initial infusion, and only 2/10 RMs had detectable PGDM1400 levels. All animals experienced viral rebound following ATI.). Importantly, the average time to virus rebound (7 weeks, range 3-10 weeks) was significantly longer than a s control group of RMs without any immune-based intervention (~2.5 weeks). By the time of viral rebound, most animals had undetectable plasma levels of 3BNC117, PGDM1400 or PGT151. However, in some animals, rebound occurs when passively administered antibodies were still detectable, suggesting the emergence of bnAb resistant variants. Conclusion Passive immunization with a triple-bnAb combination of 3BNC117, PGT151 and PGDM1400 was associated with delayed viral rebound, but more investigation is needed to understand the potential role of this strategy in the HIV therapeutic portfolio. Future work will investigate the sensitivity of the rebound viruses to the bnAb combination to evaluate the contribution of bnAb escape to viral rebound.

Exploration of newly synthesized transition metal(II) complexes for infectious diseases.

Aim: In the annals of human history, infectious diseases significantly influencing the collective well-being of people worldwide. Consequently, to identify effective agents for infectious ailments, the octahedral Co(II), Ni(II), Cu(II), Zn(II) complexes of 4-(3-methoxyphenyl)pyrimidin-2-amine and 2-methoxy-1-napthaldehyde based ligand were synthesized and well characterized in the current investigation.Results & methodology: The synthesized compounds were evaluated for anti-TB, anti-inflammatory, antibacterial, antifungal activities by microplate Alamar blue, bovine serum albumin, serial dilution assays. The [Zn(L1)2(H2O)2] complex (5) demonstrates robust potency with 0.0040±0.0007 and 0.0038μmol/ml MIC value in anti-tuberculosis and antimicrobial activities, correspondingly while 06.57±0.03μM IC50 value in anti-inflammatory investigation.Conclusion: Complex (5) show promising potential as targets for pathogen deformities, supported by rigorous biological and computational investigations including pharmacophore modelling, molecular docking (binding score -121.018 and -59.8662kcal/mol for 6H53 and 1CX2 proteins, respectively), DFT (Density functional theory), MESP (MolecularElectrostaticPotential) and ADMET (absorption, distribution, metabolism, excretion and toxicity).

A thermoresponsive metabolic nanomodulator for achieving photochemotherapy-sensitized cancer immunotherapy

The largest obstacle that impedes the successful clinical use of anticancer nanodrugs lies in their unsatisfactory therapeutic performance even if multiple drug treatments are applied, which may cause tumor relapse and drug resistance. Therefore, it is challenging to develop effective nanodrugs that can eradicate tumors with only single injection. Herein, we construct a metabolic nanomodulator (denoted as ISM liposome), where the phototherapeutic dye IR825-NH2, the chemodrug shikonin, and the mitochondrial respiration inhibitor metformin are encapsulated, to closely synergize phototherapy, chemotherapy, and immunotherapy via the laser-triggered on-demand metabolism regulation for realizing robust anticancer potency after only single injection and irradiation. Upon near-infrared laser irradiation, IR825-NH2 can generate reactive oxygen species and mild heat to enable the phototherapeutic cytotoxcity and thermoresponsive release of the loaded drugs. Then, cellular oxygen can be spared by the released metformin via metabolic modulation and be utilized by shikonin to further amplify the oxidative stress in cancer cells. More importantly, the ISM liposome-mediated photochemotherapy can subsequently elicit a strong immunogenic cell death (ICD) effect and sensitize the immunosupressive tumor to the ICD-induced immunotherapy, thereby achieving strong tumor inhibition and even eradication. The metabolic nanomodulator-based photochemotherapy-sensitized cancer immunotherapy may represent a new solution to fight against cancer.

Abstract 5727: ISM9682A, a novel and potent KIF18A inhibitor, shows robust antitumor effects against chromosomally unstable cancers

Abstract KIF18A belongs to the kinesin-8 subfamily and is a plus-end-directed motor, which regulates kinetochore microtubules dynamics to control mitotic chromosome alignment and spindle tension. Studies have shown that genetic depletion or pharmacological inhibition of KIF18A is a promising therapeutic approach for cancers, exhibiting high chromosomal instability (CIN-high), without having a significant impact on the normal division of euploid cells. Here, we report the discovery and preclinical evaluation of ISM9682A, a novel KIF18A inhibitor with excellent potency and high selectivity. ISM9682A is a potent KIF18A inhibitor with a single-digit nanomolar IC50 value and exhibits high selectivity over other kinesin family members. In addition, ISM9682A demonstrated excellent monotherapy anti-proliferative activity, with single-digit nanomolar IC50 values, in high-grade serous ovarian carcinoma (HGSOC) as well as in triple-negative breast cancer (TNBC) aneuploid cell lines with p53 mutation. Notably, ISM9682A exhibited a prolonged drug-target residence time indicated by both enzymatic and cellular assays. Consistent with the in vitro findings, ISM9682A displayed significant anti-tumor efficacy with a robust pharmacodynamic response (pH3, mitotic marker; γH2AX, DNA damage marker) in OVCAR3 and HCC1806 CDX models in a dose-dependent manner. In addition to its robust biological potency and high selectivity, ISM9682A exhibits favorable drug-like properties, including desirable in vitro ADMET characteristics and excellent in vivo exposure, clearance, as well as good oral bioavailability across multiple preclinical species. Collectively, these findings highlight the potential of ISM9682A as a novel and highly potent KIF18A inhibitor for the treatment of CIN-high cancers. Citation Format: Jie Zhang, Feng Gao, Wei Zhu, Chenxi Xu, Xiaoyu Ding, Jing Shang, Junwen Qiao, Shan Chen, Xin Cai, Xiao Ding, Supriya Bavadekar, Sujata Rao, Man Zhang, Feng Ren, Alex Zhavoronkov. ISM9682A, a novel and potent KIF18A inhibitor, shows robust antitumor effects against chromosomally unstable cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5727.

Abstract 4539: ZM-2311 a novel and highly potent Polθ inhibitor demonstrates synergistically anti-tumor activities in combination with different therapeutics

Abstract Homologous recombination (HR) is a high-fidelity DNA double-strand break (DSB) repair pathway, and its dysfunction leads to cell genome instability and can result in tumorigenesis. In the situation of HR deficiency, DNA polymerase theta (Polθ) mediated microhomology-mediated end joining (MMEJ) is up-regulated to serve as a backup pathway for DSB repair. Several studies have proved that the inhibition of Polθ causes synthetic lethality with HR deficiency, and Polθ emerges as a potential DNA damage repair (DDR) target for the treatment of HR deficient tumors. In addition, there is evidence showing that the depletion of Polθ improves the sensitivity of some other DDR-related targeted therapies, chemotherapy, and radiotherapy. Here we identify a novel small molecular Polθ inhibitor, ZM-2311, which inhibits Polθ activity with an IC50 of 24 nM, and strongly inhibits cellular MMEJ pathway with a single nanomolar IC50. ZM-2311 illustrates a robust potency against tumor growth in a MDA-MB-436 SHLD2-/- xenograft model, suggesting potential monotherapy application in BRCA1 mutation patients with TP53BP1/Shieldin complex deficiency. Besides monotherapy, ZM-2311 elicits strong synergetic anti-proliferation activities in combination with PARP inhibitors on BRCA2-/- DLD-1 and endogenous HR deficient tumor cells in vitro and in vivo. Tumor regression is observed even at a low dosage, indicating a high potency of ZM-2311. As reversion mutation of HR genes such as BRCA1/2 mediated by the MMEJ pathway is reported to be a mechanism of PARP inhibitor resistance, ZM-2311 is evaluated and demonstrates its potential to overcome the resistance to PARP inhibitors. Furthermore, the combination of ZM-2311 with other DDR agents, such as ATR inhibitors, induces fatal DNA damage to HR deficient cells. Considering a low risk of hematotoxicity of Polθ inhibition, ZM-2311 may provide a new combination option for DDR agents. Besides, ZM-2311 was assessed in combination with different therapies respectively which functionally induce DNA damage, such as radiotherapy and chemotherapy. ZM-2311 renders tumor cells more sensitive to radiotherapy in both HR deficient cells and proficient cells in vitro, accompanying with the upregulation of Polθ expression after radiotherapy and an enhanced phosphorylation level of H2AX in the combination treatment. Similarly, ZM-2311 also achieves a robust synergetic effect in combination with chemotherapy in cell lines with different HR statuses. These results indicate a potential application expansion of ZM-2311 to HR-proficient tumors, which is expected to bring better efficacy and reduced hematotoxicity. Taken together, ZM-2311 has proved to be a highly potent Polθ inhibitor and has promising combination potential with multiple therapy strategies. Citation Format: Feng Zhou, Lu Liu, Lei Jiang, Shuo Qian, Liting Xue, Mengying Li, Zhengtao Li, Zhen Li, Jian Li, Renhong Tang. ZM-2311 a novel and highly potent Polθ inhibitor demonstrates synergistically anti-tumor activities in combination with different therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4539.

Development of a robust cell-based potency assay for a coxsackievirus A21 oncolytic virotherapy

Oncolytic viruses (OV) are part of a burgeoning field of investigational oncolytic therapy (OT), in which lytic viruses dissolve advanced tumors productively and specifically. One such OT is a Coxsackievirus A21 (CVA21) based OV that is currently under clinical evaluation. A tissue culture infectious dose (TCID50) assay was used for CVA21 potency release and stability testing in early clinical development. The titer measured in this method was an extrapolated value from cytopathic effect (CPE) observed during the serial dilution but doesn't represent direct viral killing of cells. Moreover, the assay was not deemed to be optimal to carry into late phase clinical development due to limitations in assay precision, turn-around time, and sample throughput. To address these points, we developed a plaque assay to measure viral plaque forming units to measure the potency value for drug substance (DS), drug product (DP) and virus seed (master and working) stocks. In this manuscript, we describe the steps taken to develop this plaque assay for the late-stage clinical development, which include the assay qualification, validation, and robustness protocols, and describe statistical methods for data analysis. Moreover, the method was validated for linearity, accuracy, precision, and specificity. Furthermore, the plaque assay quantifies OV infectivity with better precision (32% vs 58%), with higher sample throughput (22 samples/week vs 3 samples/week) and shorter assay turnaround time (4 days vs 7 days) than the TCID50 method. This assay development strategy can provide guidance for the development of robust cell-based potency methods for OVs and other infectious viral products.

Potency assay to predict the anti-inflammatory capacity of a cell therapy product for macrophage-driven diseases: overcoming the challenges of assay development and validation

BackgroundGiven the high level of product complexity and limited regulatory guidance, designing and implementing appropriate potency assays is often the most challenging part of establishing a quality control testing matrix for a cell-based medicinal product. Among the most elusive tasks are the selection of suitable read-out parameters, the development of assay designs that most closely model the pathophysiological conditions, and the validation of the methods. Here we describe these challenges and how they were addressed in developing an assay that measures the anti-inflammatory potency of mesenchymal stromal cells (MSCs) in an M1 macrophage-dominated inflammatory environment. MethodsAn in vitro inflammation model was established by coculturing skin-derived ABCB5+ MSCs with THP-1 monocyte-derived M1-polarized macrophages. Readout was the amount of interleukin 1 receptor antagonist (IL-1RA) secreted by the MSCs in the coculture, measured by an enzyme-linked immunosorbent assay. ResultsIL-1RA was quantified with guideline-concordant selectivity, accuracy and precision over a relevant concentration range. Consistent induction of the macrophage markers CD36 and CD80 indicated successful macrophage differentiation and M1 polarization of THP-1 cells, which was functionally confirmed by release of proinflammatory tumor necrosis factor α. Testing a wide range of MSC/macrophage ratios revealed the optimal ratio for near-maximal stimulation of MSCs to secrete IL-1RA, providing absolute maximum levels per individual MSC that can be used for future comparison with clinical efficacy. Batch release testing of 71 consecutively manufactured MSC batches showed a low overall failure rate and a high comparability between donors. ConclusionsWe describe the systematic development and validation of a therapeutically relevant, straightforward, robust and reproducible potency assay to measure the immunomodulatory capacity of MSCs in M1 macrophage-driven inflammation. The insights into the challenges and how they were addressed may also be helpful to developers of potency assays related to other cellular functions and clinical indications.

Design, Synthesis, and Biological Evaluation of Hydrophobic-Tagged Glutathione Peroxidase 4 (GPX4) Degraders

Ferroptosis is an iron-dependent form of oxidative cell death induced by lipid peroxidation accumulation. Glutathione peroxidase 4 (GPX4) plays a key role in the regulation of ferroptosis and is considered to be a promising therapeutic target for cancer and other human diseases. Herein, we describe our design, synthesis, and biological evaluation of a series of HyT-based degraders of the GPX4. One of the most promising compounds, 7b (ZX782), effectively induces dose- and time-dependent degradation of GPX4 protein and potently suppresses the growth of human fibrosarcoma HT1080 cells, which are highly sensitive to ferroptosis and widely used for evaluating compound specificity in ferroptosis. Mechanism investigation indicated that 7b depletes GPX4 through both the ubiquitin–proteasome and the autophagy-lysosome. Furthermore, the degradation of GPX4 induced by 7b could significantly increase the accumulation of lipid reactive oxygen species (ROS) in HT1080 cells, ultimately leading to ferroptosis. Overall, compound 7b exhibits robust potency in depleting endogenous GPX4, thereby modulating ferroptosis in cancer cells.

Evaluation of multi-target iridium(iii)-based metallodrugs in combating antimicrobial resistance and infections caused by Staphylococcus aureus.

The rapid emergence and spread of multidrug-resistant bacteria pose a serious challenge to human life and health, necessitating the development of novel antibacterial agents. Herein, to address this challenge, three iridium-based antibacterial agents were prepared and their antimicrobial activity were explored. Importantly, the three complexes all showed robust potency against S. aureus with MIC values in the range of 1.9-7.9 μg mL-1. Notably, the most active complex Ir3 also exhibited relative stability in mammalian fluids and a significant antibacterial effect on clinically isolated drug-resistant bacteria. Mechanism studies further demonstrated that the complex Ir3 can kill S. aureus by disrupting the integrity of the bacterial membrane and inducing ROS production. This multi-target advantage allows Ir3 to not only effectively combat bacterial resistance but also efficiently clear the bacterial biofilm. In addition, when used together, complex Ir3 could enhance the antibacterial potency of some clinical antibiotics against S. aureus. Moreover, both G. mellonella wax worms and mouse infection model demonstrated that Ir3 has low toxicity and robust anti-infective efficacy in vivo. Overall, complex Ir3 can serve as a new antibacterial agent for combating Gram-positive bacterial infections.

Discovery, Optimization, and Evaluation of Potent and Selective DNA-PK Inhibitors in Combination with Chemotherapy or Radiotherapy for the Treatment of Malignancies.

Given the multifaceted biological functions of DNA-PK encompassing DNA repair pathways and beyond, coupled with the susceptibility of DNA-PK-deficient cells to DNA-damaging agents, significant strides have been made in the pursuit of clinical potential for DNA-PK inhibitors as synergistic adjuncts to chemo- or radiotherapy. Nevertheless, although substantial progress has been made with the discovery of potent inhibitors of DNA-PK, the clinical trial landscape requires even more potent and selective molecules. This necessitates further endeavors to expand the repertoire of clinically accessible DNA-PK inhibitors for the ultimate benefit of patients. Described herein are the obstacles that were encountered and the solutions that were found, which eventually led to the identification of compound 31t. This compound exhibited a remarkable combination of robust potency and exceptional selectivity along with favorable in vivo profiles as substantiated by pharmacokinetic studies in rats and pharmacodynamic assessments in H460, BT474, and A549 xenograft models.

Flexible Fabrication and Hybridization of Bioactive Hydrogels with Robust Osteogenic Potency.

Osteogenic scaffolds reproducing the natural bone composition, structures, and properties have represented the possible frontier of artificially orthopedic implants with the great potential to revolutionize surgical strategies against the bone-related diseases. However, it is difficult to achieve an all-in-one formula with the simultaneous requirement of favorable biocompatibility, flexible adhesion, high mechanical strength, and osteogenic effects. Here in this work, an osteogenic hydrogel scaffold fabricated by inorganic-in-organic integration between amine-modified bioactive glass (ABG) nanoparticles and poly(ethylene glycol) succinimidyl glutarate-polyethyleneimine (TSG-PEI) network was introduced as an all-in-one tool to flexibly adhere onto the defective tissue and subsequently accelerate the bone formation. Since the N-hydroxysuccinimide (NHS)-ester of tetra-PEG-SG polymer could quickly react with the NH2-abundant polyethyleneimine (PEI) polymer and ABG moieties, the TSG-PEI@ABG hydrogel was rapidly formed with tailorable structures and properties. Relying on the dense integration between the TSG-PEI network and ABG moieties on a nano-scale level, this hydrogel expressed powerful adhesion to tissue as well as durable stability for the engineered scaffolds. Therefore, its self-endowed biocompatibility, high adhesive strength, compressive modulus, and osteogenic potency enabled the prominent capacities on modulation of bone marrow mesenchymal stem cell (BMSCs) proliferation and differentiation, which may propose a potential strategy on the simultaneous scaffold fixation and bone regeneration promotion for the tissue engineering fields.

Green synthesis of zinc oxide nanoparticles from Phoenix dactylifera and their anti-microbial potentiality – An in vitro study

Introduction and Aim: The consumption of date palm (Phoenix dactylifera) fruit holds a widespread presence globally and holds notable importance in the diets of various Arabian nations. The adoption of green synthesis for nanoparticle production presents multiple advantages over conventional physio-chemical methods. Furthermore, these nanoparticles have found diverse applications within the medical field. In light of this, the synthesis of zinc oxide nanoparticles using extracts from Phoenix dactylifera (dates) was undertaken. The primary objective of this research was to employ a biosynthesis approach for the creation of zinc oxide nanoparticles utilizing extracts from Phoenix dactylifera (Mabroom dates), and subsequently assessing their potential antimicrobial efficacy. Materials and Methods: In the present study, the Dates extract and zinc sulphate solution was mixed and centrifuged to synthesise zinc oxide nanoparticles. Characterization was assessed using visual identification and UV visual spectrometry analysis. The anti-bacterial activity was analyzed in Mueller Hinton agar against the S. mutans pathogen, whereas anti-fungal activity was analyzed in Rose Bengal agar against the C. albicans pathogen to rule out the zone of inhibition. Results: UV-Visible spectrophotometric spectra acquired after 40 hours of incubation revealed an absorption peak at 275 nm, indicating the existence of zinc oxide nanoparticles. The assessment of antibacterial activity against S. mutans exhibited heightened efficacy with 100 µl of dates and ZnO nanoparticles, demonstrating stronger antibacterial effects. Similarly, the evaluation of antifungal activity against C. albicans displayed robust antifungal potency with 100 µl of dates and ZnO nanoparticles. Conclusion: Zinc oxide nanoparticles synthesized using dates exhibited potent anti-microbial activity and thus future clinical trials can use this for Oral potentially malignant disorders such as candidal leukoplakia, erosive lichen planus etc.

Identification of a novel PAK1/HDAC6 dual inhibitor ZMF-23 that triggers tubulin-stathmin regulated cell death in triple negative breast cancer

Triple-negative breast cancer (TNBC) is the most poorly treated subtype of breast cancer, and targeting the heterogeneity of TNBC has emerged as a fascinating therapeutic strategy. In this study, we propose for the first time that dual-targeting PAK1 and HDAC6 is a promising novel strategy for TNBC treatment due to their essential roles in the regulation of energy metabolism and epigenetic modification. We discovered a novel dual-targeting PAK1/HDAC6 inhibitor, 6 - (2-(cyclopropylamino) - 6 - (2,4-dichlorophenyl) - 7 - oxopyrido [2,3-d] pyrimidin - 8 (7H) -yl) - N-hydroxyhexanamide (ZMF-23), which presented profound inhibitory activity against PAK1 and HDAC6 and robust antiproliferative potency in MDA-MB-231 cells. In addition, SPR and CETSA assay demonstrated the targeted binding of ZMF-23 with PAK1/HDAC6. Mechanically, ZMF-23 strongly inhibited the cellular PAK1 and HDAC6 activity, impeded PAK1 and HDAC6 regulated aerobic glycolysis and migration. By RNA-seq analysis, ZMF-23 was found to induce TNF-α-regulated necroptosis, which further enhanced apoptosis. Additionally, ZMF-23 triggered PAK1-tubulin/HDAC6-Stathmin regulated microtubule structure changes, which further induced the G2/M cycle arrest. Moreover, prominent anti-proliferative effect of ZMF-23 was confirmed in the TNBC xenograft zebrafish and mouse model via PAK1 and HDAC6 inhibition. Collectively, ZMF-23 is a novel dual PAK1/HDAC6 inhibitor with TNBC treatment potential.

Molecule Flips Switch on Gene Regulation.

A new class of designer drugs can reprogram gene expression by linking a transcription factor that silences genes involved in cell death with an epigenetic regulator that stimulates gene activity. A prototype molecule showed robust potency in flipping the function of BCL6, a protein that supports lymphoma cell proliferation, offering a promising therapeutic approach to combat cancer cell growth.

Functional design of experiment for potency assay optimization and in-silico simulation

For biotherapeutic analytics, robust and reliable potency assays are required. Design of experiment (DoE) approaches are used to investigate the impact of multiple assay parameters. Currently, specific assay features (e.g., half effective concentration) are modelled independently from each other. A joint interpretation of several assay features is thus difficult. In our functional DoE approach, we use the functional relationship of the assay features to describe the sigmoidal dose-response curve. With the composed functional form, the direct impact of assay parameters on the dose-response curve shape was modelled. Moreover, a multivariate desirability was defined and used for assay optimization. We believe that functional modelling contributes to understanding the joint influence of assay parameters and helps to design robust biotherapeutic analytics.

Development and Characterization of an In Vitro Cell-Based Assay to Predict Potency of mRNA-LNP-Based Vaccines.

Messenger RNA (mRNA) vaccines have emerged as a flexible platform for vaccine development. The evolution of lipid nanoparticles as effective delivery vehicles for modified mRNA encoding vaccine antigens was demonstrated by the response to the COVID-19 pandemic. The ability to rapidly develop effective SARS-CoV-2 vaccines from the spike protein genome, and to then manufacture multibillions of doses per year was an extraordinary achievement and a vaccine milestone. Further development and application of this platform for additional pathogens is clearly of interest. This comes with the associated need for new analytical tools that can accurately predict the performance of these mRNA vaccine candidates and tie them to an immune response expected in humans. Described here is the development and characterization of an imaging based in vitro assay able to quantitate transgene protein expression efficiency, with utility to measure lipid nanoparticles (LNP)-encapsulated mRNA vaccine potency, efficacy, and stability. Multiple biologically relevant adherent cell lines were screened to identify a suitable cell substrate capable of providing a wide dose-response curve and dynamic range. Biologically relevant assay attributes were examined and optimized, including cell monolayer morphology, antigen expression kinetics, and assay sensitivity to LNP properties, such as polyethylene glycol-lipid (or PEG-lipid) composition, mRNA mass, and LNP size. Collectively, this study presents a strategy to quickly optimize and develop a robust cell-based potency assay for the development of future mRNA-based vaccines.