Unique Mechanism Of Action Research Articles

Providing insight into the mechanism of action of cationic lipidated oligomers using metabolomics.

The increasing resistance of clinically relevant microbes against current commercially available antimicrobials underpins the urgent need for alternative and novel treatment strategies. Cationic lipidated oligomers (CLOs) are innovative alternatives to antimicrobial peptides and have reported antimicrobial potential. An understanding of their antimicrobial mechanism of action is required to rationally design future treatment strategies for CLOs, either in monotherapy or synergistic combinations. In the present study, metabolomics was used to investigate the potential metabolic pathways involved in the mechanisms of antibacterial activity of one CLO, C12-o-(BG-D)-10, which we have previously shown to be effective against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. The metabolomes of MRSA ATCC 43300 at 1, 3, and 6 h following treatment with C12-o-(BG-D)-10 (48 µg/mL, i.e., 3× MIC) were compared to those of the untreated controls. Our findings reveal that the studied CLO, C12-o-(BG-D)-10, disorganized the bacterial membrane as the first step toward its antimicrobial effect, as evidenced by marked perturbations in the bacterial membrane lipids and peptidoglycan biosynthesis observed at early time points, i.e., 1 and 3 h. Central carbon metabolism and the biosynthesis of DNA, RNA, and arginine were also vigorously perturbed, mainly at early time points. Moreover, bacterial cells were under osmotic and oxidative stress across all time points, as evident by perturbations of trehalose biosynthesis and pentose phosphate shunt. Overall, this metabolomics study has, for the first time, revealed that the antimicrobial action of C12-o-(BG-D)-10 may potentially stem from the dysregulation of multiple metabolic pathways.IMPORTANCEAntimicrobial resistance poses a significant challenge to healthcare systems worldwide. Novel anti-infective therapeutics are urgently needed to combat drug-resistant microorganisms. Cationic lipidated oligomers (CLOs) show promise as new antibacterial agents against Gram-positive pathogens like methicillin-resistant Staphylococcus aureus (MRSA). Understanding their molecular mechanism(s) of antimicrobial action may help design synergistic CLO treatments along with monotherapy. Here, we describe the first metabolomics study to investigate the killing mechanism(s) of CLOs against MRSA. The results of our study indicate that the CLO, C12-o-(BG-D)-10, had a notable impact on the biosynthesis and organization of the bacterial cell envelope. C12-o-(BG-D)-10 also inhibits arginine, histidine, central carbon metabolism, and trehalose production, adding to its antibacterial characteristics. This work illuminates the unique mechanism of action of C12-o-(BG-D)-10 and opens an avenue to design innovative antibacterial oligomers/polymers for future clinical applications.

Trace amine-associated receptor 1 (TAAR1) agonism for psychosis: a living systematic review and meta-analysis of human and non-human data.

Trace amine-associated receptor 1 (TAAR1) agonism shows promise for treating psychosis, prompting us to synthesise data from human and non-human studies. We co-produced a living systematic review of controlled studies examining TAAR1 agonists in individuals (with or without psychosis/schizophrenia) and relevant animal models. Two independent reviewers identified studies in multiple electronic databases (until 17.11.2023), extracted data, and assessed risk of bias. Primary outcomes were standardised mean differences (SMD) for overall symptoms in human studies and hyperlocomotion in animal models. We also examined adverse events and neurotransmitter signalling. We synthesised data with random-effects meta-analyses. Nine randomised trials provided data for two TAAR1 agonists (ulotaront and ralmitaront), and 15 animal studies for 10 TAAR1 agonists. Ulotaront and ralmitaront demonstrated few differences compared to placebo in improving overall symptoms in adults with acute schizophrenia (N=4 studies, n=1291 participants; SMD=0.15, 95%CI: -0.05, 0.34), and ralmitaront was less efficacious than risperidone (N=1, n=156, SMD=-0.53, 95%CI: -0.86, -0.20). Large placebo response was observed in ulotaront phase-III trials. Limited evidence suggested a relatively benign side-effect profile for TAAR1 agonists, although nausea and sedation were common after a single dose of ulotaront. In animal studies, TAAR1 agonists improved hyperlocomotion compared to control (N=13 studies, k=41 experiments, SMD=1.01, 95%CI: 0.74, 1.27), but seemed less efficacious compared to dopamine D 2 receptor antagonists (N=4, k=7, SMD=-0.62, 95%CI: -1.32, 0.08). Limited human and animal data indicated that TAAR1 agonists may regulate presynaptic dopaminergic signalling. TAAR1 agonists may be less efficacious than dopamine D 2 receptor antagonists already licensed for schizophrenia. The results are preliminary due to the limited number of drugs examined, lack of longer-term data, publication bias, and assay sensitivity concerns in trials associated with large placebo response. Considering their unique mechanism of action, relatively benign side-effect profile and ongoing drug development, further research is warranted. PROSPERO-ID: CRD42023451628.

Abstract LB267: Disrupting cytoplasmic proton motive force induces ferroptotic cell death in colorectal cancer: A novel anti-cancer mechanism

Abstract Introduction: Chemotherapeutic resistance and tumor recurrence remain the leading causes of poor prognosis in colorectal cancer (CRC). Herein, we evaluated the anti-cancer effect of a novel repurposed drug HLN* in CRC. Methods: The changes in membrane potential were studied using DiSC(3) fluorescence by confocal microscopy. Cytotoxic activity of compound was studied using Sulphorhodamine-B (SRB) assay. Changes in mitochondrial membrane potential were studied using JC-1 dye. ROS accumulation was analyzed using DCFDA fluorescence using FACS analysis. Ferrorange assay was used to check the ferrous ion influx within the cell. Western blot was used to identify mode of cell death enacted. Results: Our results show that HLN treatment reduced the survival of human and murine CRC cells (CT26, HCT116, Colo 205 and MC38) with IC50 ranging 1-4 µM. HLN further inhibited proton motive force (PMF) by sequentially depolarizing and hyperpolarizing the cell membrane, as examined by cellular membrane potential using DiSC(3) dye by confocal microscopy. Disruption of the PMF leads to changes in various cellular signaling cascades including ROS production and mitochondrial membrane potential (MMP) changes. The MMP was significantly reduced by HLN treatment in CRC cells. Furthermore, we also observed an increase in ROS production by HLN. Pretreatment with N-acetyl cysteine (NAC), an antioxidant, blocked the effects of HLN. Interestingly, ferroptotic cell death markers GPX4, GPX1, SLC7A11/xCT, NRF2 and KEAP1 were significantly inhibited by HLN treatment in CRC cells as observed by western blotting. Ferroptotic induction was then confirmed using ferrorange assay wherein we observed a concentration dependent increase in ferrorange intensity indicating an increase in Fe+2. Overall, HLN dissipates ΔpH by modulating iron influx leading to ferroptotic cell death through ROS. Conclusion: Taken together, our results demonstrate the anti-cancer activity of a novel repurposed drug HLN and its unique mechanism of action. Our results provide a promise for developing this drug as a potential treatment option for CRC, through a novel anti-cancer mechanism. * Due to Intellectual property issued, the name of the drug is not provided Citation Format: Shreyas R. Gaikwad, Mohamed A. Eltokhy, Morgan Moore, Keerthi Kailath, Sanjay K. Srivastava. Disrupting cytoplasmic proton motive force induces ferroptotic cell death in colorectal cancer: A novel anti-cancer mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB267.

D-Allulose Reduces Hypertrophy and Endoplasmic Reticulum Stress Induced by Palmitic Acid in Murine 3T3-L1 Adipocytes.

Natural rare sugars are an alternative category of sweeteners with positive physiologic and metabolic effects both in in vitro and animal models. D-allulose is a D-fructose epimer that combines 70% sucrose sweetness with the advantage of an extremely low energy content. However, there are no data about the effect of D-allulose against adipose dysfunction; thus, it remains to be confirmed whether D-allulose is useful in the prevention and in treatment of adipose tissue alterations. With this aim, we evaluated D-allulose's preventive effects on lipid accumulation in 3T3-L1 murine adipocytes exposed to palmitic acid (PA), a trigger for hypertrophic adipocytes. D-allulose in place of glucose prevented adipocyte hypertrophy and the activation of adipogenic markers C/EBP-β and PPARγ induced by high PA concentrations. Additionally, D-allulose pretreatment inhibited the NF-κB pathway and endoplasmic reticulum stress caused by PA, through activation of the Nrf2 pathway. Interestingly, these effects were also observed as D-allulose post PA treatment. Although our data need to be confirmed through in vivo models, our findings suggest that incorporating D-allulose as a glucose substitute in the diet might have a protective role in adipocyte function and support a unique mechanism of action in this sugar as a preventive or therapeutic compound against PA lipotoxicity through the modulation of pathways connected to lipid transport and metabolism.

Fosfomycin Susceptibility Among Multidrug-Resistant Extended-Spectrum β-Lactamase and Metallo-β-Lactamase–Producing Uropathogens in a Tertiary Care Hospital

Background Urinary tract infections (UTIs) affect an estimated 150 million people each year worldwide involving women more than men. Because of unrestricted use of antimicrobials and accelerating antimicrobial resistance, therapeutic options for treatment of UTIs are becoming limited. Fosfomycin, a broad-spectrum, rapidly absorbable, antimicrobial agent with good bioavailability and substantial activity against multidrug-resistant (MDR) isolates has antimicrobial activity against a substantial percentage of these MDR isolates. Methods A cross-sectional study over a period of 20 months on urine samples of all patients of all age groups clinically suspected of UTI was conducted in a tertiary care center of southern Rajasthan. Samples were processed immediately as per standard microbiological techniques, followed by culture by a semiquantitative method and antimicrobial susceptibility testing using the Kirby-Bauer disk diffusion method. All gram-negative pathogens were screened for extended-spectrum β-lactamase and further metallo-β-lactamase production using standard disk diffusion tests. Results Of the 1858 positive samples isolated, the most common uropathogen was Escherichia coli (59.96%), with 8.97% confirmed extended-spectrum β-lactamase–producing isolates. Fosfomycin had susceptibility of 96.69%, with its sensitivity and specificity being 98.15% and 6.98% more than nitrofurantoin, respectively. Among the metallo-β-lactamase producers, fosfomycin had susceptibility of 78.58%, with sensitivity and specificity being 91.67% and 25% more than nitrofurantoin, respectively. Conclusion With an increasing threat due to MDR uropathogens in both hospitals and in the community, fosfomycin, with its unique mechanism of action, appears to be a better oral, effective, and safe therapeutic option and a viable potential alternative over other drugs for treatment of UTIs.

Inactivation of kindlin-3 increases human melanoma aggressiveness through the collagen-activated tyrosine kinase receptor DDR1.

The role of the focal adhesion protein kindlin-3 as a tumor suppressor and its interaction mechanisms with extracellular matrix constitute a major field of investigation to better decipher tumor progression. Besides the well-described role of kindlin-3 in integrin activation, evidence regarding modulatory functions between melanoma cells and tumor microenvironment are lacking and data are needed to understand mechanisms driven by kindlin-3 inactivation. Here, we show that kindlin-3 inactivation through knockdown or somatic mutations increases BRAFV600mut melanoma cells oncogenic properties via collagen-related signaling by decreasing cell adhesion and enhancing proliferation and migration in vitro, and by promoting tumor growth in mice. Mechanistic analysis reveals that kindlin-3 interacts with the collagen-activated tyrosine kinase receptor DDR1 (Discoidin domain receptor 1) modulating its expression and its interaction with β1-integrin. Kindlin-3 knockdown or mutational inactivation disrupt DDR1/β1-integrin complex in vitro and in vivo and its loss improves the anti-proliferative effect of DDR1 inhibition. In agreement, kindlin-3 downregulation is associated with DDR1 over-expression in situ and linked to worse melanoma prognosis. Our study reveals a unique mechanism of action of kindlin-3 in the regulation of tumorigenesis mediated by the collagen-activated tyrosine kinase receptor DDR1 thus paving the way for innovative therapeutic targeting approaches in melanoma.

Fabrication and evaluation of ribavirin-loaded electrospun nanofibers as an antimicrobial wound dressing

BackgroundSkin is regarded as an essential first line of defense against harmful pathogens and it hosts an ecosystem of microorganisms that create a widely diverse skin microbiome. In chronic wounds, alterations in the host-microbe interactions occur forming polymicrobial biofilms that hinder the process of wound healing. Ribavirin, an antiviral drug, possesses antimicrobial activity, especially against Pseudomonas aeruginosa and Candida albicans, which are known as the main opportunistic pathogens in chronic wounds. RationaleIn this study, electrospun nanofiber systems loaded with ribavirin were developed as a potential wound dressing for topical application in chronic wounds. Ribavirin was chosen in this study owing to the emerging cases of antimicrobial (antibiotics and antifungal) resistance and the low attempts to discover new antimicrobial agents, which encouraged the repurposing use of current medication as an alternative solution in case of resistance to the available agents. Additionally, the unique mechanism of action of ribavirin, i.e., perturbing the bacterial virulence system without killing or stopping their growth and rendering the pathogens disarmed, might be a promising choice to prevent drug resistance. Cyclodextrin (CD) was utilized to formulate ribavirin as an electrospun nanofibers delivery system to enhance the absorption and accelerate the release of ribavirin for topical use. ResultsThe results demonstrated a successful ribavirin nanofibers fabrication that lacked beads and pores on the nanofibrous surfaces. Ribavirin underwent a physical transformation from crystalline to amorphous form, as confirmed by X-ray diffraction analysis. This change occurred due to the molecular dispersion after the electrospinning process. Additionally, the CD enhanced the encapsulation efficiency of ribavirin in the nanofibers as observed from the drug-loading results. Polyvinylpyrrolidone (PVP) and CD increased ribavirin released into the solution and the disintegration of fibrous mats which shrank and eventually dissolved into a gel-like substance as the ribavirin-loaded fibers began to break down from their border toward the midpoint. Cytotoxicity of ribavirin and CD was evaluated against human dermal fibroblasts (HFF-1) and the results showed a relatively safe profile of ribavirin upon 24-hour cell exposure, while CD was safe within 24- and 48-hour. ConclusionThis study provides valuable insights into the potential application of our nanofibrous system for treating chronic wounds; however, further antimicrobial and in-vivo studies are required to confirm its safety and effectiveness.

A two-stage computational framework for identifying antiviral peptides and their functional types based on contrastive learning and multi-feature fusion strategy.

Antiviral peptides (AVPs) have shown potential in inhibiting viral attachment, preventing viral fusion with host cells and disrupting viral replication due to their unique action mechanisms. They have now become a broad-spectrum, promising antiviral therapy. However, identifying effective AVPs is traditionally slow and costly. This study proposed a new two-stage computational framework for AVP identification. The first stage identifies AVPs from a wide range of peptides, and the second stage recognizes AVPs targeting specific families or viruses. This method integrates contrastive learning and multi-feature fusion strategy, focusing on sequence information and peptide characteristics, significantly enhancing predictive ability and interpretability. The evaluation results of the model show excellent performance, with accuracy of 0.9240 and Matthews correlation coefficient (MCC) score of 0.8482 on the non-AVP independent dataset, and accuracy of 0.9934 and MCC score of 0.9869 on the non-AMP independent dataset. Furthermore, our model can predict antiviral activities of AVPs against six key viral families (Coronaviridae, Retroviridae, Herpesviridae, Paramyxoviridae, Orthomyxoviridae, Flaviviridae) and eight viruses (FIV, HCV, HIV, HPIV3, HSV1, INFVA, RSV, SARS-CoV). Finally, to facilitate user accessibility, we built a user-friendly web interface deployed at https://awi.cuhk.edu.cn/∼dbAMP/AVP/.

A Novel CaCu-Metal-Organic-Framework Based Multimodal Treatment Platform for Enhanced Synergistic Therapy of Hepatocellular Carcinoma.

Metal ions have attracted a lot of interest in antitumor therapy due to their unique mechanism of action. However, multiple death mechanisms associate with metal ions to synergistic antitumors have few studies mainly due to the serious challenges in designing and building metal-associated multimodal treatment platforms. Hence, a series of glutathione-activatable CaCu-based metal-organic-frameworks loaded with doxorubicin and ovalbumin are successfully designed and synthesized with an "all in one" strategy, which is modified by galactosamine-linked hyaluronic acid to prepare multimodal treatment platform (SCC/DOX@OVA-HG) for targeted delivery and synergistic antitumor therapy. SCC/DOX@OVA-HG can be rapidly degraded by the overexpressed glutathione and then releases the "cargoes" in the tumor microenvironment. The released Cu+ efficiently catalyzes H2O2 to produce highly toxic ROS for CDT, and the up-regulation of calcium ion concentration in tumor cells induced by the released Ca2+ enables calcium overload therapy, which synergically enhances the metal-related death pattern. Meanwhile, OVA combined with Ca2+/Cu2+ further activates macrophages into an M1-like phenotype to accelerate tumor cell death through immunotherapy. Besides, the released DOX can also insert into the DNA double helix for chemotherapy. Consequently, the developed SCC/DOX@OVA-HG reveals significantly improved antitumor efficacy through a multimodal synergistic therapy of chemotherapy, chemodynamic therapy, calcium overload, and immunotherapy.

Abstract 5318: SM2275, A tetraspecific, dual EGFR and PD-L1-targeting, conditionally activating co-stimulatory CD28 and simultaneously blocking PD-1/PD-L1 signaling to promoting fully activated T cell killing effects on tumor cells

Abstract Background: T cells require both signal 1 TCR engagement and signal 2 co-stimulatory signal to achieve complete activation and tumor recognition. CD28 is a signal 2 co-stimulatory receptor and activating CD28 increases T cell sensitivity, cytokine production and T cell survival. PD-1/PD-L1 signaling attenuates the T cells functional activity by inhibiting CD28 co-stimulation in the presence of TCR stimulation. Therefore, we developed SM2275, a tetraspecific VHH antibody against EGFR, PD-L1, CD28 and HSA, for the treatment of EGFR+/PD-L1+ solid tumors. Preclinical study demonstrated promising efficacy and favorable safety profile via a unique mechanism of action. Methods: SM2275 was developed using StarMab’s proprietary Quadbody multifunctional VHH antibody platform. It features two monovalent, low-affinity binding domains for EGFR and PD-L1, along with nanomolar low affinity CD28 binding domains arranged in a tandem format, followed by a half-life extending HSA binding domain. Differential binding affinities of SM2275 to EGFR/PD-L1 single-expressing versus double-expressing cell lines were analyzed by flow cytometry. Avidity-enhanced blockade of PD-1 on EGFR+PD-L1+ double-positive compared to PD-L1+ single-positive cells was analyzed via competitive binding assay and Jurkat PD1 NFAT Luciferase reporter assay. EGFR/PD-L1 dual-target-dependent CD28 activation was confirmed using a primary human T cell IL2 & IFN-γ release assay in the presence of TCR stimulation. In vivo efficacy against EGFR+/PD-L1+ double-positive human tumors were evaluated using a syngeneic tumor transplantation model of MC38-hEGFR/hPDL1 cell line into hPD-L1/hCD28 humanized mice. Results: SM2275 displays dramatically increased affinity for cancer cells co-expressing EGFR/PD-L1 compared to single-expressing cells. This leads to an avidity-enhanced PD-L1 blockade specifically on the surface of EGFR+PD-L1+ double-positive cancer cells, enabling localized reactivation of tumor-infiltrating T cells without systemic immune toxicity. Simultaneously blocking the PD-L1 signaling unleashed the inhibition of CD28 co-stimulation for the full T cell activation. In the presence of SM2275, EGFR/PD-L1 double-positive cancer cells induce significantly enhanced CD28 activation and cytokine secretion compared to single-antigen-expressing cells. In a syngeneic tumor transplantation model, SM2275 effectively inhibits the growth of MC38-hEGFR/hPDL1 tumors in hPD-L1-hCD28 humanized mice, with no apparent cytokine release and immune toxicity. Conclusions: SM2275 is a novel dual-EGFR & PD-L1-targeting, conditional CD28 agonistic antibody. It demonstrated promising efficacy and favorable safety profile in preclinical studies via a unique mechanism of action. Citation Format: Shihao Lu, Xiaodan Liu, Sheila Zhou, Wenjing Song, Huiqin Geng, Simin Yang, Hong Zhou, Siyao Xie, Yi Wei, Xing Zhang, Yanbin Liang. SM2275, A tetraspecific, dual EGFR and PD-L1-targeting, conditionally activating co-stimulatory CD28 and simultaneously blocking PD-1/PD-L1 signaling to promoting fully activated T cell killing effects on tumor cells [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 5318.

Abstract 1218: CDK9 inhibitors modulate the transcriptional landscape of colorectal cancer to suppress MAPK signaling and synergizes with BRAF inhibitors to treat BRAF-mutant colorectal cancer

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US, and it remains a significant public health burden. Metastatic CRC (mCRC) is usually treated with combination chemotherapy and targeted therapy regimens, but eventually becomes chemotherapy refractory. Thus, novel and more effective treatments for mCRC are urgently needed. Cyclin-dependent kinase 9 (CDK9) is a key activator of RNA Pol II transcription and promotes the expression of many cancer driver genes, making CDK9 a promising target for cancer therapy. Methods: Human CRC cell lines, patient-derived organoids (PDOs), cell line xenografts, and patient-derived xenografts were used to investigate the efficacy and mechanisms of action of the CDK9 inhibitors AZD4573, enitociclib, and NVP-2, as well as the BRAF inhibitors encorafenib and dabrafenib. In vitro viability was measured by chemical cell proliferation assays and in vivo tumor sizes were measured by calipers. In vitro mechanisms of action were assessed by RNA sequencing, real time polymerase chain reaction, and Western blotting. In vivo mechanisms of action were assessed by immunohistochemistry. Results: CDK9 inhibitors potently inhibited CRC cells and PDOs. In contrast to hematologic malignancies, we did not observe consistent suppression of the oncogenes c-MYC and MCL-1 by CDK9 inhibitor treatment. Instead, CDK9 inhibitors suppressed several other key cancer pathways in CRC, such as MAPK, mTOR, and PI3K signaling pathways. Importantly, multiple targets within the MAPK pathway were strongly suppressed, including EGFR, KRAS, and BRAF. We hypothesized that combination treatment with CKD9 inhibitors and MAPK pathway inhibitors can synergistically treat CRC. As proof-of-concept, we investigated the combination of CDK9 and BRAF inhibitors in models of BRAF-mutant CRC, a particularly aggressive type of CRC. We found this combination to synergistically suppress CRC growth in vitro and in vivo. Compared to single agents, combination treatment led to significantly stronger induction of apoptosis and suppression of MAPK pathway signaling. Our results suggest that concurrent treatment with CDK9 inhibitors plus established MAPK pathway inhibitors, such as BRAF inhibitors, can significantly improve upon single agent treatment. Conclusions: We have found CDK9 inhibitors to potently suppress CRC growth and survival through a unique mechanism of action, by vertical suppression of the MAPK signaling pathway. We demonstrate that CDK9 inhibitors can synergize with BRAF inhibitors in the treatment of BRAF-mutant CRC models. Thus, CDK9 inhibitors are a promising class of drugs warranting further investigation, including early-phase clinical trials, in mCRC. Citation Format: Chaoyuan Kuang, Ning Wei, Mahshid Mohammadi, Muzaffer A. Bhat, Terence Li, Priyanka Patil, Othon Wiltz, Renee Huang, Kohtaro Ooka, Melanie Quintal, Edward Chu. CDK9 inhibitors modulate the transcriptional landscape of colorectal cancer to suppress MAPK signaling and synergizes with BRAF inhibitors to treat BRAF-mutant colorectal cancer [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 1218.

Abstract 5802: An orally available small molecule JMBI-001 elicits MYC-synthetic lethality and anti-tumor immunity by disabling MKLP2-mediated cellular processes

Abstract The MYC oncogene, a pivotal regulator of various cellular processes, is deregulated in approximately 70 % of human malignancies. Mitotic Kinesin-like Protein 2 (MKLP2) plays a versatile role in both interphase and mitosis and has emerged as a significant prognostic indicator and therapeutic target in cancer. The undruggability of MYC and the scarcity of MKLP2 inhibitors, however, have impeded clinical translation. Our development of JMBI-001, a potent and orally bioavailable small-molecule compound, overcomes these barriers. JMBI-001 elicits loss of function phenotypes in MKLP2 and a synthetic lethal interaction with MYC overexpression. Extensive kinome and safety profiling have revealed no significant off-target effects, and the compound is well-tolerated in long-term animal studies. In preclinical models, JMBI-001 has demonstrated an average tumor growth inhibition rate of 75 % across more than 20 MYC-overexpressing tumor models, including those in the stomach, lung, colon, liver, breast, kidney, skin, and hematopoietic system. Its anti-tumor activity positively correlates with high MYC abundance, aligning with the selective eradication of cells with abundant MYC both in vitro and in vivo. Notably, JMBI-001 also robustly stimulates systemic anti-tumor immunity, enhancing NK and CD3+ T cell infiltration in tumors of syngeneic cancer models. Moreover, additive or synergistic effects have been observed when combined with anti-PD1 therapy even in tumors refractory to the immune checkpoint blockade (ICB) therapy. The dual therapeutic actions of JMBI-001 stem from its disruption of MKLP2 functionalities, leading to anomalies, such as Golgi fragmentation in interphase and multipolarity in pro-metaphase. These disruptions lead to two key outcomes: apoptosis and immunogenic cell death, marked by secreted ATP, released high mobility group protein B1 (HMGB1), and surface-exposed calreticulin. These abnormalities, primed and amplified by deregulated MYC, are not observed in non-transformed cells, suggesting their potential as pharmacodynamic markers for monitoring JMBI-001's activity in vivo. In conclusion, JMBI-001 represents a novel class of anticancer agents that simultaneously triggers MYC synthetic lethality and anticancer immunity by targeting MKLP2-mediated cellular processes. Its unique mechanisms of action, exceptional bioavailability, potency at low nanomolar concentrations, wide-spectrum efficacy, and favorable safety profile establish JMBI-001 as a promising clinical study-ready drug candidate for treating MYC-driven cancers. Citation Format: Ting Zhang, Qiong Shi, Julia Kalashova, Xumei Liu, Xiaohu Zhou, Chenglu Yang, Yan Long, Hongmei Li, Jinhua Li, Gang Lv, Duo Yu, Xuejiao Jiang, Shenqiu Zhang, Jing Zhang, Hong Liu, Dun Yang. An orally available small molecule JMBI-001 elicits MYC-synthetic lethality and anti-tumor immunity by disabling MKLP2-mediated cellular processes [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 5802.

Abstract 5974: Targeting VEGFR on tumor cells and activated vasculature through delivery of granzyme B

Abstract Anti-VEGF therapy is a key therapeutic in several cancer treatment regimens. However, none of the current therapeutics are directly cytotoxic against tumor cells. VEGF121 is the smallest isoform in the VEGF-A family of cytokines that binds to VEGF receptors R-1 and R-2. Both receptors are over-expressed on several tumor cells and the endothelium of tumor vasculature but not normal vasculature. We hypothesized that delivery of a cytotoxic payload through VEGFR-2 into tumor cells and tumor neovasculature would significantly enhance cancer therapy. Granzyme B is a key effector in immune-mediated cell killing through both caspase-dependent and -independent mechanisms. We previously reported the development of a fusion protein composed of granzyme B (GrB) and VEGF121 (GrB/VEGF121). Here, we report on GrB-Fc-VEGF121, a fusion protein incorporating a human IgG heavy chain Fc fragment for dimerization to increase the molecular weight and improve in vivo circulation and targeting potential. We expressed and purified GrB-Fc-VEGF121 from HEK-293E cells under serum-free conditions with a final yield of approximately 40 mg/L. The enzymatic activity of GrB in GrB-Fc-VEGF121 was comparable to that of commercially available human GrB. We characterized this construct against both human and mouse VEGFR+ cell lines as this ligand cross-reacts to both species. GrB-Fc-VEGF121 showed in vitro cytotoxicity in the nanomolar range against tumor and endothelial cell lines expressing high levels of VEGFR-1 or VEGFR-2, while receptor-negative cells demonstrated IC50 levels in the high micromolar range. GrB-Fc-VEGF121 internalized into VEGFR-2+ tumor and endothelial cells within 2 hours of treatment while untargeted GrB did not internalize, suggesting internalization was receptor-mediated. Treatment of VEGFR2+ cells at the respective IC50 dose resulted in >50% cell death via apoptosis and/or necrosis within 48 h. Ex vivo serum stability studies indicated a gradual protein loss of GrB-Fc-VEGF121 with an overall stability of about 50% over 96 hours. No toxicity was observed in mice treated with a total dose of 475 mg/kg (IP, QOD x 5), indicating that the maximum tolerated dose of GrB-Fc-VEGF121 exceeds this dose level. In vivo efficacy studies in an OVCAR8 tumor xenograft model (20 mg/kg GrB-Fc-VEGF121 x 5) resulted in significant growth inhibition of established tumors compared to vehicle controls. The number of CD31+ blood vessels and Ki-67+ proliferating tumor cells decreased significantly in treated tumors compared to controls. Pharmacokinetic studies are underway and will be reported. Thus, GrB-Fc-VEGF121 significantly reduces tumor growth in vivo with no observed toxicity against normal tissues. This tumor- and vascular-targeting agent appears to have significant potential as a new class of targeted therapeutic agents with a unique mechanism of action. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. Targeting VEGFR on tumor cells and activated vasculature through delivery of granzyme B [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 5974.

Abstract 2100: Novel LIPA targeted therapy for treating inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare but incredibly aggressive subtype of breast cancer (BC). IBC accounts for 2-4% of all occurrences of breast cancer and results in 7-10% of breast cancer-related deaths. IBC is only partially treatable with current therapies such as chemotherapy, surgery, and radiotherapy. Identification of novel therapeutic targets is urgently required. The main organelle for the synthesis, folding, and modification of proteins is called the endoplasmic reticulum (ER). Since elevated basal ER stress (ERS) is usually found in many cancers including IBC, our hypothesis was that the high basal ERS in IBC constitutes a serious vulnerability that can be targeted. Recently, we developed a small molecule ERX41 that promotes ER stress by blocking Lysosomal acid lipase A (LIPA) function in ER of cancer cells. The goal of the project is to test the utility of ERX-41 in treating IBC. Methods: To study the effect of ERX-41, we have used three well-established IBC model cells. We evaluated the efficacy of ERX-41 as a novel therapeutic for treating IBC using cell viability assays. The long-term effects of ERX-41 on IBC cell survival were evaluated using colony formation assays. Annexin V assays were used to measure apoptosis. Reporter assays, splicing assays, RT-qPCR, and Western blotting were used in mechanistic investigations. The effectiveness of ERX-41 was examined in vivo using KPL4 cell-based xenografts. Results: ERX-41 significantly decreased the viability of all three IBC model cells (SUM149, SUM190PT, and KPL4), with an IC50 of about 125 nM in MTT assay. Additionally, ERX-41 treatment significantly decreased IBC cells capacity to form colonies and promoted apoptosis. Specificity of the ERX41 mediated actions were confirmed using LIPA KO cells. Using RTqPCR based splicing assays, we demonstrated increased splicing of XBP1 as early as 6 hours after ERX41 treatment. Western analyses showed robust induction of stress markers (CHOP, PERK, ATF4) in IBC cells upon treatment with ERX-41. In KPL4 xenograft studies, ERX-41 showed significant reduction in the tumor volume. IHC analyses confirmed decreased proliferation marker and increased ER stress markers. Conclusions: Collectively, our results suggest that ERX-41 is a novel therapeutic agent that targets the LIPA with a unique mechanism of action and implicate ERX-41 binding to LIPA induces ER stress and promotes apoptosis of IBC cells. Citation Format: Zenaida Fuentes, Rahul Gopalam, Bianca A. Romo, Khaled Mohamed Nassar, Xue Yang, Behnam Ebrahimi, Paulina Ramirez, Chia-Yuan Chen, Scott Elmore, Harika Nagandla, Uday Pratap, Christoforos Thomas, Suryavathi Viswanadhapalli, Jung-Mo Ahn, Ganesh Raj, Ratna K. Vadlamudi. Novel LIPA targeted therapy for treating inflammatory breast cancer [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 2100.

Abstract 5925: Safety, efficacy, pharmacokinetics, and pharmacodynamics of a second generation Irofulven analog CAP-0121 in preclinical cancer models

Abstract Background: Irofulven is a semisynthetic sesquiterpene anticancer agent initially investigated for its unique mechanism of action and potential to overcome resistance observed in other chemotherapeutics. This study investigates the pharmacokinetics and pharmacodynamics of a second generation irofulven analog, CAP-121 (Califia Pharma), and its efficacy and safety in preclinical cancer models relative to irofulven. Methods: 24-hour comprehensive PK was performed at two different intravenous (IV) doses in canines for irofulven (0.2 mg/kg and 1 mg/kg; MTD) and CAP-121 (1 mg/kg and 3 mg/kg). Plasma concentrations were measured by ultra-high performance liquid chromatography multiple reaction monitoring mass spectrometry. PK parameters were determined for both compounds using noncompartmental methods (Phoenix WinNonlin, version 8.4). PK parameters included area under the concentration vs time curve (AUC), maximum observed concentration (Cmax), clearance (CL), volume of distribution (Vd), and elimination half-life (t1/2). Canine platelet and neutrophil counts were measured across 28 days to ascertain the maximum tolerated dose (MTD); platelet count reversibility was also assessed. Mice xenograft models implanted with a drug resistant human ovarian cancer (A2870), or a drug-resistant human metastatic lung cancer (MV522), were dosed with irofulven (9 mg/kg; MTD) and CAP-121 (15, 20, and 25 mg/kg) and resulting tumor mass, % change in body weight, and platelet counts were compared relative to vehicle only. Results: Systemic exposure, CL, Vd, and t1/2 were similar between irofulven and CAP-121 at a 1 mg/kg IV dose. For irofulven, exposure increased approximately proportionally at doses of 0.2 mg/kg and 1 mg/kg. For CAP-121, exposure increased less than proportionally at doses of 1 mg/kg and 3 mg/kg. MTD of CAP-121 was found to be 3-fold higher in canines compared to irofulven as 3-fold more CAP-121 was required to match the platelet reduction with irofulven; platelet nadir was rapidly reversible for CAP-121. Canine nadir platelets were 3-fold higher for CAP-121 vs irofulven at the same dose (1 mg/kg). CAP-121 was superior to irofulven (p < 0.02) with regards to final tumor weights in mice xenograft models, even though MTD was not reached for CAP-121; a change in body weight and platelet loss was not observed for CAP-121 at up 25 mg/kg in mice. Conclusion: The canine PK/PD study of CAP-121 demonstrates that CAP-121 has favorable PK parameters. With regards to in vivo anticancer cytotoxic activity, the two drug-resistant xenograft models confirm the superiority of CAP-121 over irofulven. Together, these findings indicate a wider therapeutic window in humans is expected for CAP-121 compared to irofulven, emphasizing the potential anticancer utility of CAP-121 for solid cancers without prolonged thrombocytopenia. Further investigation in clinical trials is warranted Citation Format: Raymond T. Suhandynata, T K. Nguyen, Venkata R. Kotamraju, Jeremiah D. Momper, Michael J. Kelner. Safety, efficacy, pharmacokinetics, and pharmacodynamics of a second generation Irofulven analog CAP-0121 in preclinical cancer models [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 5925.

Abstract 4079: EGL-001 is a novel immunocytokine designed to specifically target and disarm T regulatory cells in the tumor microenvironment

Abstract Introduction Immune checkpoint blockade (ICB) therapies targeting PD-1/PD-L1 or CTLA-4 have greatly improved clinical outcome in many cancers. However, multiple mechanisms of resistance to ICB severely limit the number of patients experiencing long-term survival benefits. Overcoming resistance to ICB represents a significant challenge for drug development. Regulatory T cells (Tregs) in the tumor microenvironment (TME) prevent effector T cells (Teffs) from mounting a productive immune response and have been implicated in ICB resistance. Novel therapeutic strategies aimed at disarming Tregs in the TME could restore anti-tumor responses and sensitize patients to ICB therapy. Treg survival is dependent on interleukin-2 (IL-2), which signals in Tregs via the high affinity IL-2 receptor (IL-2R). Tumor-associated Tregs differentiate from tumor-associated Teffs and from peripheral Tregs by higher expression of CD25, the IL-2R alpha chain. Tregs in the TME also express higher surface levels of CTLA-4 as compared to peripheral Tregs. Thus, CD25 and CTLA-4 overexpression could allow preferential targeting of tumor-associated Tregs. Methods and Results We used single-cell RNA sequencing data from cancer patients and flow cytometry analysis of human tumor samples to verify that CD25 and CTLA-4 are selectively overexpressed in tumor-associated Tregs as compared with peripheral Tregs or other T cells. Based on these findings, we developed EGL-001, a novel immunocytokine (ICK) comprising a humanized IgG1 directed against CTLA-4 fused to a Treg-specific IL-2R antagonist IL-2 mutein, to selectively target tumor infiltrating Tregs. EGL-001 IgG1 moiety binds membrane CTLA-4, as demonstrated by flow cytometry, and competes with CTLA-4-CD80/86 interaction, as evaluated by ELISA. EGL-001 IL-2 mutein moiety binds to CD25, as demonstrated by bio-layer interferometry, and specifically blocks high-affinity IL-2R signaling in Tregs. Signaling blockade is further potentiated by a CTLA-4 dependent CD25 downmodulation process evidenced by confocal microscopy and flow cytometry. We demonstrate that EGL-001 unique tumor-associated Treg-targeted dual mechanism of action results in increased Treg apoptosis and decreased Treg frequencies in multiple in vitro, ex vivo and in vivo systems. Importantly, EGL-001 only minimally affects IL-2 signaling and survival of Teffs, which are then expected to be unleashed from Treg cell-mediated suppression. Accordingly, EGL-001 induces anti-tumor responses in mice in combination with anti-PD1 therapy. Conclusions EGL-001 is an ICK with a novel and unique mechanism of action combining ICB and Treg selective IL-2 starvation, designed to specifically disarm and deplete tumor-associated Tregs. Based on our preclinical data, EGL-001 is being developed as a cancer immunotherapeutic to improve patients’ response to ICB and counteract ICB resistance. Citation Format: Fiorella Kotsias, Caroline Janot-Sardet, Pamela Pamela Caudana, Erica Russo, Marine Dubois, Andres Zucchetti, Mercedes Tkach, Sebastien Lemoine, Ilgar Z. Mamedov, Alvaro Lladser, Jordan Denizeau, Annalisa Meola, Christine Sedlik, Edith Borcoman, Felix Rey, Pablo Guardado-Calvo, Bernard Vanhove, Monica Gostissa, Eliane PIaggio. EGL-001 is a novel immunocytokine designed to specifically target and disarm T regulatory cells in the tumor microenvironment [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 4079.

Abstract 2058: Compound library screening to identify modulators of alternative splicing in non-small cell lung cancer (NSCLC)

Abstract Alternative splicing is a molecular mechanism that allows a single gene to encode multiple proteins. It is a complex and highly controlled process used to regulate normal gene expression but is often dysregulated in cancer. Compounds that can modulate alternative splicing are currently being explored as a potential new class of therapeutic agent in cancer. This highlights the need for a deeper understanding of the splicing process, its regulation, and its impact. The discovery of novel and specific tool compounds that modulate splicing would therefore not only be beneficial in investigating the regulation and dysregulation of splicing in cancer but could also be exploited therapeutically. To identify novel regulators of splicing, we generated a cell-based split luciferase screening assay based on alternative splicing of MCL-1 (myeloid cell leukemia-1 protein) pre-mRNA. The MCL1 gene usually produces an mRNA encoding a ‘long’ variant (MCL1L) that is an anti-apoptotic protein often highly expressed in cancers. However, in some circumstances, for example following genetic knockdown of splicing factors, a pro-apoptotic ‘short’ variant (MCL1S) is expressed as a result of exon-skipping. Here we engineered the human NSCLC cell line NCI-H1299 to express a luminescent-tagged version of the MCL1S splice variant to monitor its induction upon spliceosome modulation. Using this engineered cell line, we screened an unannotated library of 12,000 compounds selected to have low molecular weights and favorable properties for cellular uptake. The screen had an average Z’ value of 0.89 over 45 microplates, indicating high assay robustness. Hits were identified as any compound with a luminescence of greater than the average + two standard deviations of the screening dataset. The compounds were validated by repetition in the screening assay, giving 34 candidate hits that were then triaged by qPCR assay to confirm splicing modulation of MCL1. One interesting hit was found to increase mRNA and cellular protein levels of MCL1S and induced altered splicing across the transcriptome that was distinct from the splicing profiles of a diverse panel of splicing modulators, including compounds targeting SF3B1, CLK, and RBM39. In addition, treatment with this hit compound resulted in accumulation of the splicing factor SC35 in cytoplasmic granules, an unusual phenotype not seen with known splicing modulators, that could explain the distinct splicing modulation we have observed. Deconvolution and further characterization of screening hits and potentially unique new mechanisms of action could improve current understanding of alternative splicing and its regulation in NSCLC. The discovery of novel compounds to expand our current toolset of splicing modulators will be key in building the foundation for future splicing targeted drug discovery. Citation Format: Rachel Cooley, Marissa V. Powers, Adam G. Bond, Patrizia Jensen, Gary Newton, Andrea Scarpino, Juliane Braun, Christina Esdar, Paul A. Clarke. Compound library screening to identify modulators of alternative splicing in non-small cell lung cancer (NSCLC) [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 2058.

Abstract 4071: The first-in-class PD1-IL18 conjugate BPT567 induces potent anti-tumor immunity by preferentially activating PD1+IL18R+ intratumoral effector T cells in cis

Abstract Antibody-cytokine conjugates leverage orthogonal mechanisms of action (MoA) in one molecule to induce potent antitumor immune responses. PD-1-targeting conjugates are of particular interest since they preferentially target antigen-experienced PD-1+ CD8+ T cells enriched in the tumor microenvironment (TME) while simultaneously blocking the PD-(L)1 pathway and inducing potent cytokine receptor stimulation in the same CD8+ T cell in cis (cis-signaling). Interleukin 18 (IL-18) is a proinflammatory cytokine able to integrate both innate and adaptive immunity resulting in profound anti-tumor immune responses mediated by T effector and NK cells. BPT567 has been generated via chemical conjugation of the anti-PD-1 Ab LipustobartTM to an enhanced human IL-18 variant of increased potency and reduced sensitivity to IL-18 binding protein (IL-18BP). BPT567 is designed to specifically target and activate a subset of tumor-infiltrating PD-1+ IL18R+ CD8+ T effector cells recently described to exhibit superior cytotoxic and proliferative activity (Codarri Deak et al., Nature 2022). Due to its ability to signal in cis, BPT567 shows enhanced potency as well as increased resistance to IL-18BP when analyzing IFNγ release in vitro in PD-1+ cells. Subsequent PD-1 receptor occupancy analyses showed that it is fully sufficient for BPT567 to engage a low number of PD-1 receptors to induce maximum IFNγ release in NK92 cells expressing human PD-1. Strikingly, despite addressing overlapping epitopes in PD-1, even a 200-fold excess of pembrolizumab has no impact on the in vitro potency of BPT567. In vivo, BPT567 exhibits strong anti-tumor efficacy at significantly lower IL-18 doses compared to the combination of an untargeted antibody-IL-18 conjugate and an anti-PD1 Ab further corroborating the importance of cis-signaling as a unique MoA of this PD1-IL18 conjugate. Detailed analyses of tumor-infiltrating immune leukocytes (TIL) revealed that BPT567 triggers a preferential expansion of CD8+ T effector memory cells within the TME. In line with this finding, activity in the MC38 tumor model is dependent on the presence of CD8+ T cells while depletion of other immune cell subsets (e.g. NK cells, CD4+ T cells, macrophages) had no impact on efficacy. Although not required for anti-tumor efficacy, CD4+ T cells are essential for proficient formation of an immunological memory. In an effort to confirm findings generated in murine models in relevant human ex vivo systems, we analyzed the release of cytokines and chemokines in dissociated cells isolated from primary human tumor explants. Also in this human model, BPT567 is able to induce significantly stronger IFNγ release compared to either single agents or the combination of untargeted IL-18 and an anti-PD1 Ab, thus confirming the unique MoA of the PD1-IL18 conjugate triggering superior TIL activation in cis. Citation Format: Kea Martin, Thuy Luu, Jean-Philippe Carralot, Lilian Gremlich, Petra Herzig, Caoimhe Herr, Roy Meoded, Philipp Moosmann, Arnaud Goepfert, Alfred Zippelius, Vijaya Pattabiraman, Bertolt Kreft. The first-in-class PD1-IL18 conjugate BPT567 induces potent anti-tumor immunity by preferentially activating PD1+IL18R+ intratumoral effector T cells in cis [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 4071.

Low-Dose Naltrexone as an Adjuvant in Combined Anticancer Therapy.

Naltrexone (NTX) is a non-selective antagonist of opioid receptors, primarily used in the therapy of opioid and alcohol dependence. Low-dose naltrexone (LDN) exhibits antagonistic action against the opioid growth factor receptor (OGFr), whose signaling is associated with the survival, proliferation, and invasion of cancer cells. The mechanism of action of LDN depends on the dose and duration of the OGFr blockade, leading to a compensatory increase in the synthesis of the opioid growth factor (OGF), which has an inhibitory effect on carcinogenesis. Numerous studies on in vitro and in vivo models provide evidence of LDN's positive impact on inhibiting the OGF-OGFr axis in cancers. LDN's unique mechanism of action on cancer cells, lack of direct cytotoxic effect, and immunomodulating action form the basis for its use as an adjuvant in chemotherapy and immunotherapy of cancerous lesions.

Exploring Phytochemicals for Diabetes Management: Mechanisms, Efficacy, and Future Directions

In the realm of global health diplomacy, diabetes mellitus undoubtedly poses a significant challenge. Furthermore, there is a pressing need to explore alternative and complementary therapies. Phytochemicals, which are naturally occurring bioactive compounds found in plants, exhibit distinct biochemical properties, rendering them promising candidates for diabetes management. This review focuses on the potential of various classes of phytochemicals, including polyphenols, alkaloids, terpenoids, organosulfur compounds, and polysaccharides, in diabetes management. Each class demonstrates unique mechanisms of action, contributing to therapeutic effects such as enhancing insulin sensitivity, reducing carbohydrate digestion and glucose absorption, regulating antioxidant stress, inflammation, and glucose metabolism. While successful preclinical trials provide valuable insights, further rigorous clinical trials are imperative to ascertain the therapeutic efficacy, optimal dosage, bioavailability, and potential interactions with concomitant medications. Understanding the functional pathways of plant-derived substances is crucial, as it enables the formulation of precise strategies for incorporating them as dietary supplements for individuals with diabetes mellitus. Keywords: Phytochemicals, diabetes mellitus, insulin sensitivity, glucose metabolism, oxidative stress, and inflammation.