Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor.

KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.

Transport and inhibition mechanisms of the human noradrenaline transporter.

The noradrenaline transporter (also known as norepinephrine transporter) (NET) has a critical role in terminating noradrenergic transmission by utilizing sodium and chloride gradients to drive the reuptake of noradrenaline (also known as norepinephrine) into presynaptic neurons1-3. It is a pharmacological target for various antidepressants and analgesic drugs4,5. Despite decades of research, its structure and the molecular mechanisms underpinning noradrenaline transport, coupling to ion gradients and non-competitive inhibition remain unknown. Here we present high-resolution complex structures of NET in two fundamental conformations: in the apo state, and bound to the substrate noradrenaline, an analogue of the χ-conotoxin MrlA (χ-MrlAEM), bupropion or ziprasidone. The noradrenaline-bound structure clearly demonstrates the binding modes of noradrenaline. The coordination of Na+ and Cl- undergoes notable alterations during conformational changes. Analysis of the structure of NET bound to χ-MrlAEM provides insight into how conotoxin binds allosterically and inhibits NET. Additionally, bupropion and ziprasidone stabilize NET in its inward-facing state, but they have distinct binding pockets. These structures define the mechanisms governing neurotransmitter transport and non-competitive inhibition in NET, providing a blueprint for future drug design.

Structural insights into the inhibition of glycine reuptake.

The human glycine transporter 1 (GlyT1) regulates glycine-mediated neuronal excitation and inhibition through the sodium- and chloride-dependent reuptake of glycine1-3. Inhibition of GlyT1 prolongs neurotransmitter signalling, and has long been a key strategy in the development of therapies for a broad range of disorders of the central nervous system, including schizophrenia and cognitive impairments4. Here, using a synthetic single-domain antibody (sybody) and serial synchrotron crystallography, we have determined the structure of GlyT1 in complex with a benzoylpiperazine chemotype inhibitor at 3.4 Å resolution. We find that the inhibitor locks GlyT1 in an inward-open conformation and binds at the intracellular gate of the release pathway, overlapping with the glycine-release site. The inhibitor is likely to reach GlyT1 from the cytoplasmic leaflet of the plasma membrane. Our results define the mechanism of inhibition and enable the rational design of new, clinically efficacious GlyT1 inhibitors.

Structural basis of species-selective antagonist binding to the succinate receptor.

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation3-5. Because SUCNR1 senses succinate as an immunological danger signal6-which has relevance for diseases including ulcerative colitis, liver fibrosis7, diabetes and rheumatoid arthritis3,8-it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.

The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity.

KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours1,2. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity3-5. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking.

Verticillins: fungal epipolythiodioxopiperazine alkaloids with chemotherapeutic potential.

Covering: 1970 through June of 2023Verticillins are epipolythiodioxopiperazine (ETP) alkaloids, many of which possess potent, nanomolar-level cytotoxicity against a variety of cancer cell lines. Over the last decade, their in vivo activity and mode of action have been explored in detail. Notably, recent studies have indicated that these compounds may be selective inhibitors of histone methyltransferases (HMTases) that alter the epigenome and modify targets that play a crucial role in apoptosis, altering immune cell recognition, and generating reactive oxygen species. Verticillin A (1) was the first of 27 analogues reported from fungal cultures since 1970. Subsequent genome sequencing identified the biosynthetic gene cluster responsible for producing verticillins, allowing a putative pathway to be proposed. Further, molecular sequencing played a pivotal role in clarifying the taxonomic characterization of verticillin-producing fungi, suggesting that most producing strains belong to the genus Clonostachys (i.e., Bionectria), Bionectriaceae. Recent studies have explored the total synthesis of these molecules and the generation of analogues via both semisynthetic and precursor-directed biosynthetic approaches. In addition, nanoparticles have been used to deliver these molecules, which, like many natural products, possess challenging solubility profiles. This review summarizes over 50 years of chemical and biological research on this class of fungal metabolites and offers insights and suggestions on future opportunities to push these compounds into pre-clinical and clinical development.

Mesoporous polydopamine Targeting CDK4/6 Inhibitor toward Brilliant Synergistic Immunotherapy of Breast Cancer.

Immunotherapy utilizing anti-PD-L1 blockade has achieved dramatic success in clinical breast cancer management but is often hampered by the limited immune response. Increasing evidence shows that immunogenic cell death (ICD) recently arises as a promising strategy for enlarging tumor immunogenicity and eliciting systemic anti-tumor immunity effectively. However, developing simple but versatile, highly efficient but low-toxic, biosafe, and clinically available transformed ICD inducers remains a huge demand and is highly desirable. Herein, a multifunctional ICD inducer is purposefully developed A6-MPDA@PAL by integrating photothermal therapy (PTT) nanoplatforms mesoporous polydopamine (MPDA), CDK4/6 inhibitor palbociclib (PAL), and CD44-specific targeting A6 peptide in a simple way for augmenting the immune antitumor efficacy of anti-PD-L1 therapy. Remarkably, the light-inducible nanoplatforms exhibit multiple favorable therapeutic features ensuring a superior and biosafe PTT/chemotherapy efficacy. Together with stronger accumulative ICD induction, single administration of A6-MPDA@PAL can trigger robust systemic antitumor immunity and abscopal effect with the assistance of anti-PD-L1 blockade by fascinating the intratumoral infiltration of T lymphocytes and reversing the immunosuppressive tumor microenvironment simultaneously, therapy achieving brilliant synergistic immunotherapy with effective tumor ablation. This study presents a simple and smart ICD inducer opening up attractive clinical possibilities for reinforcing the anti-PD-L1 therapy against breast cancer.

Identification of 1,3,8-Triazaspiro[4.5]Decane-2,4-Dione Derivatives as a Novel δ Opioid Receptor-Selective Agonist Chemotype.

δ opioid receptors (DORs) hold potential as a target for neurologic and psychiatric disorders, yet no DOR agonist has proven efficacious in critical phase II clinical trials. The exact reasons for the failure to produce quality drug candidates for the DOR are unclear. However, it is known that certain DOR agonists can induce seizures and exhibit tachyphylaxis. Several studies have suggested that those adverse effects are more prevalent in delta agonists that share the (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)/4-[(αR*)-α-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl]-N,N-diethylbenzamide chemotype. There is a need to find novel lead candidates for drug development that have improved pharmacological properties to differentiate them from the current failed delta agonists. Our objective in this study was to identify novel DOR agonists. We used a ß-arrestin assay to screen a small G-protein coupled receptors (GPCR)-focused chemical library. We identified a novel chemotype of DOR agonists that appears to bind to the orthosteric site based of docking and molecular dynamic simulation. The most potent agonist hit compound is selective for the DOR over a panel of 167 other GPCRs, is slightly biased toward G-protein signaling and has anti-allodynic efficacy in a complete Freund's adjuvant model of inflammatory pain in C57BL/6 male and female mice. The newly discovered chemotype contrasts with molecules like SNC80 that are highly efficacious ß-arrestin recruiters and may suggest this novel class of DOR agonists could be expanded on to develop a clinical candidate drug. SIGNIFICANCE STATEMENT: δ opioid receptors are a clinical target for various neurological disorders, including migraine and chronic pain. Many of the clinically tested delta opioid agonists share a single chemotype, which carries risks during drug development. Through a small-scale high-throughput screening assay, this study identified a novel δ opioid receptor agonist chemotype, which may serve as alternative for the current analgesic clinical candidates.

Formation of Bridged Disulfide in Epidithiodioxopiperazines.

Epidithiodioxopiperazine (ETP) alkaloids, featuring a 2,5-diketopiperazine core and transannular disulfide bridge, exhibit a broad spectrum of biological activities. However, the structural complexity has prevented efficient chemical synthesis and further clinical research. In the past few decades, many achievements have been made in the biosynthesis of ETPs. Here, we discuss the biosynthetic progress and summarize them as two comprehensible metabolic principles for better understanding the complex pathways of α, α'- and α, ß'-disulfide bridged ETPs. Specifically, we systematically outline the catalytic machineries to install α, α'- and α, ß'-disulfide by flavin-containing oxygenases. This concept would contribute to the medical and industrial applications of ETPs.

Redox-Sensitive Poly(lactic-co-glycolic acid) Nanoparticles of Palbociclib: Development, Ultrasound/Photoacoustic Imaging, and Smart Breast Cancer Therapy.

Breast cancer is one of the leading causes of mortality in women globally. The efficacy of breast cancer treatments, notably chemotherapy, is hampered by inadequate localized delivery of anticancer agents to the tumor site, resulting in compromised efficacy and increased systemic toxicity. In this study, we have developed redox-sensitive poly(lactic-co-glycolic acid) (PLGA) nanoparticles for the smart delivery of palbociclib (PLB) to breast cancer. The particle size of formulated PLB@PLGA-NPs (nonredox-sensitive) and RS-PLB@PLGA-NPs (redox-sensitive) NPs were 187.1 ± 1.8 nm and 193.7 ± 1.5 nm, respectively. The zeta potentials of nonredox-sensitive and redox-sensitive NPs were +24.99 ± 2.67 mV and +9.095 ± 1.87 mV, respectively. The developed NPs were characterized for morphological and various physicochemical parameters such as SEM, TEM, XRD, DSC, TGA, XPS, etc. The % entrapment efficiency of PLB@PLGA-NPs and RS-PLB@PLGA-NPs was found to be 85.48 ± 1.29% and 87.72 ± 1.55%, respectively. RS-PLB@PLGA-NPs displayed a rapid drug release at acidic pH and a higher GSH concentration compared to PLB@PLGA-NPs. The cytotoxicity assay in MCF-7 cells suggested that PLB@PLGA-NPs and RS-PLB@PLGA-NPs were 5.24-fold and 14.53-fold higher cytotoxic compared to the free PLB, respectively. Further, the cellular uptake study demonstrated that redox-sensitive NPs had significantly higher cellular uptake compared to nonredox-sensitive NPs and free Coumarin 6 dye. Additionally, AO/EtBr assay and reactive oxygen species analysis confirmed the superior activity of RS-PLB@PLGA-NPs over PLB@PLGA-NPs and free PLB. In vivo anticancer activity in dimethyl-benz(a)anthracene-induced breast cancer rats depicted that RS-PLB@PLGA-NPs was highly effective in reducing the tumor size, hypoxic tumor, and tumor vascularity compared to PLB@PLGA-NPs and free PLB. Further, hemocompatibility study reveals that the developed NPs were nonhemolytic to human blood. Moreover, an in vivo histopathology study confirmed that both nanoparticles were safe and nontoxic to the vital organs.

Discovery of acetophenone/piperazin-2-one hybrids as selective anti-TNBC cancer agents by causing DNA damage.

Twenty-five acetophenone/piperazin-2-one (APPA) hybrids were designed and synthesized based on key pharmacophores found in anti-breast cancer drugs Neratinib, Palbociclib, and Olaparib. Compound 1j exhibited good in vitro antiproliferative activity (IC50 = 6.50 µM) and high selectivity (SI = 9.2 vs HER2-positive breast cancer cells SKBr3; SI = 7.3 vs normal breast cells MCF-10A) against triple negative breast cancer (TNBC) cells MDA-MB-468. In addition, 1j could selectively cause DNA damage, inducing the accumulation of γH2AX and P53 in MDA-MB-468 cells. It also reduced the phosphorylation level of P38 and the expression of HSP70, which further prevented the repair of DNA damage and caused cells S/G2-arrest leading to MDA-MB-468 cells death.

Exploring Structural Requirements for Sigma-1 Receptor Linear Ligands: Experimental and Computational Approaches.

Sigma-1 receptor (S1R) is involved in a large array of biological functions due to its ability to interact with various proteins and ion channels. Crystal structures of human S1R revealed the trimeric organization for which each protomer comprises the ligand binding pocket. This study applied a multistep computational procedure to develop a pharmacophore model obtained from molecular dynamics simulations of available cocrystal structures of well-known S1R ligands. Apart from the well-established positive ionizable and hydrophobic features, the obtained model included an additional specific hydrophobic feature and different excluded volumes, thus increasing the selectivity of the model as well as a more detailed determination of the distance between two essential features. The obtained pharmacophore model passed the validation test by receiver operating characteristic (ROC) curve analysis of active and inactive S1R ligands. Finally, the pharmacophoric performance was experimentally investigated through the synthesis and binding assay of new 4-phenylpiperazine-based compounds. The most active new ligand 2-(3-methyl-1-piperidyl)-1-(4-phenylpiperazin-1-yl)ethanone (3) showed an S1R affinity close to the reference compound haloperidol (Ki values of 4.8 and 2.6 nM, respectively). The proposed pharmacophore model can represent a useful tool to design and discover new potent S1R ligands.

Design, synthesis, and antiviral activity of 1-aryl-4-arylmethylpiperazine derivatives as Zika virus inhibitors with broad antiviral spectrum.

Zika virus (ZIKV) disease has been given attention due to the risk of congenital microcephaly and neurodevelopmental disorders after ZIKV infection in pregnancy, but no vaccine or antiviral drug is available. Based on a previously reported ZIKV inhibitor ZK22, a series of novel 1-aryl-4-arylmethylpiperazine derivatives was designed, synthesized, and investigated for antiviral activity by quantify cellular ZIKV RNA amount using RT-qPCR method in ZIKV-infected human venous endothelial cells (HUVECs) assay. Structure-activity relationship (SAR) analysis demonstrated that anti-ZIKV activity of 1-aryl-4-arylmethylpiperazine derivatives is not correlated with molecular hydrophobicity, multiple new derivatives with pyridine group to replace the benzonitrile moiety of ZK22 showed stronger antiviral activity, higher ligand lipophilicity efficiency as well as lower cytotoxicity. Two active compounds 13 and 33 were further identified as novel ZIKV entry inhibitors with the potential of oral available. Moreover, both ZK22 and newly active derivatives also possess of obvious inhibition on the viral replication of coronavirus and influenza A virus at low micromolar level. In summary, this work provided better candidates of ZIKV inhibitor for preclinical study and revealed the promise of 1-aryl-4-arylmethylpiperazine chemotype in the development of broad-spectrum antiviral agents.

Design, synthesis, and biological evaluation of aralkyl piperazine and piperidine derivatives targeting SSRI/5-HT1A/5-HT7.

Serotonin reuptake inhibition combined with the action targeting 5-hydroxytryptamine receptor subtypes can serve as a potential target for the development of antidepressant drugs. Herein a series of new aralkyl piperazines and piperidines were designed and synthesized by the structural modifications of the previously discovered aralkyl piperidine compound 1, targeting SSRI/5-HT1A/5-HT7. The results exhibited that compound 5a showed strong binding to 5-HT1A and 5-HT7 (Ki of 0.46 nM, 2.7 nM, respectively) and a high level of serotonin reuptake inhibition (IC50 of 1.9 nM), all of which were significantly elevated compared to 1. In particular, compound 5a showed weaker inhibitory activity against hERG than 1, and demonstrated good stability in liver microsomes in vitro. The preliminary screening using FST indicated that orally administered 5a, at a high dose, could reduce immobility time in mice markedly, indicating potential antidepressant activity.

Design, synthesis, and evaluation of formylpiperazine analogs of Ferrostatin-1 as novel improved ferroptosis inhibitors.

In this study, a series of new formylpiperazine-derived ferroptosis inhibitors were designed and synthesized based on the structure of a known ferroptosis inhibitor, ferrostatin-1 (Fer-1). The anti-ferroptosis activity of these synthetic compounds in human umbilical vein endothelial cells (HUVECs) induced by Erastin was evaluated. It was found that some of the new compounds, especially compound 26, showed potent anti-ferroptosis activity, as evidenced by its ability to restore cell viability, reduce iron accumulation, scavenge reactive oxygen species, maintain mitochondrial membrane potential, increase GSH levels, decrease LPO and MDA content, and upregulate GPX4 expression. Moreover, compound 26 exhibited superior microsomal stability than Fer-1. The present results suggest that compound 26 is a promising lead compound for the development of new ferroptosis inhibitors for the treatment of vascular diseases.

Exploring the structural activity relationship of the Osimertinib: A covalent inhibitor of double mutant EGFRL858R/T790M tyrosine kinase for the treatment of Non-Small Cell Lung Cancer (NSCLC).

The USFDA granted regular approval to Osimertinib (AZD9291) on March 2017, for treating individuals with metastatic Non-Small Cell Lung Cancer having EGFR T790M mutation. Clinically, Osimertinib stands at the forefront for the treatment of patients with Non-Small Cell Lung Cancer. Osimertinib forms a covalent bond with the Cys797 residue and predominantly spares binding to WT-EGFR, thereby reducing toxicity and enabling the administration of doses that effectively inhibit T790M. However, a high percentage of patients treated with Osimertinib (AZD9291) developed a tertiary cysteine797 to serine797 (C797S) mutation in the EGFR kinase domain, rendering resistance to it. This comprehensive review sheds light on the chemistry, computational aspects, structural features, and expansive spectrum of biological activities of Osimertinib and its analogues. The in-depth exploration of these facets serves as a valuable resource for medicinal chemists, empowering them to design better Osimertinib analogues. This exhaustive study not only provides insights into improving potency but also emphasizes considerations for mutant selectivity and optimizing pharmacokinetic properties. This review acts as a guiding beacon for the strategic design and development of next-generation Osimertinib analogues.

Piperazinium Poly(Ionic Liquid)s as Solid Electrolytes for Lithium Batteries.

Poly(ionic liquid)s combine the unique properties of ionic liquids (ILs) within ionic polymers holding significant promise for energy storage applications. It is reported here the synthesis and characterization of a new family of poly(ionic liquid)s synthesized from cationic piperazinium ionic liquid monomers. The cationic poly(acrylamide piperazinium) in combination with sulfonamide anions like bis(trifluoromethanesulfonyl) imide (TFSI) and bis(fluorosulfonyl) imide (FSI) are characterized as solid polymer electrolytes. The polymer electrolytes in combination with pyrrolidonium ILs and LiFSI show high ionic conductivity, 5×10-3 S cm-1 at 100 °C. Piperazinium polymer electrolytes show excellent compatibility with lithium metal reversible plating and stripping at high current density and low temperature 40 °C.

UPLC-Q-TOF-MS/MS-Based Targeted Discovery of Chetomin Analogues from Chaetomium cochliodes.

Chetocochliodin M (5) containing a rare cage-ring and chetocochliodin N (6) featuring an unusual piperazine-2,3-dione ring system together with known analogues chetomin (1), chetoseminudin C (2), chetocochliodin I (3), and oidioperazine E (4) were targeted for purification from the fungus Chaetomium cochliodes using a UPLC-Q-TOF-MS/MS approach. The structures of the new compounds were elucidated using HR-ESI-MS, NMR, and ECD spectra. Compounds 1, 3, and 6 exhibited strong cytotoxic activities against A549 and HeLa cancer cell lines.

Unlocking the potential of higher-molecular-weight 5-HT7R ligands: Synthesis, affinity, and ADMET examination.

An increasing number of drugs introduced to the market and numerous repositories of compounds with confirmed activity have posed the need to revalidate the state-of-the-art rules that determine the ranges of properties the compounds should possess to become future drugs. In this study, we designed a series of two chemotypes of aryl-piperazine hydantoin ligands of 5-HT7R, an attractive target in search for innovative CNS drugs, with higher molecular weight (close to or over 500). Consequently, 14 new compounds were synthesised and screened for their receptor activity accompanied by extensive docking studies to evaluate the observed structure-activity/properties relationships. The ADMET characterisation in terms of the biological membrane permeability, metabolic stability, hepatotoxicity, cardiotoxicity, and protein plasma binding of the obtained compounds was carried out in vitro. The outcome of these studies constituted the basis for the comprehensive challenge of computational tools for ADMET properties prediction. All the compounds possessed high affinity to the 5-HT7R (Ki below 250 nM for all analysed structures) with good selectivity over 5-HT6R and varying affinity towards 5-HT2AR, 5-HT1AR and D2R. For the best compounds of this study, the expression profile of genes associated with neurodegeneration, anti-oxidant response and anti-inflammatory function was determined, and the survival of the cells (SH-SY5Y as an in vitro model of Alzheimer's disease) was evaluated. One 5-HT7R agent (32) was characterised by a very promising ADMET profile, i.e. good membrane permeability, low hepatotoxicity and cardiotoxicity, and high metabolic stability with the simultaneous high rate of plasma protein binding and high selectivity over other GPCRs considered, together with satisfying gene expression profile modulations and neural cell survival. Such encouraging properties make it a good candidate for further testing and optimisation as a potential agent in the treatment of CNS-related disorders.

Novel mandelic acid derivatives containing piperazinyls as potential candidate fungicides against Monilinia fructicola: Design, synthesis and mechanism study.

Brown rot of stone fruit, a disease caused by the ascomycete fungus Monilinia fructicola, has caused significant losses to the agricultural industry. In order to explore and discover potential fungicides against M. fructicola, thirty-one novel mandelic acid derivatives containing piperazine moieties were designed and synthesized based on the amide skeleton. Among them, target compound Z31 exhibited obvious in vitro antifungal activity with the EC50 value of 11.8 mg/L, and significant effects for the postharvest pears (79.4 % protective activity and 70.5 % curative activity) at a concentration of 200 mg/L. Antifungal activity for the target compounds was found to be significantly improved by the large steric hindrance of the R1 groups and the electronegative of the piperazines in the molecular structure, according to a three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis. Further mechanism studies have demonstrated that the compound Z31 can disrupt cell membrane integrity, resulting in increased membrane permeability, release of intracellular electrolytes, and affect the normal growth of hyphae. Additional, morphological study also indicated that Z31 may disrupt the integrity of the membrane by inducing generate excess endogenous reactive oxygen species (ROS) and resulting in the peroxidation of cellular lipids, which was further verified by the detection of malondialdehyde (MDA) content. These studies have provided the basis for the creation of novel fungicides to prevent brown rot in stone fruits.