High-throughput Screening Research Articles

Expedited Aminoglutarimide C-N Cross-Coupling Enabled by High-Throughput Experimentation.

A simple protocol for the Buchwald-Hartwig cross-coupling of (hetero)aryl halides with unprotected aminoglutarimide to afford diverse cereblon binding motifs is disclosed. The development of this C-N cross-coupling method was enabled by high-throughput combinatory screening of solvents, bases, temperatures, and ligands. Scope studies revealed generality across various heteroaryl and aryl halides with the reaction proceeding under mild conditions. In comparison, this method demonstrated strategic superiority over previously reported approaches, as evidenced by a significant decrease in step count from known syntheses in the patent literature.

Droplet-Based Microfluidic Photobioreactor as a Growth Optimization Tool for Cyanobacteria and Microalgae

Microalgae and cyanobacteria are photosynthetic microorganisms with significant biotechnological potential for the production of bioactive compounds, making them a promising resource for diverse industrial applications. This study presents the development and validation of a modular, droplet-based microfluidic photobioreactor (µPBR) designed for high-throughput screening and cultivation under controlled light conditions. The µPBR, based on polytetrafluoroethylene (PTFE) tubing and a 4-channel LED illumination system, enables precise modulation of light intensity, wavelength, and photoperiod, facilitating dose–response experiments. Synechococcus elongatus UTEX 2973 and Chlorella vulgaris were used to demonstrate the system’s capacity to support photosynthetic growth under various conditions. The results indicate that continuous illumination, particularly under blue and mixed blue-red light, promotes higher autofluorescence and chlorophyll a content in cyanobacteria Synechococcus elongatus UTEX2973, while Chlorella vulgaris achieved optimal growth under a 16:8 light-dark cycle with moderate light intensity. This µPBR offers not only a flexible, scalable platform for optimizing growth parameters but also allows for the investigation of highly resolved dose response screenings of environmental stressors such as salinity. The presented findings highlight its potential for advancing microalgal biotechnology research and applications.

A Phenotypic High-Throughput Screen Identifies Small Molecule Modulators of Endogenous RGS10 in BV-2 Cells.

Chronic dysregulation of microglial phenotypic balance contributes to prolonged neuroinflammation and neurotoxicity, which is a hallmark of neurodegenerative diseases. Thus, targeting microglial inflammatory signaling represents a promising therapeutic strategy for neurodegenerative diseases. Regulator of G protein Signaling 10 (RGS10) is highly expressed in microglia, where it suppresses pro-inflammatory signaling. However, RGS10 is silenced following microglial activation, augmenting inflammatory responses. While modulating RGS10 expression is a promising strategy to suppress pro-inflammatory microglial activation, no chemical tools with this ability exist. We developed a phenotypic high-throughput assay to screen for compounds with the ability to reverse interferon-γ (IFNγ)-induced RGS10 silencing in BV-2 cells. Identified hits had no effect on RGS10 expression in the absence of stimulus or in response to lipopolysaccharide (LPS). Furthermore, the hits reversed some of the inflammatory gene expression induced by IFNγ. This is the first demonstration of the potential for small molecule intervention to modulate the RGS10 expression in microglia.

Small Molecule Inhibitors of Mycobacterium tuberculosis Topoisomerase I Identified by Machine Learning and In Vitro Assays

Tuberculosis (TB) caused by Mycobacterium tuberculosis is a leading infectious cause of death globally. The treatment of patients becomes much more difficult for the increasingly common multi-drug resistant TB. Topoisomerase I is essential for the viability of M. tuberculosis and has been validated as a new target for the discovery of novel treatment against TB resistant to the currently available drugs. Virtual high-throughput screening based on machine learning was used in this study to identify small molecules that target the binding site of divalent ion near the catalytic tyrosine of M. tuberculosis topoisomerase I. From the virtual screening of more than 2 million commercially available compounds, 96 compounds were selected for testing in topoisomerase I relaxation activity assay. The top hit that has IC50 of 7 µM was further investigated. Commercially available analogs of the top hit were purchased and tested with the in vitro enzyme assay to gain further insights into the molecular scaffold required for topoisomerase inhibition. Results from this project demonstrated that novel small molecule inhibitors of bacterial topoisomerase I can be identified starting with the machine-learning-based virtual screening approach.

Scalable Thiol Reactivity Profiling Identifies Azetidinyl Oxadiazoles as Cysteine-Targeting Electrophiles.

Cysteine reactive groups are a mainstay in the design of covalent drugs and probe molecules, yet only a handful of electrophiles are routinely used to target this amino acid. Here, we report the development of scalable thiol reactivity (STRP), a method which enables the facile interrogation of large chemical libraries for intrinsic reactivity with cysteine. High throughput screening using STRP identified the azetidinyl oxadiazole as a moiety that selectively reacts with cysteine through a ring opening-based mechanism, capable of covalently engaging cysteine residues broadly across the human proteome. We show the utility of this reactive group with the discovery of an azetidinyl oxadiazole containing a small molecule that augments the catalytic activity of the deubiquitinase UCHL1 in vitro and in cells by covalently modifying a cysteine distal to its enzymatic active site. This study adds a novel cysteine targeting group to the electrophilic lexicon and provides robust methodology to rapidly surveil libraries for reactivity with cysteine.

Discovery of 1,2,4-Triazole-3-thione Derivatives as Potent and Selective DCN1 Inhibitors for Pathological Cardiac Fibrosis and Remodeling.

DCN1, a critical co-E3 ligase during the neddylation process, is overactivated in many diseases, such as cancers, heart failure as well as fibrotic diseases, and has been regarded as a new target for drug development. Herein, we designed and synthesized a new class of 1,2,4-triazole-3-thione-based DCN1 inhibitors based the hit HD1 identified from high-throughput screening and optimized through numerous structure-activity-relationship (SAR) explorations. HD2 (IC50= 2.96 nM) was finally identified and represented a highly potent and selective DCN1 inhibitor with favorable PK properties and low toxicity. Amazingly, HD2 effectively relieved Ang II/TGFβ-induced cardiac fibroblast activation in vitro, and reduced ISO-induced cardiac fibrosis as well as remodeling in vivo, which was linked to the inhibition of cullin 3 neddylation and its substrate Nrf2 accumulation. Our findings unveil a novel 1,2,4-triazole-3-thione-based derivative HD2, which can be recognized as a promising lead compound targeting DCN1 for cardiac fibrosis and remodeling.

Abstract B001: CircRNA-derived neoantigen identification in cancer vaccine development

Abstract Immunotherapy has been a popular topic in combating cancer for its great performance in specificity, versatility and durability recently. Cancer vaccines targeting neoantigens have shown clinical efficacy against cancer, eliciting long-lasting immune response and offering a more selective therapeutic approach with less side effects. However, rare mutation rates and low expression levels of neoantigen make it challenging to identify suitable neoantigens. Traditionally, researchers focus on the canonical mRNA transcriptome and proteome to find potential targets. Recent studies suggest that exploring the non-canonical transcriptome and proteome, particularly circular RNAs (circRNAs), can expand the search. CircRNAs, endogenously formed by the unique back-splicing events during pre-RNA processing, can serve as templates for protein synthesis. Some of these non-canonical peptides are specifically expressed in cancer cells, making circRNAs a valid source for neoantigen discovery. We have developed high-throughput circRNA reporter screening methods and mass spectrometry-based peptidomic workflow to pinpoint translating circRNAs and their derived peptides. Using these techniques, we have identified circRNA-derived neoantigens in breast cancer (BC) cell lines that can be used as possible targets for cancer vaccines. These circRNA-derived neoantigens are immunogenic and can trigger dendritic cell (DC) maturation and T cell activation in vitro. Next, we will test these findings by transferring to in vivo mouse system. We will assess cancer vaccine efficacy by targeting the neoantigens using syngeneic mouse tumor models. Our long- term goal is to apply the circRNA neoantigen identification technology to BC patient samples to facilitate cancer vaccine development and improve cancer immunotherapy. Our work will demonstrate the potential of circRNA-derived non canonical neoantigens in cancer vaccines, serving as a crucial first step to prove their effectiveness in therapy. By expanding our search beyond the traditional transcriptome and proteome, we will explore new targets and transform the neoantigen-based treatment. This innovative effort shifts the current understanding of neoantigen sources and opens up new possibilities for targeted cancer therapy strategies. Citation Format: Yi-Cian Zheng, Chun-Kan Chen. CircRNA-derived neoantigen identification in cancer vaccine development [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B001.

Accelerated hit identification with target evaluation, deep learning and automated labs: prospective validation in IRAK1.

Target identification and hit identification can be transformed through the application of biomedical knowledge analysis, AI-driven virtual screening and robotic cloud lab systems. However there are few prospective studies that evaluate the efficacy of such integrated approaches. We synergistically integrate our in-house-developed target evaluation (SpectraView) and deep-learning-driven virtual screening (HydraScreen) tools with an automated robotic cloud lab designed explicitly for ultra-high-throughput screening, enabling us to validate these platforms experimentally. By employing our target evaluation tool to select IRAK1 as the focal point of our investigation, we prospectively validate our structure-based deep learning model. We can identify 23.8% of all IRAK1 hits within the top 1% of ranked compounds. The model outperforms traditional virtual screening techniques and offers advanced features such as ligand pose confidence scoring. Simultaneously, we identify three potent (nanomolar) scaffolds from our compound library, 2 of which represent novel candidates for IRAK1 and hold promise for future development. This study provides compelling evidence for SpectraView and HydraScreen to provide a significant acceleration in the processes of target identification and hit discovery. By leveraging Ro5's HydraScreen and Strateos' automated labs in hit identification for IRAK1, we show how AI-driven virtual screening with HydraScreen could offer high hit discovery rates and reduce experimental costs. We present an innovative platform that leverages Knowledge graph-based biomedical data analytics and AI-driven virtual screening integrated with robotic cloud labs. Through an unbiased, prospective evaluation we show the reliability and robustness of HydraScreen in virtual and high-throughput screening for hit identification in IRAK1. Our platforms and innovative tools can expedite the early stages of drug discovery.

Studying Exoribonuclease Activity Using Fluorescence Anisotropy Assay.

Fluorescence anisotropy is a powerful technique, widely used for investigating ligand-macromolecule binding and high-throughput screens for drugs. Here, we employ fluorescence anisotropy to quantitatively study the activity of exoribonucleases exemplified by the Xrn2 enzyme. Recording changes in the fluorescence anisotropy over time allows real-time detection of enzymatic activity and provides a framework that can be tailored to particular questions. We discuss the experimental setup, the potential substrate RNAs and highlight data analysis. We envision that this assay can be applied to study other nucleic acid-degrading enzymes and further expanded to include competition and inhibitor screens.

Enhancement of non-oleaginous green microalgae Ulothrix for bio-fixing CO2 and producing biofuels by ARTP mutagenesis.

Oleaginous green microalgae are often mentioned in algae-based biodiesel industry, but most of them belong to specific genus (Chlorella, Scenedesmus, Botryococcus and Desmodesmus). Thus, the microalgal germplasm resources for biodiesel production are limited. Mutagenesis is regarded as an important technology for expanding germplasm resources. The main purpose of this study is to screen microalgae strains with high carbon dioxide tolerance and high lipid content from mutants derived from indigenous non-oleaginous green microalgae species-Ulothrix SDJZ-17. Two mutants with high CO2 tolerance and high lipid content genetic stability were obtained from the mutants by high-throughput screening, named Ulothrix SDJZ-17-A20 and Ulothrix SDJZ-17-A23. In order to evaluate the potential of CO2 fixation and biofuel production, A20 and A23 were cultured under air and 15% CO2 (v/v) conditions, and their wild-type strains (WT) were used as controls. Under the condition of high CO2 concentration, the growth performance and lipid production capacity of mutant strains A20 and A23 were not only significantly better than those of wild strains, but also better than those of their own cultured under air conditions. Among them, A23 obtained the highest LCE (light conversion efficiency) (14.79%), Fv/Fm (maximal photochemical efficiency of photosystem II) (71.04%) and biomass productivity (81.26mgL-1d-1), while A20 obtained the highest lipid content (22.45%). Both mutants can be used as candidate strains for CO2 fixation and biofuel production. By ARTP (atmospheric and room temperature plasma) mutagenesis with high-throughput screening, the mutants with higher CO2 tolerance, photosynthetic efficiency and lipid productivity can be obtained, even if they are derived from non-oleaginous microalgae, which is of great significance for enriching the energy microalgae germplasm bank, alleviating the global warming and energy crisis.

An iridium(III) complex-based luminogenic probe for high-throughput screening of hydrogen sulfide donors in living cells.

The scarcity of suitable high-throughput screening technology for hydrogen sulfide (H2S) donors has hampered the discovery of H2S donors. In this study, a long-lived cyclometalated iridium complex was rationally designed as a mitochondria-targeted H2S probe to monitor the real-time dynamic change of H2S. By using the time-resolved emission spectroscopy (TRES) technique, an anti-interference high-throughput screening system was developed to monitor H2S in living cells with decreased false negative results. As a proof-of-concept, three natural products were identified as potential H2S donors from a natural product library using the developed TRES probe. Notably, the discovery of allicin and diallyl trisulfide demonstrated the feasibility of this screening platform, while garlic-derived allyl methyl sulfide was explored as a H2S donor candidate. The results were further validated by a commercial assay. We anticipate this high-throughput platform could facilitate the discovery of H2S donors by discriminating the endogenous interfering fluorescence from biological systems.

Oncogenic cholesterol rewires lipid metabolism in hepatocellular carcinoma via the CSNK2A1-IGF2R Ser2484 axis.

Alcohol consumption and hepatitis B virus (HBV) infection are common risk factors for hepatocellular carcinoma (HCC). However, few studies have focused on elucidating the mechanisms of HCC with combined alcohol and HBV etiology. We aimed to investigate the molecular features of alcohol and HBV on HCC and to seek out potential therapeutic strategies. Two independent cohorts of HCC patients (n = 539 and n = 140) were included to investigate HCC with synergetic alcohol and HBV (AB-HCC) background. Patient-derived cell lines, organoids, and xenografts were used to validate the metabolic fragile. High-throughput drug screening (1181 FDA-approved anticancer drugs) was leveraged to explore the potential therapeutic agents. Here, we delineated AB-HCC as a distinctive metabolic subtype, hallmarked by oncogenic cholesterol, through the integration of clinical cohorts, proteomics, phosphoproteomics, and spatial transcriptome. Mechanistically, our findings revealed that cholesterol directly binds to CSNK2A1 (Casein Kinase 2 Alpha 1), augmenting its kinase activity and leading to phosphorylation of IGF2R (Insulin-Like Growth Factor 2 Receptor) at Ser2484. This cascade rewires lipid-driven mitochondrial oxidative phosphorylation, spawns reactive oxygen species measured by malondialdehyde assay, and perpetuates a positive feedback loop for cholesterol biosynthesis, ultimately culminating in tumorigenesis. Initial transcriptional activation of CSNK2A1 is driven by upregulation of RAD21 in AB-HCC. Our cholesterol profiling exposes AB-HCC's compensatory mechanism of AB-HCC, which capitalizes on both uptake and biosynthesis of cholesterol to confer survival edge. Moreover, high-throughput drug screening coupled with in vivo validation has uncovered the susceptibilities of AB-HCC, which can be effectively addressed by a combination of dietary cholesterol restriction and oral administration of Fostamatinib. The CSNK2A1-mediated cholesterol biosynthesis pathway has been implicated in various cancers characterized by cholesterol metabolism. These findings not only pinpoint the oncogenic metabolite cholesterol as a hidden culprit in AB-HCC subtype, but also enlighten a novel combination strategy to rejuvenate tumor metabolism.

High-throughput screening for optimizing adoptive T cell therapies.

Adoptive T cell therapy is a pivotal strategy in cancer immunotherapy, demonstrating potent clinical efficacy. However, its limited durability often results in primary resistance. High-throughput screening technologies, which include both genetic and non-genetic approaches, facilitate the optimization of adoptive T cell therapies by enabling the selection of biologically significant targets or substances from extensive libraries. In this review, we examine advancements in high-throughput screening technologies and their applications in adoptive T cell therapies. We highlight the use of genetic screening for T cells, tumor cells, and other promising combination strategies, and elucidate the role of non-genetic screening in identifying small molecules and targeted delivery systems relevant to adoptive T cell therapies, providing guidance for future research and clinical applications.

Enhancing Manganese Peroxidase: Innovations in Genetic Modification, Screening Processes, and Sustainable Agricultural Applications.

Manganese peroxidase (MnP), a vital extracellular enzyme for the degradation of lignin and other organic pollutants, has demonstrated immense potential for agricultural and environmental applications, including straw pretreatment, feed fermentation, mycotoxin degradation, and water treatment. However, current research remains in its exploratory phase, with naturally sourced MnP unable to meet industrial-scale demands and no mature commercial enzyme preparations available on the market. This comprehensive review innovatively constructs a framework for MnP research, probing into its molecular conformation and catalytic principles, while providing an overview of the advancements in high-throughput screening and In silco designing strategies. Specifically, this review focuses on the practical applications of MnP in sustainable agriculture, elaborating on its potential and challenges in straw resource utilization, efficient feed fermentation, mycotoxin control, and water quality improvement. Furthermore, this review summarizes the recent achievements in optimizing MnP activity through enzyme engineering techniques and discuss customized mutation strategies tailored to specific agricultural and environmental requirements, thereby laying a solid theoretical foundation and scientific basis for the industrial production and commercialization of MnP.

High-throughput structural screening and property study of noncentrosymmetric superconductors in Th-based ZrNiAl-like compounds

Noncentrosymmetric superconductors (NCSs) are emerging as potential materials for the investigation of nontraditional phenomena, including both the presence of a spin-singlet pairing configuration and the occurrence of a spin-triplet pairing configuration, as well as the manifestation of topological superconductivity. In this work, we constructed a high-throughput theoretical design and performed a computational screening of Th-based NCSs with ZrNiAl-like structures based on the first principles method. Through systematic stability evaluations, we expanded the range of Th-based ZrNiAl-like compounds with superior stability to 45 materials. In this group of substances, we have pinpointed 17 prospective entities exhibiting notable band separation in the vicinity of the Fermi level. Electron–phonon coupling calculations indicated that 14 of these compounds are superconducting. By performing an analysis of electronic properties, we proposed that ThAlRu and ThAlOs are two promising NCSs in this family. This work may aid in the exploration of the design and synthesis of Th-based ZrNiAl-like materials, leading to the discovery of more NCSs with unique superconducting properties.

The Genomic Landscape of Wilson Disease in a Pan India Disease Cohort and Population-Scale Data.

Wilson's disease (WD) results from pathogenic ATP7B gene variations, causing copper accumulation mainly in the liver, brain, and kidneys. In India, despite studies on ATP7B variants, WD often goes undiagnosed, with the prevalence, carrier rate, and mutation spectrum remaining unknown. A multicenter study examined genetic variations in WD among individuals of Indian origin via whole exome sequencing. The study used the InDelible structural variants calling pipeline and conducted molecular dynamic simulations on variants of uncertain significance (VUS) in ATP7B AlphaFold protein structures. Additionally, a high-throughput gene screening panel for WD was developed. This study examined 128 clinically diagnosed cases of WD, revealing 74 genetically confirmed cases, 22 with ATP7B variants, and 32 without. Twenty-two novel ATP7B gene variants were identified, including a 322 bp deletion classified as a structural variant. Molecular dynamics simulations highlighted the potential deleterious effects of 11 ATP7B VUS. Gene burden analysis suggested associations with ANO8, LGR4, and CDC7. ATP7B gene hotspots for pathogenic variants were identified. Prevalence and carrier rates were determined as one in 18,678 and one in 67, respectively. A multiplex sequencing panel showed promise for accurate WD diagnosis. This study offers crucial insights into WD's genetic variations and prevalence in India, addressing its underdiagnosis. It highlights the novel genetic variants in the ATP7B gene, the involvement of other genes, a scalable, cost-effective multiplex sequencing panel for WD diagnosis and management and promising advancements in WD care.

Yeast metabolism adaptation for efficient terpenoids synthesis via isopentenol utilization.

Microbial biosynthesis has become the leading commercial approach for large-scale production of terpenoids, a valuable class of natural products. Enhancing terpenoid production, however, requires complex modifications on the host organism. Recently, a two-step isopentenol utilization (IU) pathway relying solely on ATP as the cofactor has been proposed as an alternative to the mevalonate (MVA) pathway, streamlining the synthesis of the common terpenoid precursors. Herein, we find that isopentenol inhibits energy metabolism, leading to reduced efficiency of the IU pathway in Saccharomyces cerevisiae. To overcome this, we engineer an IU pathway-dependent (IUPD) strain, designed for growth-coupled production. The IUPD strain is compelled to enhance the ATP supply, essential for the IU pathway, and incorporates a high-throughput screening method for enzyme evolution. The refined IU pathway surpasses the MVA pathway in synthesizing complex terpenoids. Our work offers valuable insights into developing growth-coupled strains adapted to efficient natural product synthesis.

Abstract 4135476: The Cardiomyocyte Hypertrophy Inhibitor RFN-409, Identified by High Throughput Screening Assay, Suppresses Pressure Overload-induced Systolic Dysfunction in Mice by Suppressing p38 Activity

Purpose: When the heart is exposed to stresses such as myocardial infarction or hypertension, it undergoes compensatory hypertrophy in response. However, continuation of the stress causes this compensatory mechanism to fail, and eventually systolic dysfunction or decompensated heart failure occur. As the hypertrophy of individual cardiomyocytes has been observed in this process, controlling cardiomyocyte hypertrophy is a potential target the prevention and treatment of heart failure. In this study, we constructed a high throughput screening (HTS) assay using cardiomyocyte hypertrophy as an index parameter. Compounds that inhibit cardiomyocyte hypertrophy were selected from our low molecular compound library. Methods and Results: In the primary screening, cultured rat primary cardiomyocytes were treated with each compound at a final concentration of 1 µM and then stimulated with 30 µM phenylephrine (PE) for 48 hours. These cells were subjected to fluorescent immunostaining with α-actinin, and cardiomyocyte area was measured using an ArrayScan™ system. The hypertrophy inhibition rate (%) of each compound was calculated as [(PE(+) - compound) / (PE(+) - PE(-))] × 100. The compounds with a hypertrophy inhibition rate greater than 50% and less than 150% were selected as hit compounds. In the secondary screening, these hit compounds were evaluated based on the dose-dependency of cardiomyocyte hypertrophy inhibition and the inhibition of the mRNA levels of the cardiac hypertrophy response genes ANF and BNP using real-time PCR. From the 269 low molecular-weight compounds in the original compound library, eight were selected through the primary and secondary screenings. Among them, we focused on Reference Number 409 (RFN-409). Western blotting indicated that RFN-409 inhibited PE-induced p38 activation. Next, we investigated the effect of RFN-409 on heart failure. Eight-week-old male C57 BL/6J mice were subjected to transverse aortic constriction (TAC) surgery and then randomly assigned to intraperitoneal treatment with RFN-409 (3, 10 mg/kg) or vehicle for eight weeks. RFN-409 at 10 mg/kg significantly prevented TAC-induced increase in left ventricular posterior wall thickness and decrease in left ventricular fractional shortening. Discussion: RFN-409 suppressed TAC-induced development of heart failure, at least partially by inhibiting p38 activity. These findings suggest that RFN-409 may be an effective agent for heart failure therapy.

Identification and characterization of the Bacillus subtilis spore germination protein GerY.

Pathogens in the orders Bacillales and Clostridiales resist sterilization by differentiating into stress-resistant spores. Spores are metabolically inactive and can remain dormant for decades, yet upon exposure to nutrients, they rapidly resume growth, causing food spoilage, food-borne illness, or life-threatening disease. The exit from dormancy, called germination, is a key target in combating these important pathogens. Here, we report a high-throughput genetic screen using transposon sequencing to identify novel germination factors that ensure the efficient exit from dormancy. We identify several new factors and characterize one in greater detail. This factor, renamed GerY, is part of the proteinaceous coat that encapsulates the dormant spore. Our data suggest that GerY enables efficient transit of nutrients through the coat to trigger germination.

Gen-AI Methods, Molecular Docking and Molecular Dynamics Simulations for Identification of Novel Inhibitors of MmPL3 Transporter of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), the bacterium responsible for tuberculosis (TB), employs mycolic acids to build its cell wall. This robust structure plays a vital role in protecting the bacterium from external threats and contributes to its resistance against antibiotics. Mycobacterial membrane protein Large 3 (MmpL3), a secondary resistance nodulation division transporter, is essential in mycolic acid biosynthesis, transporting mycolic acid precursors into the periplasm using the proton motive force. Due to its role in bacterial cell wall formation, it is a promising target for new tuberculosis treatments. In this study, starting with 85 known MmPL3 compounds, the artificial intelligence (AI)-assisted tool “Design of Druglike Analogues (DeLA-Drug)” was employed to generate about 15,000 novel molecules. These compounds were then subjected to structure-based high-throughput virtual screening and molecular dynamics (MD) simulations to identify potential novel inhibitors of MmpL3. The binding affinity was obtained by docking the above molecules at the SQ109 binding site in MmPL3, followed by pharmacokinetics and toxicity, which were used to reduce the chemical space. Finally, five ligands were subjected to 100 ns MD simulations to investigate the binding energetics of inhibitors to MmpL3. These compounds demonstrated stable binding and favorable drug-like properties, indicating that they could serve as potential novel inhibitors of MmpL3 for Mtb.