High-throughput Screening Techniques Research Articles

Virtual screening, ADMET prediction, molecular docking, and dynamic simulation studies of natural products as BACE1 inhibitors for the management of Alzheimer’s disease

Alzheimer’s disease (AD) is a degenerative neurological disorder that chronically and irreversibly affects memory, cognitive function, learning ability, and organizational skills. Numerous studies have demonstrated BACE1 as a critical therapeutic target for AD, emphasizing the need for specific inhibition of BACE1 to develop effective therapeutics. However, current BACE1 inhibitors have certain limitations. Therefore, the aim of this study was to identify potential novel candidates derived from natural products that can be utilized for the treatment of AD. To achieve this, 80,617 natural compounds from the ZINC database were subjected to virtual screening and subsequently filtered according to the rule of five (RO5), leading to the identification of 1,200 compounds. Subsequently, the 1,200 compounds underwent molecular docking studies against the BACE1 receptor, utilizing high-throughput virtual screening (HTVS), standard precision (SP), and extra precision (XP) techniques to identify high-affinity ligands. Of the 50 ligands that exhibited the highest G-Scores in HTVS, further analysis was conducted using SP docking and scoring methods. This analysis led to the identification of seven ligands with enhanced binding affinities, which were then subjected to additional screening via XP docking and scoring. Finally, the stability of the most promising ligand in relation to BACE1 was assessed through molecular dynamics (MD) simulations. The computational screening demonstrated that the docking energy values for seven ligands binding to target enzymes ranged between − 6.096 and − 7.626 kcal/mol. Among these, ligand 2 (L2) exhibited the best binding energy at -7.626 kcal/mol with BACE1. MD simulations further confirmed the stability of the BACE1-L2 complex, emphasizing the formation of a robust interaction between L2 and the target enzymes. Additionally, pharmacokinetic and drug-likeness evaluations indicated that L2 is non-carcinogenic and able to permeate the blood-brain barrier (BBB). The findings of this study will contribute to narrowing down the selection of candidates for subsequent in vitro and in vivo testing.

NOVAsort for error-free droplet microfluidics

High-throughput screening techniques are pivotal to unlocking the mysteries of biology. Yet, the promise of droplet microfluidics in enabling single-cell resolution, ultra-high-throughput screening remains largely unfulfilled. Droplet sorting errors caused by polydisperse droplet sizes that are often inevitable in multi-step assays have severely limited the effectiveness and utility of this technique, especially when screening large libraries. Even a relatively low 1% sorting error results in 10,000 false calls in a 1,000,000 droplet screen, imposing an unreasonably large burden on downstream validation. Here, we present NOVAsort (Next-generation Opto-Volume-based Accurate droplet sorter), a device capable of discerning droplets based on both size and fluorescence intensity. With a 1000- and 10,000-fold reduction in false positives and false negatives, respectively. NOVAsort addresses the challenges of conventional droplet sorting approaches and sets standards for accuracy and throughput in droplet microfluidic assays.

Elemene as a binding stabilizer of microRNA-145-5p suppresses the growth of non-small cell lung cancer

Elemene is widely recognized as an effective anti-cancer compound and is routinely administered in Chinese clinical settings for the management of several solid tumors, including non-small cell lung cancer (NSCLC). However, its detailed molecular mechanism has not been adequately demonstrated. In this research, it was demonstrated that elemene effectively curtailed NSCLC growth in the patient-derived xenograft (PDX) model. Mechanistically, employing high-throughput screening techniques and subsequent biochemical validations such as microscale thermophoresis (MST), microRNA-145-5p (miR-145-5p) was pinpointed as a critical target through which elemene exerts its anti-tumor effects. Interestingly, elemene serves as a binding stabilizer for miR-145-5p, demonstrating a strong binding affinity (KD = 0.39 ± 0.17 μg/mL) and preventing its degradation both in vitro and in vivo, while not interfering with the synthesis of the primary microRNA transcripts (pri-miRNAs) and precursor miRNAs (pre-miRNAs). The stabilization of miR-145-5p by elemene resulted in an increased level of this miRNA, subsequently suppressing NSCLC progression through the miR-145-5p/mitogen-activated protein kinase kinase kinase 3 (MAP3K3)/nuclear factor kappaB (NF-κB) pathway. Our findings provide a new perspective on revealing the interaction patterns between clinical anti-tumor drugs and miRNAs.

Intracellular zinc protects tumours from T cell-mediated cytotoxicity.

Tumour immune evasion presents a significant challenge to the effectiveness of cancer immunotherapies. Recent advances in high-throughput screening techniques have uncovered that loss of antigen presentation and cytokine signalling pathways are central mechanisms by which tumours evade T cell immunity. To uncover additional vulnerabilities in tumour cells beyond the well-recognized antigen presentation pathway, we conducted a genome-wide CRISPR/Cas9 screen to identify genes that mediate resistance to chimeric-antigen receptor (CAR)-T cells, which function independently of classical antigen presentation. Our study revealed that loss of core-binding factor subunit beta (CBFβ) enhances tumour cell resistance to T cell killing, mediated through T cell-derived TNF. Mechanistically, RNA-sequencing and elemental analyses revealed that deletion of CBFβ disrupts numerous pathways including those involved in zinc homoeostasis. Moreover, we demonstrated that modulation of cellular zinc, achieved by supplementation or chelation, significantly altered tumour cell susceptibility to TNF by regulating the levels of inhibitor of apoptosis proteins. Consistent with this, treatment of tumour cells with a membrane-permeable zinc chelator had no impact on tumour cell viability alone, but significantly increased tumour cell lysis by CD8+ T cells in a TNF-dependent but perforin-independent manner. These results underscore the crucial role of intracellular zinc in regulating tumour cell susceptibility to T cell-mediated killing, revealing a novel vulnerability in tumour cells that might be exploited for the development of future cancer immunotherapeutics.

Transition-metal-doped biphenylene for enhanced CO detection: A high-throughput first-principles study

Our study examines the CO detection capabilities of pristine biphenylene (BP) and transition-metal-doped BP (M–BPx, x = 1 or 2), a novel two-dimensional nano-carbon material. Despite BP’s potential in toxic gas detection, its low CO adsorption energy (Eads) of 0.134 eV limits its application. Utilizing a combined strategy of high-throughput computational screening and DFT technique, we accurately identified the ground state configurations of CO-adsorbed M–BPx (M = Sc, Ti, Fe, Co, Ni, and Zn) and confirmed M doping significantly boosts BP’s CO adsorption ability, with Eads values ranging from 2.809 to 8.206 eV. This improvement is supported by ab initio molecular dynamics simulations and ionic d-p bonding interactions observed in electron localization functions, densities of state (DOS), and bond population analysis. Additionally, CO adsorption induces notable DOS changes in the M–BPx systems, validating the potential of M–BPx as suitable materials for CO detection. Notably, Zn-BP2 show notable work function shift after CO adsorption (0.36 eV), outperforming pristine BP’s 0.30 eV shift, and favorable recovery time (8.940 s). These shifts underscore the potential of Zn-BP2 for work function-based high-temperature CO sensor, marking it as a promising material for sensitive CO detection.

Accelerated Discovery of Halide Perovskite Materials via Computational Methods: A Review.

Halide perovskites have gained considerable attention in materials science due to their exceptional optoelectronic properties, including high absorption coefficients, excellent charge-carrier mobilities, and tunable band gaps, which make them highly promising for applications in photovoltaics, light-emitting diodes, synapses, and other optoelectronic devices. However, challenges such as long-term stability and lead toxicity hinder large-scale commercialization. Computational methods have become essential in this field, providing insights into material properties, enabling the efficient screening of large chemical spaces, and accelerating discovery processes through high-throughput screening and machine learning techniques. This review further discusses the role of computational tools in the accelerated discovery of high-performance halide perovskite materials, like the double perovskites A2BX6 and A2BB'X6, zero-dimensional perovskite A3B2X9, and novel halide perovskite ABX6. This review provides significant insights into how computational methods have accelerated the discovery of high-performance halide perovskite. Challenges and future perspectives are also presented to stimulate further research progress.

Unraveling the separation mechanism of gas mixtures in MOFs by combining the breakthrough curve with machine learning and high-throughput calculation

In the field of metal–organic frameworks (MOFs) screening studies, the batch calculation of the mixed gas breakthrough time difference (ΔTi) in MOFs and its intricate correlation with various descriptors remain underexplored. This research undertook batch calculations of the breakthrough curves (BC) for different gases within a simulated natural gas environment, designating ΔTi as the performance metric for MOFs in gas separation. The separation performance of computation-ready experimental MOFs for CH4/C2H6 and CH4/CO2 mixtures was analyzed in depth utilizing machine learning (ML)-assisted high-throughput computational screening (HTCS) techniques. Then, five ML algorithms were used to quantify the relationship between MOF descriptors and performance, and the effect of the metal center site on the separation performance was further explored. Ultimately, the top ten MOFs were selected for each system. Combining HTCS, ML, and BC, this work provides fresh insights for understanding and designing MOFs with customized adsorption and separation properties.

Enhancing Crop Resilience: Advances and Challenges in Marker-Assisted Selection for Disease Resistance

Marker-assisted selection (MAS) has emerged as a pivotal technique in crop improvement programs, enabling the identification and incorporation of disease-resistant traits in crops. This review highlights the significance of MAS in enhancing disease resistance, the current state of research, and the challenges faced in its implementation. Various case studies, including successful applications in crops such as rice, wheat, and maize, demonstrate the potential of MAS in sustainable agriculture. The review also discusses the integration of MAS with other modern breeding techniques and emphasizes the need for advanced genomic tools for future advancements in this field. The development of advanced molecular markers, high-throughput screening techniques, and the incorporation of genomic selection approaches have all contributed to recent developments in MAS. Even in intricate polygenic situations, these developments have significantly improved t+he precision and speed of discovering disease-resistant characteristics. Case studies involving important crops such as maize, wheat, and rice show how MAS can be successfully applied to create varieties with improved resistance to certain diseases, significantly lowering crop losses and reduced use of pesticides. These developments have consequences that go beyond the purely technical; they could have a big influence on environmentally friendly agriculture methods, cost effectiveness, and sustainable development. This study addresses the possible future path of MAS in crop breeding and summarizes the major discoveries from current research, highlighting both the successes and difficulties in the field. The body of data highlights MAS as a critical instrument in the toolbox of the contemporary breeder, essential for satisfying the ever-increasing demands of a world that is changing quickly.

Biomarkers for aging in Caenorhabditis elegans high throughput screening.

Aging is characterized by a functional decline in organism fitness over time due to a complex combination of genetic and environmental factors [ 1-4]. With an increasing elderly population at risk of age-associated diseases, there is a pressing need for research dedicated to promoting health and longevity through anti-aging interventions. The roundworm Caenorhabditis elegans is an established model organism for aging studies due to its short life cycle, ease of culture, and conserved aging pathways. These benefits also make the worm well-suited for high-throughput screening (HTS) methods to study biomarkers of the molecular changes, cellular dysfunction, and physiological decline associated with aging. Within this review, we offer a summary of recent advances in HTS techniques to study biomarkers of aging in C. elegans.

Abstract B014: Unveiling the future: Exploring cutting-edge technologies for synthetic lethality discovery

Abstract Synthetic lethality discovery is an emerging field of research that aims to reshape the landscape of cancer treatment, offering new hope and possibilities for patients faced with challenging diagnoses. In this article, we explored the cutting-edge technologies shaping the future of synthetic lathality research. Artificial intelligence and machine learning are not just buzzwords but indispensable tools that are shaping the field of precision medicine and personalized cancer treatment. By leveraging the power of computational tools, scientists can accelerate the identification of synthetic lethal interactions and pave the way for the development of more effective targeted cancer therapies. Through case studies, researchers can uncover the functional genomics of complex diseases and identify novel drug targets for cancer therapy. The integration of multi-omics approaches not only enhances the depth of analysis but also offers a more nuanced understanding of the molecular mechanisms driving synthetic lethal interactions, paving the way to unlock novel therapeutics and personalized treatment strategies. In addition, the integration of high-throughput screening techniques, such as CRISPR-Cas9 gene editing, opens up new avenues for identifying new drug targets and therapeutic strategies, ultimately paving a new era where personalized and targeted therapies redefine the way we approach cancer care, ushering in a novel era of precision and efficacy in medicine. Citation Format: Peter Oloche David. Unveiling the future: Exploring cutting-edge technologies for synthetic lethality discovery [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B014.

High-throughput screening unveils nitazoxanide as a potent PRRSV inhibitor by targeting NMRAL1

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) poses a major threat to the global swine industry, yet effective prevention and control measures remain elusive. This study unveils Nitazoxanide (NTZ) as a potent inhibitor of PRRSV both in vitro and in vivo. Through High-Throughput Screening techniques, 16 potential anti-PRRSV compounds are identified from a library comprising FDA-approved and pharmacopeial drugs. We show that NTZ displays strong efficacy in reducing PRRSV proliferation and transmission in a swine model, alleviating viremia and lung damage. Additionally, Tizoxanide (TIZ), the primary metabolite of NTZ, has been identified as a facilitator of NMRAL1 dimerization. This finding potentially sheds light on the underlying mechanism contributing to TIZ’s role in augmenting the sensitivity of the IFN-β pathway. These results indicate the promising potential of NTZ as a repurposed therapeutic agent for Porcine Reproductive and Respiratory Syndrome (PRRS). Additionally, they provide valuable insights into the antiviral mechanisms underlying NTZ’s effectiveness.

Fluorescence Thermal Shift Assay based High Through-put Screening in Small Molecule Drug Discovery: A Brief Overview

Background: The Early drug discovery process was majorly phenotypic, lacking target specificity, and mechanism of action causing short and/or long-term toxicological impacts on the patients leading to several drug withdrawals from the market. Biochemical procedures and methods used in drug discovery and development make it lengthy in terms of time requiring approx. 15 years with the investment of billions of dollars for a new drug molecule. Technological advancement leading to the identification of protein structures through crystallography paved a new path toward targeted drug discovery. Objective: This paper provides a brief overview of the use of the fluorescence thermal shift assay (FTSA) as a high throughput (HTS) screening technique in small molecule drug discovery. Methods: The article discusses the principles of thermal denaturation techniques and how they are applied in drug discovery. It highlights the advantages of FTSA over other biophysical methods, including its label-free, cost-effective, and high HTS nature. The review also presents a comparative analysis of FTSA with techniques such as isothermal titration calorimetry (ITC), mass spectrometry (MS), nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), X-ray crystallography(X-RD), circular dichroism (CD), and differential scanning calorimetry (DSC). Results: The FTSA technique is described as a powerful tool that addresses the limitations of larger protein requirements, specific assay development, and low HTS associated with other thermal denaturation methods. Unlike biochemical and cellular assays that measure secondary effects, FTSA directly estimates the binding of small molecules to target proteins. Conclusion: The review concludes that FTSA is the only biophysical method that satisfies the three key prerequisites of being label-free, cost-effective, and suitable for high HTS screening in small molecule drug discovery.

MOF-derived nanozyme CuOx@C and its application for cascade colorimetric detection of phytosterols.

Phytosterols (PSs), a class of naturally occurring bioactive lipid compounds, have been found to possess a significant cholesterol-lowering effect. In developing countries, the consumption of rapeseed oil is the primary pathway of PS intake for the general population. However, developing low-cost, real-time, and high-throughput screening techniques for PSs remains a challenge. Here, a Cu-based nanocomposite CuOx@C was synthesized via a simple method of the calcination of HKUST-1 and systematically characterized by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The CuOx@C demonstrated excellent peroxidase-like (POD-like) activity, functioning as a peroxidase mimic to facilitate the catalysis of 3,3',5,5'-tetramethylbenzidine (TMB) into its oxidized form (oxTMB), thereby initiating a discernible color response. On the basis of this discovery, a CuOx@C-based colorimetric method for detecting total sterols in rapeseed was successfully constructed via cascade reactions. After optimizing the conditions, the high-throughput screening of total sterols in rapeseed could be completed in only 21min, which significantly facilitated the sensing of PSs. A linear range of 0.6-6mg/g was achieved for the detection of total sterols in rapeseed samples, thereby satisfying the requirements for detection. In addition, due to the high stability of CuOx@C and the specificity of cholesterol oxidase, the developed method had excellent stability and selectivity toward PSs, indicating that this work has huge prospects for commercial application. This innovative work overcomes the limitation of the instrumental method and provides a portable and reliable tool for total sterols detection. It can also facilitate the development of oilseeds with a high content of PSs.

Development of a fluorescence-based excipient screening for improved stability and shelf-life of recombinant chitin deacetylase

Chitin deacetylase (CDA) modifies chitin into chitosan by removing acetyl groups, but its inherent instability poses a challenge for successful crystallisation. Despite limited successes in crystallizing CDAs, prior attempts with recombinant chitin deacetylase (BaCDA) failed due to poor stability. To address this, we propose an enzyme buffer formulation as a cost-effective strategy to enhance stability, prolong shelf life, and increase the likelihood of crystallisation. Utilizing the high-throughput screening technique FTSA, we developed a screening method correlating BaCDA stability with its activity. The optimised formulation comprises 50 mM Tris-HCl buffer pH 7, 1 M NaCl, 20 % glycerol, and 1 mM Mg2+ as excipients. This formulation significantly improves BaCDA's thermostability (140.47 % increase) and enzyme activity (2.9-fold enhancement). BaCDA remains stable in the formulated buffer at −20 °C and −80 °C for 30 days and at 4 °C for 15 days. The current study has designed a high-throughput screening method approach to assess the stability of CDA enzyme formulations. The results of this study could contribute to the exploration of formulation elements that enhance the structural stability of CDA, thereby facilitating investigations into the enzyme's structure-function relationships.

Improving the Synthesis Efficiency of Amino Acids by Analyzing the Key Sites of Intracellular Self-Assembly of Artificial Cellulosome

To explore the key sites affecting the intracellular assembly of key components of cellulosomes and obtain DocA mutants independent of Ca2+, Swiss-model, GROMACS, PyMOL, and other molecular dynamics simulation software were used for modeling and static and dynamic combination analysis. Site-specific mutation technology was used to mutate DocA, and Biacore was used to test the dependence of Ca2+ on the binding ability of protein DocA mutants and protein Coh, and to analyze the interaction and binding effect of mutant proteins in vitro. Forward intracellular mutant screening was performed based on semi-rational design and high throughput screening techniques. The orientation of mutations suitable for intracellular assembly was determined, and three directional mutant proteins, DocA-S1, DocA-S2, and DocA-S3, were obtained. Ca2+ independent DocA mutants were obtained gradually and their potential interaction mechanisms were analyzed. In the present study, intracellular self-assembly of key components of cellulosomes independent of Ca2+ was achieved, and DocA-S3 was applied to the assembly of key enzymes of L-lysine biosynthesis, in which DapA and DapB intracellular assembly increased L-lysine accumulation by 29.8% when compared with the control strains, providing a new strategy for improving the intracellular self-assembly of cellulosomes and amino acid fermentation efficiency.

A hybrid RNA-protein biosensor for high-throughput screening of adenosylcobalamin biosynthesis

Genetically encoded circuits have been successfully utilized to assess and characterize target variants with desirable traits from large mutant libraries. Adenosylcobalamin is an essential coenzyme that is required in many intracellular physiological reactions and is widely used in the pharmaceutical and food industries. High-throughput screening techniques capable of detecting adenosylcobalamin productivity and selecting superior adenosylcobalamin biosynthesis strains are critical for the creation of an effective microbial cell factory for the production of adenosylcobalamin at an industrial level. In this study, we developed an RNA-protein hybrid biosensor whose input part was an endogenous RNA riboswitch to specifically respond to adenosylcobalamin, the inverter part was an orthogonal transcriptional repressor to obtain signal inversion, and the output part was a fluorescent protein to be easily detected. The hybrid biosensor could specifically and positively correlate adenosylcobalamin concentrations to green fluorescent protein expression levels in vivo. This study also improved the operating concentration and dynamic range of the hybrid biosensor by systematic optimization. An individual cell harboring the hybrid biosensor presented over 20-fold higher fluorescence intensity than the negative control. Then, using such a biosensor combined with fluorescence-activated cell sorting, we established a high-throughput screening platform for screening adenosylcobalamin overproducers. This study demonstrates that this platform has significant potential to quickly isolate high-productive strains to meet industrial demand and that the framework is acceptable for various metabolites.

A Review on Unlocking Toxicity: How Zebrafish embryos Help Ensure Safe Chemicals

Aquatic ecotoxicity testing has benefited greatly from zebrafish embryo toxicity testing (ZFET) because of its high throughput, low cost, sensitivity to a variety of toxicants, and ethical issues. This study highlights the benefits of ZFET over conventional fish toxicity testing, such as its capacity to detect teratogenic defects and possible long-term impacts. The limitations of ZFET are also addressed, notably its emphasis on acute dosages and difficulties with interspecies extrapolation. In addition to the generation of in vitro alternatives utilizing zebrafish cell lines, recent developments in ZFET technology are also highlighted, including the use of transgenic zebrafish lines and high-throughput screening techniques. The paper's conclusion includes a discussion of potential prospects for ZFET research. These include improving interspecies extrapolation methods, optimizing chronic toxicity assessment procedures, and integrating ZFET with other ecotoxicity testing strategies. ZFET is a great instrument for environmental protection and a crucial component in avoiding chemical contamination of aquatic ecosystems, so long as it is acknowledged for what it is and is open to future improvement

Advances in succinic acid production: the enhancement of CO2 fixation for the carbon sequestration benefits.

Succinic acid (SA), one of the 12 top platform chemicals produced from biomass, is a precursor of various high value-added derivatives. Specially, 1mol CO2 is assimilated in 1mol SA biosynthetic route under anaerobic conditions, which helps to achieve carbon reduction goals. In this review, methods for enhanced CO2 fixation in SA production and utilization of waste biomass for SA production are reviewed. Bioelectrochemical and bioreactor coupling systems constructed with off-gas reutilization to capture CO2 more efficiently were highlighted. In addition, the techno-economic analysis and carbon sequestration benefits for the synthesis of bio-based SA from CO2 and waste biomass are analyzed. Finally, a droplet microfluidics-based high-throughput screening technique applied to the future bioproduction of SA is proposed as a promising approach.

Advances in hydrogen storage materials: harnessing innovative technology, from machine learning to computational chemistry, for energy storage solutions

The demand for clean and sustainable energy solutions is escalating as the global population grows and economies develop. Fossil fuels, which currently dominate the energy sector, contribute to greenhouse gas emissions and environmental degradation. In response to these challenges, hydrogen storage technologies have emerged as a promising avenue for achieving energy sustainability. This review provides an overview of recent advancements in hydrogen storage materials and technologies, emphasizing the importance of efficient storage for maximizing hydrogen's potential. The review highlights physical storage methods such as compressed hydrogen (reaching pressures of up to 70 MPa) and material-based approaches utilizing metal hydrides and carbon-containing substances. It also explores design considerations, computational chemistry, high-throughput screening, and machine-learning techniques employed in developing efficient hydrogen storage materials. This comprehensive analysis showcases the potential of hydrogen storage in addressing energy demands, reducing greenhouse gas emissions, and driving clean energy innovation.

Abstract 4: Identifying novel patient-derived T cell receptors targeting TP53 public neoantigens

Abstract Adoptive cell transfer (ACT) is a promising immunotherapeutic strategy to bolster reactivity against immunologically cold tumors through infusion of T cells that can recognize and eliminate cancer cells bearing mutation-derived neoantigens (NeoAgs). T cell receptors (TCRs) that confer recognition of public NeoAgs–an elite subset of Human leukocyte antigen (HLA)-restricted epitopes arising from recurrent hotspot mutations in driver genes that enhance cancer cell fitness and are clonally conserved–are highly desirable and would be effective against many tumors sharing the common hotspot mutation. Tumor suppressor TP53 is a supreme target for this approach: it is the most frequently mutated gene in cancer yet remains untargetable with FDA-approved therapies. The most common TP53 alteration (R175H) generates a public NeoAg in the context of HLA-A*02, an allele expressed by ~40% of the North American and Western European populations. Earlier work established the immunogenicity of this public NeoAg by isolating a TP53(R175H)-specific clone from a colorectal cancer patient. ACT of this TCR in a single patient produced an objective anti-tumor response, but also resulted in multisystem organ failure indicating this receptor might possess off-target reactivity to unrelated proteins in healthy tissue. To discover safe and effective TP53(R175H)-specific TCRs, we assembled a unique biorepository of peripheral blood mononuclear cells (PBMCs) from n=26 cancer patients with diverse tumors that co-express HLA-A*02/TP53(R175H) and applied a novel high-throughput screening technique to retrieve candidate TCRs. These efforts resulted in a panel of n=9 genetically distinct and functionally validated receptors, each with NeoAg-specific anti-tumor activity against a panel of cancer cell lines. To define the cross-reactivity profile of these novel TCRs and the benchmark TCR that caused toxicity, we performed amino acid scanning mutagenesis (X-Scan) to identify potential off-targets. Importantly, we identified significant off-target reactivity to normal human proteins for the benchmark TCR, which may explain the lung and kidney toxicities observed in the trial patient. By contrast, a subset of the novel TCRs exhibited exquisite specificity for the TP53 public NeoAg with no predicted off-targets. Finally, we performed ACT of all TCRs in a mouse xenograft model and identified several novel TCRs that potently eliminated TP53(R175H)+/HLA*A02+ tumors in vivo. Based on these data, we are now proceeding with the clinical development of a best-in-class TCR. This work is poised to bring an important new class of precision immunotherapeutics to patients with TP53-mutant tumors that presently lack effective molecularly targeted agents. Citation Format: Michael V. Gormally, Smita S. Chandran, Inaki Etxebarria, Christopher Klebanoff. Identifying novel patient-derived T cell receptors targeting TP53 public neoantigens [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 4.