Off-target Activity Research Articles

RAG1/2 induces double-stranded DNA breaks at non-Ig loci in the proximity of single sequence repeats in developing B cells.

In developing B cells, V(D)J gene recombination is initiated by the RAG1/2 endonuclease complex, introducing double-stranded DNA breaks (DSBs) in V, D, and J genes and resulting in the formation of the hypervariable parts of immunoglobulins (Ig). Persistent or aberrant RAG1/2 targeting is a potential threat to genome integrity. While RAG1 and RAG2 have been shown to bind various regions genome-wide, the in vivo off-target DNA damage instigated by RAG1/2 endonuclease remains less well understood. In the current study, we identified regions containing RAG1/2-induced DNA breaks in mouse pre-B cells on a genome-wide scale using a global DNA DSB detection strategy. We detected 1489 putative RAG1/2-dependent DSBs, most of which were located outside the Ig loci. DNA sequence motif analysis showed a specific enrichment of RAG1/2-induced DNA DSBs at GA- and CA-repeats and GC-rich motifs. These findings provide further insights into RAG1/2 off-target activity. The ability of RAG1/2 to introduce DSBs on the non-Ig loci during the endogenous V(D)J recombination emphasizes its genotoxic potential in developing lymphocytes.

CavitOmiX Drug Discovery: Engineering Antivirals with Enhanced Spectrum and Reduced Side Effects for Arboviral Diseases.

Advancing climate change increases the risk of future infectious disease outbreaks, particularly of zoonotic diseases, by affecting the abundance and spread of viral vectors. Concerningly, there are currently no approved drugs for some relevant diseases, such as the arboviral diseases chikungunya, dengue or zika. The development of novel inhibitors takes 10-15 years to reach the market and faces critical challenges in preclinical and clinical trials, with approximately 30% of trials failing due to side effects. As an early response to emerging infectious diseases, CavitOmiX allows for a rapid computational screening of databases containing 3D point-clouds representing binding sites of approved drugs to identify candidates for off-label use. This process, known as drug repurposing, reduces the time and cost of regulatory approval. Here, we present potential approved drug candidates for off-label use, targeting the ADP-ribose binding site of Alphavirus chikungunya non-structural protein 3. Additionally, we demonstrate a novel in silico drug design approach, considering potential side effects at the earliest stages of drug development. We use a genetic algorithm to iteratively refine potential inhibitors for (i) reduced off-target activity and (ii) improved binding to different viral variants or across related viral species, to provide broad-spectrum and safe antivirals for the future.

VEXP: A virtual enhanced cross screen panel for off-target pharmacology alerts

We describe the development of the GSK vEXP (virtual enhanced cross screen panel) for off-target pharmacology alerts. The derivation of a panel of machine learning classification models or QSAR models (Quantitative Structure-Activity Relationship) for off-target safety assessment allows early alerting to risk factors in candidate drugs. The models are matched to an internal in-vitro biochemical screening panel described previously with some updates reported here. The extreme imbalance of some internal GSK datasets and most of the related external ChEMBL datasets is shown when considering potency thresholds relevant to in-vitro screening. The small size and bias to the active class make many ChEMBL datasets un-modellable using such thresholds. Although larger, many GSK datasets remain too imbalanced to give a performant model. The value of merging internal and external data to help rebalance datasets and improve the domain of applicability is demonstrated with improvements in model performance frequently seen on merged data. Efforts to collate public datasets with a far better balance of the missing in-actives would likely do more to improve opensource models than simply increasing dataset size. We investigate the use of moving the probability threshold and applying imbalanced learners to help overcome the imbalance problem. Both methods can produce models with improved performance when applied to imbalanced datasets. Datasets with class imbalance 95:5 % or with <100 compounds were un-modellable. Where datasets had a class imbalance of 90:10 % the imbalanced learn methods were often more performant than standard tree-based classifiers. No one classification algorithm consistently out-performed all others and our approach emphasises a standardised, automated build and evaluate approach across all classifiers to identify the best model. The application of vEXP includes ranking of hit compounds for fast prioritisation, flagging of hit series that contain systematic scaffold or functional group related risks and the confirmation that late-stage optimisation is not introducing new off-target activities in established chemical series.

Site-Specific Antibody Prodrugs via S-Arylation: a Bioconjugation Approach Toward Masked Tyrosine Analogues.

The utility of antibody therapeutics is hampered by potential cross-reactivity with healthy tissue. Over the past decade, significant advances have been made in the design of activatable antibodies, which increase, or create altogether, the therapeutic window of a parent antibody. Of these, antibody prodrugs (pro-antibodies) are masked antibodies that have advanced the most for therapeutic use. They are designed to reveal the active, parent antibody only when encountering proteases upregulated in the microenvironment of the targeted disease tissue, thereby minimizing off-target activity. However, current pro-antibody designs are relegated to fusion proteins that append masking groups restricted to the use of only canonical amino acids, offering excellent control of the site of introduction, but with no authority over where the masking group is installed other than the N-terminus of the antibody. Here, we present a palladium-based bioconjugation approach for the site-specific introduction of a masked tyrosine mimic in the complementary determining region of the FDA approved antibody therapeutic ipilimumab used as a model system. The approach enables the introduction of a protease cleavable group tethered to noncanonical polymers (polyethylene glycol (PEG)) resulting in 47-fold weaker binding to cells expressing CTLA-4, the target antigen of ipilimumab. Upon exposure to tumor-associated proteases, the masking group is cleaved, unveiling a tyrosine-mimic (dubbed hydroxyphenyl cysteine (HPC)) that restores (>90% restoration) binding affinity to its target antigen.

Use of Drug Sensitisers to Improve Therapeutic Index in Cancer.

The clinical management of malignant tumours is challenging, often leading to severe adverse effects and death. Drug resistance (DR) antagonises the effectiveness of treatments, and increasing drug dosage can worsen the therapeutic index (TI). Current efforts to overcome DR predominantly involve the use of drug combinations, including applying multiple anti-cancerous drugs, employing drug sensitisers, which are chemical agents that enhance pharmacokinetics (PK), including the targeting of cellular pathways and regulating pertinent membrane transporters. While combining multiple compounds may lead to drug-drug interactions (DDI) or polypharmacy effect, the use of drug sensitisers permits rapid attainment of effective treatment dosages at the disease site to prevent early DR and minimise side effects and will reduce the chance of DDI as lower drug doses are required. This review highlights the essential use of TI in evaluating drug dosage for cancer treatment and discusses the lack of a unified standard for TI within the field. Commonly used benefit-risk assessment criteria are summarised, and the critical exploration of the current use of TI in the pharmaceutical industrial sector is included. Specifically, this review leads to the discussion of drug sensitisers to facilitate improved ratios of effective dose to toxic dose directly in humans. The combination of drug and sensitiser molecules might see additional benefits to rekindle those drugs that failed late-stage clinical trials by the removal of detrimental off-target activities through the use of lower drug doses. Drug combinations and employing drug sensitisers are potential means to combat DR. The evolution of drug combinations and polypharmacy on TI are reviewed. Notably, the novel binary weapon approach is introduced as a new opportunity to improve TI. This review emphasises the urgent need for a criterion to systematically evaluate drug safety and efficiency for practical implementation in the field.

Single-base tiled screen unveils design principles of PspCas13b for potent and off-target-free RNA silencing.

The development of precise RNA-editing tools is essential for the advancement of RNA therapeutics. CRISPR (clustered regularly interspaced short palindromic repeats) PspCas13b is a programmable RNA nuclease predicted to offer superior specificity because of its 30-nucleotide spacer sequence. However, its design principles and its on-target, off-target and collateral activities remain poorly characterized. Here, we present single-base tiled screening and computational analyses that identify key design principles for potent and highly selective RNA recognition and cleavage in human cells. We show that the de novo design of spacers containing guanosine bases at precise positions can greatly enhance the catalytic activity of inefficient CRISPR RNAs (crRNAs). These validated design principles (integrated into an online tool, https://cas13target.azurewebsites.net/ ) can predict highly effective crRNAs with ~90% accuracy. Furthermore, the comprehensive spacer-target mutagenesis revealed that PspCas13b can tolerate only up to four mismatches and requires ~26-nucleotide base pairing with the target to activate its nuclease domains, highlighting its superior specificity compared to other RNA or DNA interference tools. On the basis of this targeting resolution, we predict an extremely low probability of PspCas13b having off-target effects on other cellular transcripts. Proteomic analysis validated this prediction and showed that, unlike other Cas13 orthologs, PspCas13b exhibits potent on-target activity and lacks collateral effects.

Safety and efficacy studies of CRISPR-Cas9 treatment of sickle cell disease highlights disease-specific responses

Fetal hemoglobin (HbF) reactivation expression through CRISPR/Cas9 is a promising strategy for the treatment of sickle cell disease (SCD). Here, we describe a genome editing strategy leading to reactivation of HbF expression by targeting the binding sites (BSs) for the LRF repressor in the γ-globin promoters. CRISPR/Cas9 treatment in healthy donor (HD) and patient-derived HSPCs resulted in a high frequency of LRF BS disruption and potent HbF synthesis in their erythroid progeny. LRF BS disruption did not impair HSPC engraftment and differentiation, but was more efficient in SCD than in HD cells. However, SCD HSPCs showed a reduced engraftment and a myeloid bias compared to HD cells. We detected off-target activity and chromosomal rearrangements, particularly in SCD samples (likely because of the higher overall editing efficiency), but did not impact the target gene expression and HSPC engraftment and differentiation. Transcriptomic analyses showed that the editing procedure results in the upregulation of genes involved in DNA damage and inflammatory responses, which was more evident in SCD HSPCs. This study provides evidences of efficacy and safety for an editing strategy based on HbF reactivation and highlights the need of performing safety studies in clinically relevant conditions, i.e., in patient-derived HSPCs.

2,8-Disubstituted-1,5-naphthyridines as Dual Inhibitors of Plasmodium falciparum Phosphatidylinositol-4-kinase and Hemozoin Formation with In Vivo Efficacy.

Structure-activity relationship studies of 2,8-disubstituted-1,5-naphthyridines, previously reported as potent inhibitors of Plasmodium falciparum (Pf) phosphatidylinositol-4-kinase β (PI4K), identified 1,5-naphthyridines with basic groups at 8-position, which retained Plasmodium PI4K inhibitory activity but switched primary mode of action to the host hemoglobin degradation pathway through inhibition of hemozoin formation. These compounds showed minimal off-target inhibitory activity against the human phosphoinositide kinases and MINK1 and MAP4K kinases, which were associated with the teratogenicity and testicular toxicity observed in rats for the PfPI4K inhibitor clinical candidate MMV390048. A representative compound from the series retained activity against field isolates and lab-raised drug-resistant strains of Pf. It was efficacious in the humanized NSG mouse malaria infection model at a single oral dose of 32 mg/kg. This compound was nonteratogenic in the zebrafish embryo model of teratogenicity and has a low predicted human dose, indicating that this series has the potential to deliver a preclinical candidate for malaria.

CRISPR-Cas9 screens reveal common essential miRNAs in human cancer cell lines

BackgroundGenome-wide functional screening using the CRISPR-Cas9 system is a powerful tool to uncover tumor-specific and common genetic dependencies across cancer cell lines. Current CRISPR-Cas9 knockout libraries, however, primarily target protein-coding genes. This limits functional genomics-based investigations of miRNA function.MethodsWe designed a novel CRISPR-Cas9 knockout library (lentiG-miR) of 8107 distinct sgRNAs targeting a total of 1769 human miRNAs and benchmarked its single guide RNA (sgRNA) composition, predicted on- and off-target activity, and screening performance against previous libraries. Using a total of 45 human cancer cell lines, representing 16 different tumor entities, we performed negative selection screens to identify miRNA fitness genes. Fitness miRNAs in each cell line were scored using a combination of supervised and unsupervised essentiality classifiers. Common essential miRNAs across distinct cancer cell lines were determined using the 90th percentile method. For subsequent validation, we performed knockout experiments for selected common essential miRNAs in distinct cancer cell lines and gene expression profiling.ResultsWe found significantly lower off-target activity for protein-coding genes and a higher miRNA gene coverage for lentiG-miR as compared to previously described miRNA-targeting libraries, while preserving high on-target activity. A minor fraction of miRNAs displayed robust depletion of targeting sgRNAs, and we observed a high level of consistency between redundant sgRNAs targeting the same miRNA gene. Across 45 human cancer cell lines, only 217 (12%) of all targeted human miRNAs scored as a fitness gene in at least one model, and fitness effects for most miRNAs were confined to small subsets of cell lines. In contrast, we identified 49 common essential miRNAs with a homogenous fitness profile across the vast majority of all cell lines. Transcriptional profiling verified highly consistent gene expression changes in response to knockout of individual common essential miRNAs across a diverse set of cancer cell lines.ConclusionsOur study presents a miRNA-targeting CRISPR-Cas9 knockout library with high gene coverage and optimized on- and off-target activities. Taking advantage of the lentiG-miR library, we define a catalogue of miRNA fitness genes in human cancer cell lines, providing the foundation for further investigation of miRNAs in human cancer.

Library-Assisted Evolution in Eukaryotic Cells Yield Adenine Base Editors with Enhanced Editing Specificity.

The current-generation adenine base editor (ABE) ABE8e, which has evolved from the prokaryotic evolution system, exhibits high efficiency in mediating A-to-Gconversion and is presumed to be promising for gene therapy. However, its much wider editing window and substantially higher off-target editing activity restricted its applications in precise base editing for therapeutic use. This study uses a library-assistedprotein evolution approach using eukaryotic cells to generate ABE variants with improved specificity and reduced off-target editing while maintaining high activity in human cells. The study generated an expanded set of ABEs with efficient editing activities and chose fourevolved variants thatoffered either similar or modestly higher efficiency within a narrower editing window of protospacer position ≈4-7 compared to that of ABE8e in human cells, which would enable minimized bystander editing. Moreover, these variants resulted in reduced off-target editing events when delivered as plasmid or mRNA into human cells. Finally, these variants can install both disease-suppressing mutations and disease-correcting mutations efficiently with minimal undesired bystander editing making them promising approaches for specific therapeutic edits. In summary, the work establishes a mutant-library-assisted protein evolution method in eukaryotic cells and generates alternative ABE variants as efficient tools for precise human genome editing.

Discovery of Potent and Selective Covalent Inhibitors of HER2WT and HER2YVMA.

HER2 overexpression and amplification have been identified as oncogenic drivers, and the development of therapies to treat tumors harboring these markers has received considerable attention. Activation of HER2 signaling and subsequent cell growth can also be induced by HER2 mutations, including the common YVMA insertion in exon 20 within the kinase domain. Enhertu is currently the only approved treatment for HER2 mutant tumors in NSCLC. TKIs tested in this space have suffered from off-target activity, primarily due to EGFRWT inhibition or attenuated activity against HER2 mutants. The goal of this work was to identify a TKI that would provide robust inhibition of oncogenic HER2WT and HER2 mutants while sparing EGFRWT activity. Herein, we describe the development of a potent, covalent inhibitor of HER2WT and the YVMA insertion mutant while providing oral bioavailability and avoiding the inhibition of EGFRWT.

Progress and pitfalls of gene editing technology in CAR-T cell therapy: a state-of-the-art review.

CAR-T cell therapy has shown remarkable promise in treating B-cell malignancies, which has sparked optimism about its potential to treat other types of cancer as well. Nevertheless, the Expectations of CAR-T cell therapy in solid tumors and non-B cell hematologic malignancies have not been met. Furthermore, safety concerns regarding the use of viral vectors and the current personalized production process are other bottlenecks that limit its widespread use. In recent years the use of gene editing technology in CAR-T cell therapy has opened a new way to unleash the latent potentials of CAR-T cell therapy and lessen its associated challenges. Moreover, gene editing tools have paved the way to manufacturing CAR-T cells in a fully non-viral approach as well as providing a universal, off-the-shelf product. Despite all the advantages of gene editing strategies, the off-target activity of classical gene editing tools (ZFNs, TALENs, and CRISPR/Cas9) remains a major concern. Accordingly, several efforts have been made in recent years to reduce their off-target activity and genotoxicity, leading to the introduction of advanced gene editing tools with an improved safety profile. In this review, we begin by examining advanced gene editing tools, providing an overview of how these technologies are currently being applied in clinical trials of CAR-T cell therapies. Following this, we explore various gene editing strategies aimed at enhancing the safety and efficacy of CAR-T cell therapy.

Off-target autophagy inhibition by SHP2 allosteric inhibitors contributes to their antitumor activity in RAS-driven cancers.

Aberrant activation of RAS/MAPK signaling is common in cancer, and efforts to inhibit pathway components have yielded drugs with promising clinical activities. Unfortunately, treatment-provoked adaptive resistance mechanisms inevitably develop, limiting their therapeutic potential. As a central node essential for receptor tyrosine kinase-mediated RAS activation, SHP2 has emerged as an attractive cancer target. Consequently, many SHP2 allosteric inhibitors are now in clinical testing. Here we discovered a previously unrecognized off-target effect associated with SHP2 allosteric inhibitors. We found that these inhibitors accumulate in the lysosome and block autophagic flux in an SHP2-independent manner. We showed that off-target autophagy inhibition by SHP2 allosteric inhibitors contributes to their antitumor activity. We also demonstrated that SHP2 allosteric inhibitors harboring this off-target activity not only suppress oncogenic RAS signaling but also overcome drug resistance such as MAPK rebound and protective autophagy in response to RAS/MAPK pathway blockage. Finally, we exemplified a therapeutic framework that harnesses both the on- and off-target activities of SHP2 allosteric inhibitors for improved treatment of mutant RAS-driven and drug-resistant malignancies such as pancreatic and colorectal cancers.

A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9

The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we have focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we have designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation (RMSD) values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability plays a crucial role.

Synthetic abnormal mast cell particles successfully mimic neoplastic mast cells by flow cytometry.

Clinical flow cytometry laboratories require quality control materials for assay development, validation, and performance monitoring, including new reagent lot qualification. However, finding suitable controls for populations with uncommonly expressed antigens or for rare populations, such as mast cells, can be difficult. To that end, we evaluated synthetic abnormal mast cell particles (SAMCP), developed together with, and manufactured by, Slingshot Biosciences. The SAMCP's were designed to phenotypically mimic abnormal neoplastic mast cells: they were customized to have the same light scatter and autofluorescence properties of mast cells, along with surface antigen levels of CD45, CD33, CD117, CD2, CD25, and CD30 consistent with that seen in mast cell disease. We evaluated several performance characteristics of these particles using ARUP's high sensitivity clinical mast cell assay, including limit of detection, off-target activity and FMO controls, precision, scatter properties of the particles utilizing several different cytometer platforms, and particle antigen stability. The phenotype of the SAMCP mimicked abnormal mast cells, and they could be distinguished from normal native mast cells. FMO controls demonstrated specificity of each of the markers, and no off-target binding was detected. The limit of detection of the particles spiked into normal bone marrow was found to be ≤0.003% in a limiting dilution assay. The mast cell particles were found to perform similarly on Becton Dickinson Lyric, Cytek Aurora, and Beckman Coulter Navios and CytoFLEX platforms. Within run and between run precision were less than 10% CV. SAMCP were stable up to 13 days with minimal loss of antigen fluorescence intensity. The SAMCP's were able to successfully mimic neoplastic mast cells based on the results of our high sensitivity mast cell flow cytometry panel. These synthetic cell particles represent an exciting and innovative technology, which can fulfill vital needs in clinical flow cytometry such as serving as standardized control materials for assay development and performance monitoring.

The stateoftheart in secondary pharmacology and its impact on the safety of new medicines.

Secondary pharmacology screening of investigational small-molecule drugs for potentially adverse off-target activities has become standard practice in pharmaceutical research and development, and regulatory agencies are increasingly requesting data on activity against targets with recognized adverse effect relationships. However, the screening strategies and target panels used by pharmaceutical companies may vary substantially. To help identify commonalities and differences, as well as to highlight opportunities for further optimization of secondary pharmacology assessment, we conducted a broad-ranging survey across 18 companies under the auspices of the DruSafe leadership group of the International Consortium for Innovation and Quality in Pharmaceutical Development. Based on our analysis of this survey and discussions and additional research within the group, we present here an overview of the current state of the art in secondary pharmacology screening. We discuss best practices, including additional safety-associated targets not covered by most current screening panels, and present approaches for interpreting and reporting off-target activities. We also provide an assessment of the safety impact of secondary pharmacology screening, and a perspective on opportunities and challenges in this rapidly developing field.

An Improved PDE6D Inhibitor Combines with Sildenafil To Inhibit KRAS Mutant Cancer Cell Growth.

The trafficking chaperone PDE6D (or PDEδ) was proposed as a surrogate target for K-Ras, leading to the development of a series of inhibitors that block its prenyl binding pocket. These inhibitors suffered from low solubility and suspected off-target effects, preventing their clinical development. Here, we developed a highly soluble, low nanomolar PDE6D inhibitor (PDE6Di), Deltaflexin3, which has the lowest off-target activity as compared to three prominent reference compounds. Deltaflexin3 reduces Ras signaling and selectively decreases the growth of KRAS mutant and PDE6D-dependent cancer cells. We further show that PKG2-mediated phosphorylation of Ser181 lowers K-Ras binding to PDE6D. Thus, Deltaflexin3 combines with the approved PKG2 activator Sildenafil to more potently inhibit PDE6D/K-Ras binding, cancer cell proliferation, and microtumor growth. As observed previously, inhibition of Ras trafficking, signaling, and cancer cell proliferation remained overall modest. Our results suggest reevaluating PDE6D as a K-Ras surrogate target in cancer.

Unexpected Noncovalent Off-Target Activity of Clinical BTK Inhibitors Leads to Discovery of a Dual NUDT5/14 Antagonist.

Cofactor mimicry represents an attractive strategy for the development of enzyme inhibitors but can lead to off-target effects due to the evolutionary conservation of binding sites across the proteome. Here, we uncover the ADP-ribose (ADPr) hydrolase NUDT5 as an unexpected, noncovalent, off-target of clinical BTK inhibitors. Using a combination of biochemical, biophysical, and intact cell NanoBRET assays as well as X-ray crystallography, we confirm catalytic inhibition and cellular target engagement of NUDT5 and reveal an unusual binding mode that is independent of the reactive acrylamide warhead. Further investigation of the prototypical BTK inhibitor ibrutinib also revealed potent inhibition of the largely unstudied NUDIX hydrolase family member NUDT14. By exploring structure-activity relationships (SARs) around the core scaffold, we identify a potent, noncovalent, and cell-active dual NUDT5/14 inhibitor. Cocrystallization experiments yielded new insights into the NUDT14 hydrolase active site architecture and inhibitor binding, thus providing a basis for future chemical probe design.

The physiological interactome of TCR-like antibody therapeutics in human tissues.

Selective binding of TCR-like antibodies that target a single tumour-specific peptide antigen presented by human leukocyte antigens (HLA) is the absolute prerequisite for their therapeutic suitability and patient safety. To date, selectivity assessment has been limited to peptide library screening and predictive modeling. We developed an experimental platform to de novo identify interactomes of TCR-like antibodies directly in human tissues using mass spectrometry. As proof of concept, we confirm the target epitope of a MAGE-A4-specific TCR-like antibody. We further determine cross-reactive peptidesequences for ESK1, a TCR-like antibody with known off-target activity, in human liver tissue. We confirm off-target-induced T cell activation and ESK1-mediated liver spheroid killing. Off-target sequences feature an amino acid motif that allows a structural groove-coordination mimicking that of the target peptide, therefore allowing the interaction with the engager molecule. We conclude that our strategy offers an accurate, scalable route for evaluating the non-clinical safety profile of TCR-like antibody therapeutics prior to first-in-human clinical application.

The 5HT2b Receptor in Alzheimer's Disease: Increased Levels in Patient Brains and Antagonist Attenuation of Amyloid and Tau Induced Dysfunction.

Background: Neurodegenerative diseases manifest behavioral dysfunction with disease progression. Intervention with neuropsychiatric drugs is part of most multi-drug treatment paradigms. However, only a fraction of patients responds to the treatments and those responding must deal with drug-drug interactions and tolerance issues generally attributed to off-target activities. Recent efforts have focused on the identification of underexplored targets and exploration of improved outcomes by treatment with selective molecular probes. As part of ongoing efforts to identify and validate additional targets amenable to therapeutic intervention, we examined levels of the serotonin 5-HT2b receptor (5-HT2bR) in Alzheimer's disease (AD) brains and the potential of a selective 5-HT2bR antagonist to counteract synaptic plasticity and memory damage induced by AD-related proteins, amyloid-β, and tau. This work used a combination of biochemical, chemical biology, electrophysiological, and behavioral techniques. Biochemical methods included analysis of protein levels. Chemical biology methods included the use of an in vivo molecular probe MW071, a selective antagonist for the 5HT2bR. Electrophysiological methods included assessment of long-term potentiation (LTP), a type of synaptic plasticity thought to underlie memory formation. Behavioral studies investigated spatial memory and associative memory. 5HT2bR levels are increased in brain specimens of AD patients compared to controls. 5HT2bR antagonist treatment rescued amyloid-β and tau oligomer-induced impairment of synaptic plasticity and memory. The increased levels of 5HT-2bR in AD patient brains and the attenuation of disease-related synaptic and behavioral dysfunctions by MW071 treatment suggest that the 5HT-2bR is a molecular target worth pursuing as a potential therapeutic target.