Use of in vitro methods combined with in silico analysis to identify potential skin sensitizers in the Tox21 10K compound library.

Introduction: Skin sensitization, which leads to allergic contact dermatitis, is a key toxicological endpoint with high occupational and consumer prevalence. This study optimized several in vitro assays listed in OECD skin sensitization test guidelines for use on a quantitative high-throughput screening (qHTS) platform and performed in silico model predictions to assess the skin sensitization potential of prioritized compounds from the Tox21 10K compound library. Methods: First, we screened the entire Tox21 10K compound library using a qHTS KeratinoSensTM (KS) assay and built a quantitative structure-activity relationship (QSAR) model based on the KS results. From the qHTS KS screening results, we prioritized 288 compounds to cover a wide range of structural chemotypes and tested them in the solid phase extraction-tandem mass spectrometry (SPE-MS/MS) direct peptide reactivity assay (DPRA), IL-8 homogeneous time-resolved fluorescence (HTRF) assay, CD86 and CD54 surface expression in THP1 cells, and predicted in silico sensitization potential using the OECD QSAR Toolbox (v4.5). Results: Interpreting tiered qHTS datasets using a defined approach showed the effectiveness and efficiency of in vitro methods. We selected structural chemotypes to present this diverse chemical collection and to explore previously unidentified structural contributions to sensitization potential. Discussion: Here, we provide a skin sensitization dataset of unprecedented size, along with associated tools, and analysis designed to support chemical assessments.

Comparative Analysis of Drug-like EP300/CREBBP Acetyltransferase Inhibitors.

The human acetyltransferase paralogues EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potencies of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize the binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of the relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.

The dose-related plateau effect of surviving fraction in normal tissue during the ultra-high-dose-rate radiotherapy.

Objective.Ultra-high-dose-rate radiotherapy, referred to as FLASH therapy, has been demonstrated to reduce the damage of normal tissue as well as inhibiting tumor growth compared with conventional dose-rate radiotherapy. The transient hypoxia may be a vital explanation for sparing the normal tissue. The heterogeneity of oxygen distribution for different doses and dose rates in the different radiotherapy schemes are analyzed. With these results, the influence of doses and dose rates on cell survival are evaluated in this work.Approach.The two-dimensional reaction-diffusion equations are used to describe the heterogeneity of the oxygen distribution in capillaries and tissue. A modified linear quadratic model is employed to characterize the surviving fraction at different doses and dose rates.Main results.The reduction of the damage to the normal tissue can be observed if the doses exceeds a minimum dose threshold under the ultra-high-dose-rate radiation. Also, the surviving fraction exhibits the 'plateau effect' under the ultra-high dose rates radiation, which signifies that within a specific range of doses, the surviving fraction either exhibits minimal variation or increases with the dose. For a given dose, the surviving fraction increases with the dose rate until tending to a stable value, which means that the protection in normal tissue reaches saturation.Significance.The emergence of the 'plateau effect' allows delivering the higher doses while minimizing damage to normal tissue. It is necessary to develop appropriate program of doses and dose rates for different irradiated tissue to achieve more efficient protection.

Hemoporfin-Mediated Photodynamic Therapy for Port-Wine Stains on Extremities.

INTRODUCTION: Pulsed dye laser (PDL) is currently considered to be the first-line treatment for port-wine stains (PWSs) on the extremities despite its less than satisfactory therapeutic efficacy. Hemoporfin-mediated photodynamic therapy (HMME-PDT) is a vascular-targeted therapy that has rarely been used to treat PWSs on the extremities. Here, we evaluate the clinical efficacy and safety of HMME-PDT for the treatment of PWSs on the extremities. METHODS: Clinical data and dermoscopic images of PWSs on the extremities were obtained from 65 patients who underwent HMME-PDT between February 2019 and December 2022. The clinical efficacy of HMME-PDT was analyzed by comparing the pre- and post-treatment images. The safety of HMME-PDT was evaluated through observation during the treatment period and post-treatment follow-up. RESULTS: The efficacy rate of a single HMME-PDT session was 63.0% and that of two and three to six sessions was 86.7% and 91.3%, respectively. A positive correlation was found between therapeutic efficacy and the number of HMME-PDT sessions. The therapeutic efficacy of HMME-PDT was better on the proximal extremities than on other parts of the extremities (P = 0.038), and the efficacy of treating PWSs in each site was relatively improved with an increase of treatment time. The clinical efficacy of HMME-PDT differed across four PWS vascular patterns identified by dermoscopy (P = 0.019). However, there was no statistical difference in the therapeutic efficacy based on age, sex, type of PWS, and treatment history (P > 0.05), which may be partly attributed to the relatively small sample size or poor cooperation of infant patients. No obvious adverse reactions were observed during the follow-up period. CONCLUSIONS: HMME-PDT is a very safe and effective treatment for PWSs on the extremities. Multiple HMME-PDT treatments, lesions located in proximal limbs, and PWSs with type I and IV vascular patterns under dermoscopy were associated with higher efficacy of HMME-PDT. Dermoscopy may help predict the clinical efficacy of HMME-PDT. TRIAL REGISTRATION NO: 2020KJT085.

Comparative analysis of drug-like EP300/CREBBP acetyltransferase inhibitors.

The human acetyltransferase paralogs EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potency of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.

Discovery of Novel Small-Molecule Scaffolds for the Inhibition and Activation of WIP1 Phosphatase from a RapidFire Mass Spectrometry High-Throughput Screen.

Wild-type P53-induced phosphatase 1 (WIP1), also known as PPM1D or PP2Cδ, is a serine/threonine protein phosphatase induced by P53 after genotoxic stress. WIP1 inhibition has been proposed as a therapeutic strategy for P53 wild-type cancers in which it is overexpressed, but this approach would be ineffective in P53-negative cancers. Furthermore, there are several cancers with mutated P53 where WIP1 acts as a tumor suppressor. Therefore, activating WIP1 phosphatase might also be a therapeutic strategy, depending on the P53 status. To date, no specific, potent WIP1 inhibitors with appropriate pharmacokinetic properties have been reported, nor have WIP1-specific activators. Here, we report the discovery of new WIP1 modulators from a high-throughput screen (HTS) using previously described orthogonal biochemical assays suitable for identifying both inhibitors and activators. The primary HTS was performed against a library of 102 277 compounds at a single concentration using a RapidFire mass spectrometry assay. Hits were further evaluated over a range of 11 concentrations with both the RapidFire MS assay and an orthogonal fluorescence-based assay. Further biophysical, biochemical, and cell-based studies of confirmed hits revealed a WIP1 activator and two inhibitors, one competitive and one uncompetitive. These new scaffolds are prime candidates for optimization which might enable inhibitors with improved pharmacokinetics and a first-in-class WIP1 activator.

Hemoporfin-Mediated Photodynamic Therapy for Port-Wine Stains: Multivariate Analysis of Clinical Efficacy and Optical Coherence Tomography Appearance.

Background: Hemoporfin-mediated photodynamic therapy (HMME-PDT) is reported to be effective and safe for port-wine stains (PWS). However, its efficacy is influenced by several factors and there is no appropriate method to evaluate efficacy so far. Therefore, this study explored the clinical efficacy of HMME-PDT for PWS on the face and neck and the feasibility of evaluating treatment potency with optical coherence tomography (OCT). Methods: A total of 211 PWS patients subjected to HMME-PDT were recruited for study and correlations of therapeutic effect with treatment sessions, age, gender, lesion distribution and treatment history analyzed. OCT was utilized for quantitative analysis of PWS lesions of 36 selected patients before and after HMME-PDT. Results: The efficacy of two consecutive treatments was significantly higher than that of single treatment (P < 0.05). In multivariate analysis, after the first treatment, age, lesion distribution and treatment history were correlative factors affecting treatment efficacy (P < 0.05). The improvement effect on central facial lesions was lower than that on lateral facial lesions (P < 0.05). The efficacy of therapy on the group with no history of pulsed dye laser (PDL) treatment was greater than that on effective and ineffective treatment groups (P < 0.05). After the second session, age remained the only factor correlated with efficacy (P < 0.05). Dilated vessel diameter and depth before and after treatment were significantly different (P < 0.05). With increasing treatment times, age was the most significant factor influencing treatment efficacy. Conclusions: Our collective findings indicate that HMME-PDT therapy is effective and safe for PWS and support the utility of OCT in objective assessment of the efficacy of HMME-PDT.

Structure-Based Optimization of Small Molecule Human Galactokinase Inhibitors.

Classic galactosemia is a rare disease caused by inherited deficiency of galactose-1 phosphate uridylyltransferase (GALT). Accumulation of galactose-1 phosphate (gal-1P) is thought to be the major cause of the chronic complications associated with this disease, which currently has no treatment. Inhibiting galactokinase (GALK1), the enzyme that generates galactose-1 phosphate, has been proposed as a novel strategy for treating classic galactosemia. Our previous work identified a highly selective unique dihydropyrimidine inhibitor against GALK1. With the determination of a co-crystal structure of this inhibitor with human GALK1, we initiated a structure-based structure-activity relationship (SAR) optimization campaign that yielded novel analogs with potent biochemical inhibition (IC50 < 100 nM). Lead compounds were also able to prevent gal-1P accumulation in patient-derived cells at low micromolar concentrations and have pharmacokinetic properties suitable for evaluation in rodent models of galactosemia.

A Universal and High-Throughput Proteomics Sample Preparation Platform.

Major advances have been made to improve the sensitivity of mass analyzers, spectral quality, and speed of data processing enabling more comprehensive proteome discovery and quantitation. While focus has recently begun shifting toward robust proteomics sample preparation efforts, a high-throughput proteomics sample preparation is still lacking. We report the development of a highly automated universal 384-well plate sample preparation platform with high reproducibility and adaptability for extraction of proteins from cells within a culture plate. Digestion efficiency was excellent in comparison to a commercial digest peptide standard with minimal sample loss while improving sample preparation throughput by 20- to 40-fold (the entire process from plated cells to clean peptides is complete in ∼300 min). Analysis of six human cell types, including two primary cell samples, identified and quantified ∼4,000 proteins for each sample in a single high-performance liquid chromatography (HPLC)-tandem mass spectrometry injection with only 100-10K cells, thus demonstrating universality of the platform. The selected protein was further quantified using a developed HPLC-multiple reaction monitoring method for HeLa digests with two heavy labeled internal standard peptides spiked in. Excellent linearity was achieved across different cell numbers indicating a potential for target protein quantitation in clinical research.

Quantitative analysis of vascular changes during photoimmunotherapy using speckle variance optical coherence tomography (SV-OCT).

Near-infrared (NIR) photoimmunotherapy (NIR-PIT) is an emerging cancer therapy based on a monoclonal antibody and phthalocyanine dye conjugate. Direct tumor necrosis and immunogenic cell death occur during NIR irradiation. However, the alteration of tumor blood vessels and blood volume inside the blood vessels induced by the NIR-PIT process is still unknown. In our study, a speckle variance (SV) algorithm combined with optical coherence tomography (OCT) technology was applied to monitor the change of blood vessels and the alterations of the blood volume inside the blood vessels during and after NIR-PIT treatment. Vascular density and the measurable diameter of the lumen in the blood vessel (the diameter of the region filled with blood) were extracted for quantitively uncovering the alterations of blood vessels and blood volume induced by NIR-PIT treatment. The results indicate that both the density and the diameter of the lumen in the blood vessels decrease during the NIR-PIT process, while histological results indicated the blood vessels were dilated. The increase of permeability of blood vessels could lead to the increase of the blood pool volume within the tumor (shown in histology) and results in the decrease of free-moving red blood cells inside the blood vessels (shown in SV-OCT).

A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells.

Human pluripotent stem cells (hPSCs) are capable of extensive self-renewal yet remain highly sensitive to environmental perturbations in vitro, posing challenges to their therapeutic use. There is an urgent need to advance strategies that ensure safe and robust long-term growth and functional differentiation of these cells. Here, we deployed high-throughput screening strategies to identify a small-molecule cocktail that improves viability of hPSCs and their differentiated progeny. The combination of chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. CEPT provided strong improvements for several key applications in stem-cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body (EB) and organoid formation, single-cell cloning, and genome editing. Thus, CEPT represents a unique poly-pharmacological strategy for comprehensive cytoprotection, providing a rationale for efficient and safe utilization of hPSCs.

Discovery and Optimization of 2H-1λ2-Pyridin-2-one Inhibitors of Mutant Isocitrate Dehydrogenase 1 for the Treatment of Cancer.

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are oncogenic for a number of malignancies, primarily low-grade gliomas and acute myeloid leukemia. We report a medicinal chemistry campaign around a 7,7-dimethyl-7,8-dihydro-2H-1λ2-quinoline-2,5(6H)-dione screening hit against the R132H and R132C mutant forms of isocitrate dehydrogenase (IDH1). Systematic SAR efforts produced a series of potent pyrid-2-one mIDH1 inhibitors, including the atropisomer (+)-119 (NCATS-SM5637, NSC 791985). In an engineered mIDH1-U87-xenograft mouse model, after a single oral dose of 30 mg/kg, 16 h post dose, between 16 and 48 h, (+)-119 showed higher tumoral concentrations that corresponded to lower 2-HG concentrations, when compared with the approved drug AG-120 (ivosidenib).

A direct peptide reactivity assay using a high-throughput mass spectrometry screening platform for detection of skin sensitizers.

Chemical-peptide conjugation is the molecular initiating event in skin sensitization. The OECD test guideline uses a high-performance liquid chromatography/ultraviolet (HPLC/UV) detection method to quantify chemical-peptide conjugation in a direct peptide reactivity assay (DPRA), which measures the depletion of two synthetic peptides containing lysine or cysteine residues. To improve assay throughput, sensitivity and accuracy, an automated 384-well plate-based RapidFire solid-phase extraction (SPE) system coupled with tandem mass spectrometry (MS/MS) DPRA was developed and validated in the presence of a newly designed internal standard. Compared to the HPLC/UV-based DPRA, the automated SPE-MS/MS-based DPRA improved throughput from 16 min to 10 s per sample, and substrate peptides usage was reduced from 100 mM to 5 µM. When implementing the SPE-MS/MS-based DPRA into a high-throughput platform, we found 10 compounds that depleted lysine peptide and 24 compounds that depleted cysteine peptide (including 7 unreported chemicals from 55 compounds we tested) in a concentration-response manner. The adduct formation between cysteine and cinnamic aldehyde and ethylene glycol dimethacrylate were further analyzed using high-performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF-MS) to confirm the conjugation. Overall, the automated SPE-MS/MS-based platform is an efficient, economic, and accurate way to detect skin sensitizers.

Pyrazole-Based Lactate Dehydrogenase Inhibitors with Optimized Cell Activity and Pharmacokinetic Properties.

Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate, with concomitant oxidation of reduced nicotinamide adenine dinucleotide as the final step in the glycolytic pathway. Glycolysis plays an important role in the metabolic plasticity of cancer cells and has long been recognized as a potential therapeutic target. Thus, potent, selective inhibitors of LDH represent an attractive therapeutic approach. However, to date, pharmacological agents have failed to achieve significant target engagement in vivo, possibly because the protein is present in cells at very high concentrations. We report herein a lead optimization campaign focused on a pyrazole-based series of compounds, using structure-based design concepts, coupled with optimization of cellular potency, in vitro drug-target residence times, and in vivo PK properties, to identify first-in-class inhibitors that demonstrate LDH inhibition in vivo. The lead compounds, named NCATS-SM1440 (43) and NCATS-SM1441 (52), possess desirable attributes for further studying the effect of in vivo LDH inhibition.

Toward image quality assessment in optical coherence tomography (OCT) of rat kidney.

BACKGROUND: Optical coherence tomography (OCT) is a useful tool for the evaluation of structure and function of the kidney, but the image quality can be effected by many factors. OBJECTIVE: The objective of this study was to assess the image quality of different OCT systems in OCT imaging of the living kidney. METHODS: One swept-source OCT (SSOCT) of 1300 nm, one spectral domain OCT (SDOCT) of 1300 nm and another of 900 nm were used. A FeO phantom was used to establish the point spread function (PSF). Rat kidneys were imaged for image quality assessment. Light penetration in the kidney and the optical attenuation coefficient were also evaluated. The quantification of uriniferous tubules was carried out via the threshold segmentation of 3D OCT images. RESULTS: The quality of kidney images was resolution dependent. SDOCT of 900 nm showed higher peak signal-to noise ratio and dynamic range. The spatial resolution in the light field could be derived from the PSF distribution along three mutually orthogonal axes. In conjunction with the PSF, the Lucy-Richardson algorithm could improve image quality but could not reveal more microstructural information. The penetration depth of 1300 nm was deeper than that of 900 nm. The attenuation coefficient of the kidney was 29 cm-1 at 1300 nm and 50 cm-1 at 900 nm (P < 0.001). More accurate measurement of uriniferous tubules was achieved with the SDOCT-900 due to its higher resolution. CONCLUSIONS: Both SSOCT and SDOCT systems could be useful for imaging uriniferous tubules in the superficial layers of the cortex. The OCT image quality was highly correlated with the spatial resolution of OCT system.

Correction to: Bioaccumulation and growth characteristics of Vallisneria natans (Lour.) Hara after chronic exposure to metal-contaminated sediments.

The correct name of the 9th Author is shown in this paper.

Physiologically relevant orthogonal assays for the discovery of small-molecule modulators of WIP1 phosphatase in high-throughput screens.

WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, Pi We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens.

Quantitative assessment of skin swelling using optical coherence tomography.

BACKGROUND: Preclinical and clinical studies suggest that optical coherence tomography (OCT) is a useful tool to visualize inflammatory conditions. OBJECTIVE: The objective of this study was to evaluate the usefulness of combining OCT and various image processing techniques for quantitative assessment of histamine-induced tissue swelling. METHODS: Both time-domain and frequency-domain OCT were used on a mouse ear model. The ear thickness and volume before and after histamine challenge were determined from pixel locations in 2D scans and voxel number counting in 3D scans. Swelling kinetics was analyzed on 3D contour mapping. Microvessel network was visualized using speckle decorrelation analysis. RESULTS: OCT images showed that the thickness and volume changes were histamine dose and contact time dependent. The 3D mapping showed that the histamine-induced swelling spread slowly and directionally. OCT data indicated that microvessel opening and vessel dilation occurred prior to tissue swelling. CONCLUSION: OCT is a robust and quantitative non-invasive imaging tool for assessing skin swelling.

Bioaccumulation and growth characteristics of Vallisneria natans (Lour.) Hara after chronic exposure to metal-contaminated sediments.

Metal-contaminated sediments in lakes is a global concern that poses toxicological risk to aquatic organisms. This study performed bioassays using the submerged macrophyte, Vallisneria natans (Lour.) Hara, exposed to contaminated sediments collected from five locations in Dianchi Lake, Yunnan, China. Among the sediments collected, Igeo showed enrichment of As and Cd in Dianchi Lake sediments. In spite of enriched toxic metals at some locations, laboratory bioassays found no significant difference in leaf biomass or leaf photosynthesis rate between the sites. Root biomass and root activity showed significant differences between locations and were negatively correlated with the concentration of As, Cd, Hg, and Pb in sediment but not related to Cr. The above correlations were strongest for Hg and As, respectively. Accumulation of Cd and Pb to leaves of bioassay plants was observed, but this was not evident for As and Cr. Overall, the results indicate that V. natans can be used as a bioassay organism and measures of root toxicity are sensitive to metal concentrations present in Dianchi Lake sediments. Furthermore, the study species holds promise for use as a biomonitor of Cd and Pb sediment metal content.

Insights into the Action of Inhibitor Enantiomers against Histone Lysine Demethylase 5A.

Isomers of chiral drugs can exhibit marked differences in biological activities. We studied the binding and inhibitory activities of 12 compounds against KDM5A. Among them are two pairs of enantiomers representing two distinct inhibitor chemotypes, namely, ( R)- and ( S)-2-((2-chlorophenyl)(2-(piperidin-1-yl)ethoxy)methyl)-1 H-pyrrolo[3,2- b]pyridine-7-carboxylic acid (compounds N51 and N52) and ( R) - and ( S) -N-(1-(3-isopropyl-1 H-pyrazole-5-carbonyl)pyrrolidin-3-yl)cyclopropanecarboxamide (compounds N54 and N55). In vitro, the S enantiomer of the N51/N52 pair (N52) and the R enantiomer of the N54/N55 pair (N54) exhibited about 4- to 5-fold greater binding affinity. The more potent enzyme inhibition of KDM5A by the R-isoform for the cell-permeable N54/N55 pair translated to differences in growth inhibitory activity. We determined structures of the KDM5A catalytic domain in complex with all 12 inhibitors, which revealed the interactions (or lack thereof) responsible for the differences in binding affinity. These results provide insights to guide improvements in binding potency and avenues for development of cell permeable inhibitors of the KDM5 family.