Translatability of In Vitro Stress for Predicting Deamidation and Oxidation Biotransformation on Biotherapeutics.

Biotransformation leading to single residue modifications (e.g., deamidation, oxidation) can contribute to decreased efficacy/potency, poor pharmacokinetics, and/or toxicity/immunogenicity for protein therapeutics. Identifying and characterizing such liabilities in vivo are emerging needs for biologics drug discovery. In vitro stress assays involving PBS for deamidation or AAPH for oxidation are commonly used for predicting liabilities in manufacturing and storage and are sometimes considered a predictive tool for in vivo liabilities. However, reports discussing their in vivo translatability are limited. Herein, we introduce a mass spectrometry workflow that characterizes in vivo oxidation and deamidation in pharmacokinetically relevant compartments for diverse protein therapeutic modalities. The workflow has low bias of <10% in quantitating degradation in the relevant pharmacokinetic concentration range for monkey and rabbit serum/plasma (1-100 µg/mL) and allows for high sequence coverage (∼85%) for discovery/monitoring of amino acid modifications. For oxidation and deamidation, the assay was precise, with percent coefficient of variation of <8% at 1-100 µg/mL and ≤6% method-induced artifacts. A high degree of in vitro and in vivo correlation was observed for deamidation on the six diverse protein therapeutics (seven liability sites) tested. In vivo translatability for oxidation liabilities were not observed for the 11 molecules tested using in vitro AAPH stress. One of the molecules dosed in eyes resulted in a false positive and a false negative prediction for in vivo oxidation following AAPH stress. Finally, peroxide stress was also tested but resulted in limited success (1 out of 4 molecules) in predicting oxidation liabilities.

Quantifying KRAS G12C Covalent Drug Inhibitor Activity in Mouse Tumors Using Mass Spectrometry.

The growing opportunities recognized for covalent drug inhibitors, like KRAS G12C inhibitors, are driving the need for mass spectrometry methods that can quickly and robustly measure therapeutic drug activity in vivo for drug discovery research and development. Effective front-end sample preparation is critical for proteins extracted from tumors but is generally labor intensive and impractical for large sample numbers typical in pharmacodynamic (PD) studies. Herein, we describe an automated and integrated sample preparation method for the measurement of activity levels of KRAS G12C drug inhibitor alkylation from complex tumor samples involving high throughput detergent removal and preconcentration followed by quantitation using mass spectrometry. We introduce a robust assay with an average intra-assay coefficient of variation (CV) of 4% and an interassay CV of 6% obtained from seven studies, enabling us to understand the relationship between KRAS G12C target occupancy and the therapeutic PD effect from mouse tumor samples. Further, the data demonstrated that the drug candidate GDC-6036, a KRAS G12C covalent inhibitor, shows dose-dependent target inhibition (KRAS G12C alkylation) and MAPK pathway inhibition, which correlate with high antitumor potency in the MIA PaCa-2 pancreatic xenograft model.

Using a peptide-based mass spectrometry approach to quantitate proteolysis of an intact heterogeneous procollagen substrate by BMP1 for antagonistic antibody screening.

Proteases are critical proteins involved in cleaving substrates that may impact biological pathways, cellular processes, or disease progression. In the biopharmaceutical industry, modulating the levels of protease activity is an important strategy for mitigating many types of diseases. While a variety of analytical tools exist for characterizing substrate cleavages, in vitro functional screening for antibody inhibitors of protease activity using physiologically relevant intact protein substrates remains challenging. In addition, detecting such large protein substrates with high heterogeneity using high-throughput mass spectrometry screening has rarely been reported in the literature with concerns for assay robustness and sensitivity. In this study, we established a peptide-based in vitro functional screening assay for antibody inhibitors of mouse bone morphogenic protein 1 (mBMP1) metalloprotease using a heterogeneous recombinant 66-kDa mouse Procollagen I alpha 1 chain (mProcollagen) substrate. We compared several analytical tools including capillary gel electrophoresis Western blot (CE-Western blot), as well as both intact protein and peptide-based mass spectrometry (MS) to quantitate the mBMP1 proteolytic activity and its inhibition by antibodies using this heterogeneous mProcollagen substrate. We concluded that the peptide-based mass spectrometry screening assay was the most suitable approach in terms of throughput, sensitivity, and assay robustness. We then optimized our mBMP1 proteolysis reaction after characterizing the enzyme kinetics using the peptide-based MS assay. This assay resulted in Z' values ranging from 0.6 to 0.8 from the screening campaign. Among over 1200 antibodies screened, IC50 characterization was performed on the top candidate hits, which showed partial or complete inhibitory activities against mBMP1.

High-throughput antibody screening from complex matrices using intact protein electrospray mass spectrometry.

Toward the goal of increasing the throughput of high-resolution mass characterization of intact antibodies, we developed a RapidFire-mass spectrometry (MS) assay using electrospray ionization. We achieved unprecedented screening throughput as fast as 15 s/sample, which is an order of magnitude improvement over conventional liquid chromatography (LC)-MS approaches. The screening enabled intact mass determination as accurate as 7 ppm with baseline resolution at the glycoform level for intact antibodies. We utilized this assay to characterize and perform relative quantitation of antibody species from 248 samples of 62 different cell line clones at four time points in 2 h using RapidFire-time-of-flight MS screening. The screening enabled selection of clones with the highest purity of bispecific antibody production and the results significantly correlated with conventional LC-MS results. In addition, analyzing antibodies from a complex plasma sample using affinity-RapidFire-MS was also demonstrated and qualified. In summary, the platform affords high-throughput analyses of antibodies, including bispecific antibodies and potential mispaired side products, in cell culture media, or other complex matrices.

Implementation of the CYP Index for the Design of Selective Tryptophan-2,3-dioxygenase Inhibitors.

A class of imidazoisoindole (III) heme-binding indoleamine-2,3-dioxygenase (IDO1) inhibitors were optimized via structure-based drug design into a series of tryptophan-2,3-dioxygenase (TDO)-selective inhibitors. Kynurenine pathway modulation was demonstrated in vivo, which enabled evaluation of TDO as a potential cancer immunotherapy target. As means of mitigating the risk of drug-drug interactions arising from cytochrome P450 inhibition, a novel property-based drug design parameter, herein referred to as the CYP Index, was implemented for the design of inhibitors with appreciable selectivity for TDO over CYP3A4. We anticipate the CYP Index will be a valuable design parameter for optimizing CYP inhibition of any small molecule inhibitor containing a Lewis basic motif capable of binding heme.

Characterizing and Quantitating Therapeutic Tethered Multimeric Antibody Degradation Using Affinity Capture Mass Spectrometry.

There are many pharmacokinetic challenges associated with administering protein therapeutics, including biotransformation via clipping, deamidation, isomerization, oxidation, etc. In the case of engineered multivalent tethered antibody formats, proteolysis or deconjugation at the fusion or conjugation site present further issues. Unlike degradations associated with antibody drug conjugates, such biotransformations of tethered antibody formats usually result in degraded products with large mass differences. These large differences can result in processing or mass spectrometry response bias among the resulting product species that can lead to inaccurate stability quantitation. Herein, we describe an assay strategy for characterizing and quantitating degradations accurately for multivalent antibodies by incorporating response bias corrections. For the multivalent tethered antibody molecules selected, an ∼30-80% difference in response, compared to the cleaved product, was observed. To correct for the response bias, selected tethered multivalent antibodies and an IgG antibody (representing the stable intact and the degraded product species, respectively) were spiked in serum at known ratios for analysis. Following affinity capture, we generated calibration curves (five-parameter logistic fit p < 0.05) by plotting the measured ratios of the MS ion responses against the known spiked-in ratios (CVs < 8% for calibration standards). The qualified calibration curve (accuracy within 8% and 2% for measuring degradations of 5% and 15% product, respectively) was then used, through interpolation, to determine stability profiles for the same multivalent tethered antibody formats from both in vitro serum and pharmacokinetic study samples.

Improved translation of stability for conjugated antibodies using an in vitro whole blood assay.

For antibody-drug conjugates to be efficacious and safe, they must be stable in circulation to carry the payload to the site of the targeted cell. Several components of a drug-conjugated antibody are known to influence stability: 1) the site of drug attachment on the antibody, 2) the linker used to attach the payload to the antibody, and 3) the payload itself. In order to support the design and optimization of a high volume of drug conjugates and avoid unstable conjugates prior to testing in animal models, we wanted to proactively identify these potential liabilities. Therefore, we sought to establish an in vitro screening method that best correlated with in vivo stability. While traditionally plasma has been used to assess in vitro stability, our evaluation using a variety of THIOMABTM antibody-drug conjugates revealed several disconnects between the stability assessed in vitro and the in vivo outcomes when using plasma. When drug conjugates were incubated in vitro for 24 h in mouse whole blood rather than plasma and then analyzed by affinity capture LC-MS, we found an improved correlation to in vivo stability with whole blood (R2 = 0.87, coefficient of determination) compared to unfrozen or frozen mouse plasma (R2 = 0.34, 0.01, respectively). We further showed that this whole blood assay was also able to predict in vivo stability of other preclinical species such as rat and cynomolgus monkey, as well as in human. The screening method utilized short (24 h) incubation times, as well as a custom analysis software, allowing increased throughput and in-depth biotransformation characterization. While some instabilities that were more challenging to identify remain, the method greatly enhanced the process of screening, optimizing, and lead candidate selection, resulting in the substantial reduction of animal studies.

Novel Interface for High-Throughput Analysis of Biotherapeutics by Electrospray Mass Spectrometry.

With the rapid rise of therapeutic antibodies and antibody-drug conjugates, significant investments have been made in developing workflows that utilize mass spectrometry to detect these intact molecules, the large fragments generated by their selective digestion, and the peptides generated by traditional proteomics workflows. The resultant data is used to gain insight into a wide range of parameters, including primary sequence, disulfide bonding, glycosylation patterns, biotransformation, and more. However, many of the technologies utilized to couple these workflows to mass spectrometers have significant limitations that force nonoptimal modifications to upstream sample preparation steps, limit the throughput of high-volume workflows, and prevent the harmonization of diverse experiments onto a single hardware platform. Here, we describe a new analytical platform that enables direct and high-throughput coupling to electrospray ionization mass spectrometry. The SampleStream platform is compatible with both native and denaturing electrospray, operates with a throughput of up to 15 s/sample, provides extensive concentration of dilute samples, and affords similar sensitivity to comparable liquid chromatographic methods.

Catalytic Cleavage of Disulfide Bonds in Small Molecules and Linkers of Antibody-Drug Conjugates.

In cells, catalytic disulfide cleavage is an essential mechanism in protein folding and synthesis. However, detailed enzymatic catalytic mechanism relating cleavage of disulfide bonds in xenobiotics is not well understood. This study reports an enzymatic mechanism of cleavage of disulfide bonds in xenobiotic small molecules and antibody conjugate (ADC) linkers. The chemically stable disulfide bonds in substituted disulfide-containing pyrrolobenzodiazepine (PBD, pyrrolo[2,1-c][1,4]benzodiazepine) monomer prodrugs in presence of glutathione or cysteine were found to be unstable in incubations in whole blood of humans and rats. It was shown the enzymes involved were thioredoxin (TRX) and glutaredoxin (GRX). For a diverse set of drug-linker conjugates, we determined that TRX in the presence of TRX-reductase and NADPH generated the cleaved products that are consistent with catalytic disulfide cleavage and linker immolation. GRX was less rigorously studied; in the set of compounds studied, its role in the catalytic cleavage was also confirmed. Collectively, these in vitro experiments demonstrate that TRX as well as GRX can catalyze the cleavage of disulfide bonds in both small molecules and linkers of ADCs.

Quantitative Determination of Protein-Ligand Affinity by Size Exclusion Chromatography Directly Coupled to High-Resolution Native Mass Spectrometry.

High throughput protein-ligand interaction screening assays employing mass spectrometric detection are widely used in early stage drug discovery. Mass spectrometry-based screening approaches employ a target protein added to a pool of small-molecule compounds, and binding is assessed by measuring ligands denatured from the complexes. Direct analysis of protein-ligand interactions using native mass spectrometry has been demonstrated but is not widely used due to the detection limit on protein size, the requirement of volatile buffers, and the necessity for specialized instrumentation to preserve weak interactions under native conditions. Here we present a robust, quantitative, and automated online size-exclusion chromatography-native mass spectrometry (SEC-nMS) platform for measuring affinities of noncovalent protein-small-molecule interactions on an Orbitrap mass spectrometer. Indoleamine 2,3-dioxygenase 1, a catabolic enzyme, and inhibitory ligands were employed as a demonstration of the method. Efficient separation and elution enabled preservation of protein-ligand complexes and increased throughput. The high sensitivity and intra charge state resolution at high m/ z offered by the Exactive Plus EMR Orbitrap allowed for protein ligand affinity quantitation and resolved individual compounds close in mass. Vc50 values determined via collision-induced dissociation experiments enabled the evaluation of complex stability in the gas phase and were found to be independent of the extent of complex formation. For the first time, Vc50 determinations were achieved on an inline SEC-nMS platform. Systematic comparison of our method with optimized chip-based nanoelectrospray infusion served as a reference for ligand screening and affinity quantitation and further revealed the advantages of SEC-MS.

Automated On-tip Affinity Capture Coupled with Mass Spectrometry to Characterize Intact Antibody-Drug Conjugates from Blood.

Antibody-drug conjugates (ADCs) present unique challenges for ligand-binding assays primarily due to the dynamic changes of the drug-to-antibody ratio (DAR) distribution in vivo and in vitro. Here, an automated on-tip affinity capture platform with subsequent mass spectrometry analysis was developed to accurately characterize the DAR distribution of ADCs from biological matrices. A variety of elution buffers were tested to offer optimal recovery, with trastuzumab serving as a surrogate to the ADCs. High assay repeatability (CV 3%) was achieved for trastuzumab antibody when captured below the maximal binding capacity of 7.5 µg. Efficient on-tip deglycosylation was also demonstrated in 1 h followed by affinity capture. Moreover, this tip-based platform affords higher throughput for DAR characterization when compared with a well-characterized bead-based method. Graphical Abstract ᅟ.

Aminoisoxazoles as Potent Inhibitors of Tryptophan 2,3-Dioxygenase 2 (TDO2).

Tryptophan 2,3-dioxygenase 2 (TDO2) catalyzes the conversion of tryptophan to the immunosuppressive metabolite kynurenine. TDO2 overexpression has been observed in a number of cancers; therefore, TDO inhibition may be a useful therapeutic intervention for cancers. We identified an aminoisoxazole series as potent TDO2 inhibitors from a high-throughput screen (HTS). An extensive medicinal chemistry effort revealed that both the amino group and the isoxazole moiety are important for TDO2 inhibitory activity. Computational modeling yielded a binding hypothesis and provided insight into the observed structure-activity relationships. The optimized compound 21 is a potent TDO2 inhibitor with modest selectivity over indolamine 2,3-dioxygenase 1 (IDO1) and with improved human whole blood stability.

Modulating Antibody-Drug Conjugate Payload Metabolism by Conjugation Site and Linker Modification.

Previous investigations on antibody-drug conjugate (ADC) stability have focused on drug release by linker-deconjugation due to the relatively stable payloads such as maytansines. Recent development of ADCs has been focused on exploring technologies to produce homogeneous ADCs and new classes of payloads to expand the mechanisms of action of the delivered drugs. Certain new ADC payloads could undergo metabolism in circulation while attached to antibodies and thus affect ADC stability, pharmacokinetics, and efficacy and toxicity profiles. Herein, we investigate payload stability specifically and seek general guidelines to address payload metabolism and therefore increase the overall ADC stability. Investigation was performed on various payloads with different functionalities (e.g., PNU-159682 analog, tubulysin, cryptophycin, and taxoid) using different conjugation sites (HC-A118C, LC-K149C, and HC-A140C) on THIOMAB antibodies. We were able to reduce metabolism and inactivation of a broad range of payloads of THIOMAB antibody-drug conjugates by employing optimal conjugation sites (LC-K149C and HC-A140C). Additionally, further payload stability was achieved by optimizing the linkers. Coupling relatively stable sites with optimized linkers provided optimal stability and reduction of payloads metabolism in circulation in vivo.

High-Throughput Cysteine Scanning To Identify Stable Antibody Conjugation Sites for Maleimide- and Disulfide-Based Linkers.

THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technology has enabled the exploration of different attachment sites on the antibody in combination with small molecules, peptides, or proteins to yield antibody conjugates with unique properties. As reported previously ( Shen , B. Q. , et al. ( 2012 ) Nat. Biotechnol. 30 , 184 - 189 ; Pillow , T. H. , et al. ( 2017 ) Chem. Sci. 8 , 366 - 370 ), the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds. High stability of the linkage is usually desired to maximize the delivery of the cargo to the intended target. In the current study, cysteines were individually substituted into every position of the anti-HER2 antibody (trastuzumab), and the stabilities of drug conjugations at those sites were evaluated. We screened a total of 648 THIOMAB antibody-drug conjugates, each generated from a trastuzamab prepared by sequentially mutating non-cysteine amino acids in the light and heavy chains to cysteine. Each THIOMAB antibody variant was conjugated to either maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (MC-vc-PAB-MMAE) or pyridyl disulfide monomethyl auristatin E (PDS-MMAE) using a high-throughput, on-bead conjugation and purification method. Greater than 50% of the THIOMAB antibody variants were successfully conjugated to both MMAE derivatives with a drug to antibody ratio (DAR) of >0.5 and <50% aggregation. The relative in vitro plasma stabilities for approximately 750 conjugates were assessed using enzyme-linked immunosorbent assays, and stable sites were confirmed with affinity-capture LC/MS-based detection methods. Highly stable conjugation sites for the two types of MMAE derivatives were identified on both the heavy and light chains. Although the stabilities of maleimide conjugates were shown to be greater than those of the disulfide conjugates, many sites were identified that were stable for both. Furthermore, in vitro stabilities of selected stable sites translated across different cytotoxic payloads and different target antibodies as well as to in vivo stability.

Analysis of Cloud-Resolving Model Simulations for Scale Dependence of Convective Momentum Transport.

We use 3-D cloud-resolving model (CRM) simulations of two mesoscale convective systems at mid-latitudes and a simple statistical ensemble method to diagnose the scale dependency of convective momentum transport (CMT) and CMT-related properties, and evaluate a parameterization scheme for convection-induced pressure gradient (CIPG) developed by Gregory et al. (GKI97). GKI97 relates CIPG to a constant coefficient multiplied by mass flux and vertical mean wind shear. CRM results show that mass fluxes and CMT exhibit strong scale dependency in temporal evolution and vertical structure. The prevalent understandings of CMT characteristics in terms of upgradient/downgradient transport are applicable to updrafts but not downdrafts across a wide range of grid spacings (4-512 km). For the small-to-median grid spacings (4~64 km), GKI97 reproduces some aspects of CIPG scale dependency except for underestimating the variations of CIPG as grid spacing decreases. However, for large grid spacings (128~512 km), GKI97 might even less adequately parameterize CIPG because it omits the contribution from either the nonlinear shear or buoyancy forcings. Further diagnosis of CRM results suggests that inclusion of nonlinear shear forcing in GKI97 is needed for the large grid spacings, and use of the three-updraft and one downdraft approach proposed in an earlier study may help a modified GKI97 capture more variations of CIPG as grid spacing decreases for the small-to-median grid spacings. Further, the optimal coefficients used in GKI97 seems insensitive to grid spacings, but they might be different for updrafts and downdrafts, for different MCS types, and for zonal and meridional components.

Discovery of Peptidomimetic Antibody-Drug Conjugate Linkers with Enhanced Protease Specificity.

Antibody-drug conjugates (ADCs) have become an important therapeutic modality for oncology, with three approved by the FDA and over 60 others in clinical trials. Despite the progress, improvements in ADC therapeutic index are desired. Peptide-based ADC linkers that are cleaved by lysosomal proteases have shown sufficient stability in serum and effective payload-release in targeted cells. If the linker can be preferentially hydrolyzed by tumor-specific proteases, safety margin may improve. However, the use of peptide-based linkers limits our ability to modulate protease specificity. Here we report the structure-guided discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing linker is hydrolyzed predominantly by cathepsin B while the valine-citrulline dipeptide linker is not. ADCs bearing the nonpeptidic linker are as efficacious and stable in vivo as those with the dipeptide linker. Our results strongly support the application of the peptidomimetic linker and present new opportunities for improving the selectivity of ADCs.

Cathepsin B Is Dispensable for Cellular Processing of Cathepsin B-Cleavable Antibody-Drug Conjugates.

Antibody-drug conjugates (ADC) are designed to selectively bind to tumor antigens via the antibody and release their cytotoxic payload upon internalization. Controllable payload release through judicious design of the linker has been an early technological milestone. Here, we examine the effect of the protease-cleavable valine-citrulline [VC(S)] linker on ADC efficacy. The VC(S) linker was designed to be cleaved by cathepsin B, a lysosomal cysteine protease. Surprisingly, suppression of cathepsin B expression via CRISPR-Cas9 gene deletion or shRNA knockdown had no effect on the efficacy of ADCs with VC(S) linkers armed with a monomethyl auristatin E (MMAE) payload. Mass spectrometry studies of payload release suggested that other cysteine cathepsins can cleave the VC(S) linker. Also, ADCs with a nonprotease-cleavable enantiomer, the VC(R) isomer, mediated effective cell killing with a cysteine-VC(R)-MMAE catabolite generated by lysosomal catabolism. Based on these observations, we altered the payload to a pyrrolo[2,1-c][1,4]benzodiazepine dimer (PBD) conjugate that requires linker cleavage in order to bind its DNA target. Unlike the VC-MMAE ADCs, the VC(S)-PBD ADC is at least 20-fold more cytotoxic than the VC(R)-PBD ADC. Our findings reveal that the VC(S) linker has multiple paths to produce active catabolites and that antibody and intracellular targets are more critical to ADC efficacy. These results suggest that protease-cleavable linkers are unlikely to increase the therapeutic index of ADCs and that resistance based on linker processing is improbable. Cancer Res; 77(24); 7027-37. ©2017 AACR.

Selective Inhibitors of Dual Leucine Zipper Kinase (DLK, MAP3K12) with Activity in a Model of Alzheimer's Disease.

Significant data exists to suggest that dual leucine zipper kinase (DLK, MAP3K12) is a conserved regulator of neuronal degeneration following neuronal injury and in chronic neurodegenerative disease. Consequently, there is considerable interest in the identification of DLK inhibitors with a profile compatible with development for these indications. Herein, we use structure-based drug design combined with a focus on CNS drug-like properties to generate compounds with superior kinase selectivity and metabolic stability as compared to previously disclosed DLK inhibitors. These compounds, exemplified by inhibitor 14, retain excellent CNS penetration and are well tolerated following multiple days of dosing at concentrations that exceed those required for DLK inhibition in the brain.

Identification of Small-Molecule Noncovalent Binders Utilizing SAMDI Technology.

In recent years, the ability to unambiguously identify complex mixtures of analytes with high accuracy and resolving power in a label-free format continues to expand the application of mass spectrometry (MS) in the drug discovery process. This advantage combined with improved instrumentation makes MS suitable for targets with limited alternative assays for high-throughput screening (HTS). We describe a novel screening format using Self-Assembled Monolayers and matrix-assisted laser Desorption Ionization (SAMDI) technology. SAMDI enables affinity capture of a target protein for use in a small-molecule-binding assay format. Subsequent ionization enables the inferred identification of noncovalent compound interactions. SAMDI technology overcomes shot-to-shot variability by uniformly saturating the surface with captured protein, thereby minimizing matrix crystallization "hot spots." Furthermore, the combination with high-resolution matrix-assisted laser desorption/ionization time of flight significantly reduces interference of small-molecule detection from salt, detergent, and matrix. By using a pooled library format, the SAMDI assay can significantly improve the throughput of MS-based screening irrespective of enzyme activity. Finally, we demonstrate binding affinity rank ordering from a pool of compounds that correlates with potency data from a biochemical assay.

From a novel HTS hit to potent, selective, and orally bioavailable KDM5 inhibitors.

A high-throughput screening (HTS) of the Genentech/Roche library identified a novel, uncharged scaffold as a KDM5A inhibitor. Lacking insight into the binding mode, initial attempts to improve inhibitor potency failed to improve potency, and synthesis of analogs was further hampered by the presence of a C-C bond between the pyrrolidine and pyridine. Replacing this with a C-N bond significantly simplified synthesis, yielding pyrazole analog 35, of which we obtained a co-crystal structure with KDM5A. Using structure-based design approach, we identified 50 with improved biochemical, cell potency and reduced MW and lower lipophilicity (LogD) compared with the original hit. Furthermore, 50 showed lower clearance than 9 in mice. In combination with its remarkably low plasma protein binding (PPB) in mice (40%), oral dosing of 50 at 5mg/kg resulted in unbound Cmax ∼2-fold of its cell potency (PC9 H3K4Me3 0.96µM), meeting our criteria for an in vivo tool compound from a new scaffold.