Inhibitor Scaffolds Research Articles

Imidazo[1,2-c]quinazolines as a novel and potent scaffold of α-glucosidase inhibitors: design, synthesis, biological evaluations, and in silico studies

α-Glucosidase inhibition is an approved treatment for type 2 diabetes mellitus (T2DM). In an attempt to develop novel anti-α-glucosidase agents, two series of substituted imidazo[1,2-c]quinazolines, namely 6a–c and 11a–o, were synthesized using a simple, straightforward synthetic routes. These compounds were thoroughly characterized by IR, 1H and 13C NMR spectroscopy, as well as mass spectrometry and elemental analysis. Subsequently, the inhibitory activities of these compounds were evaluated against Saccharomyces cerevisiae α-glucosidase. In present study, acarbose was utilized as a positive control. These imidazoquinazolines exhibited excellent to great inhibitory potencies with IC50 values ranging from 12.44 ± 0.38 μM to 308.33 ± 0.06 μM, which were several times more potent than standard drug with IC50 value of 750.0 ± 1.5 μM. Representatively, compound 11j showed remarkable anti-α-glucosidase potency with IC50 = 12.44 ± 0.38 μM, which was 60.3 times more potent than positive control acarbose. To explore the potential inhibition mechanism, further evaluations including kinetic analysis, circular dichroism, fluorescence spectroscopy, and thermodynamic profile were carried out for the most potent compound 11j. Moreover, molecular docking studies and in silico ADME prediction for all imidazoquinazolines 6a–c and 11a–o were performed to reveal their important binding interactions, as well as their physicochemical and drug-likeness properties, respectively.

Biophysical Rationale for the Selective Inhibition of PTP1B over TCPTP by Nonpolar Terpenoids.

Protein tyrosine phosphatases (PTPs) are emerging drug targets for many diseases, including cancer, autoimmunity, and neurological disorders. A high degree of structural similarity between their catalytic domains, however, has hindered the development of selective pharmacological agents. Our previous research uncovered two unfunctionalized terpenoid inhibitors that selectively inhibit PTP1B over T-cell PTP (TCPTP), two PTPs with high sequence conservation. Here, we use molecular modeling, with supporting experimental validation, to study the molecular basis of this unusual selectivity. Molecular dynamics (MD) simulations suggest that PTP1B and TCPTP share a h-bond network that connects the active site to a distal allosteric pocket; this network stabilizes the closed conformation of the catalytically essential WPD loop, which it links to the L-11 loop and neighboring α3 and α7 helices on the other side of the catalytic domain. Terpenoid binding to either of two proximal C-terminal sites─an α site and a β site─can disrupt the allosteric network; however, binding to the α site forms a stable complex only in PTP1B. In TCPTP, two charged residues disfavor binding at the α site in favor of binding at the β site, which is conserved between the two proteins. Our findings thus indicate that minor amino acid differences at the poorly conserved α site enable selective binding, a property that might be enhanced with chemical elaboration, and illustrate more broadly how minor differences in the conservation of neighboring─yet functionally similar─allosteric sites can affect the selectivity of inhibitory scaffolds (e.g., fragments).

Evaluating Known Zika Virus NS2B-NS3 Protease Inhibitor Scaffolds via In Silico Screening and Biochemical Assays.

The NS2B-NS3 protease (NS2B-NS3pro) is regarded as an interesting molecular target for drug design, discovery, and development because of its essential role in the Zika virus (ZIKV) cycle. Although no NS2B-NS3pro inhibitors have reached clinical trials, the employment of drug-like scaffolds can facilitate the screening process for new compounds. In this study, we performed a combination of ligand-based and structure-based in silico methods targeting two known non-peptide small-molecule scaffolds with micromolar inhibitory activity against ZIKV NS2B-NS3pro by a virtual screening (VS) of promising compounds. Based on these two scaffolds, we selected 13 compounds from an initial library of 509 compounds from ZINC15's similarity search. These compounds exhibited structural modifications that are distinct from previously known compounds yet keep pertinent features for binding. Despite promising outcomes from molecular docking and initial enzymatic assays against NS2B-NS3pro, confirmatory assays with a counter-screening enzyme revealed an artifactual inhibition of the assessed compounds. However, we report two compounds, 9 and 11, that exhibited antiviral properties at a concentration of 50 μM in cellular-based assays. Overall, this study provides valuable insights into the ongoing research on anti-ZIKV compounds to facilitate and improve the development of new inhibitors.

Discovery of new non-covalent and covalent inhibitors targeting SARS-CoV-2 papain-like protease and main protease

Global coronavirus disease 2019 (COVID-19) pandemic still threatens human health and public safety, and the development of effective antiviral agent is urgently needed. The SARS-CoV-2 main protease (Mpro) and papain-like protease (PLpro) are vital proteins in viral replication and promising therapeutic targets. Additionally, PLpro also modulates host immune response by cleaving ubiquitin and interferon-stimulated gene product 15 (ISG15) from ISGylated host proteins. In this report, we identified [1,2]selenazolo[5,4-c]pyridin-3(2H)-one and benzo[d]isothiazol-3(2H)-one as attractive scaffolds of PLpro and Mpro inhibitors. The representative compounds 6c and 7e exhibited excellent PLpro inhibition with percent inhibition of 42.9% and 44.9% at 50 nM, respectively. The preliminary enzyme kinetics experiment and fluorescent labelling experiment results determined that 6c was identified as a covalent PLpro inhibitor, while 7e was a non-covalent inhibitor. Molecular docking and dynamics simulations revealed that 6c and 7e bound to Zn-finger domain of PLpro. Compounds 6c and 7e were also identified to potent Mpro inhibitors, and they exhibited potent antiviral activities in SARS-CoV-2 infected Vero E6 cells, with EC50 value of 3.9 μM and 7.4 μM, respectively. In addition, the rat liver homogenate half-life of 6c and 7e exceeded 24 h. These findings suggest that 6c and 7e are promising led compounds for further development of PLpro/Mpro dual-target antiviral drugs.

CoMFA and CoMSIA Analyses of Novel Chromenone Derivatives as Interleukin 5 Inhibitors

Abstract: Major allergic diseases are caused by an increased accumulation of eosinophils driven by interleukin 5 (IL-5). Chromenone analogues have been demonstrated as a promising scaffold for IL-5 inhibitors to control allergic diseases. Therefore, the molecular interaction of IL-5 inhibitors with biological targets is important for better drug discovery. In this study, we analysed in vitro data using 3D-QSAR (Quantitative Structure-Activity Relationships) modelling to find the IL-5 inhibitory activity and molecular interaction. The results of Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Field Similarity Indices (CoMSIA IB) revealed that the in vitro IL-5 inhibitory activity of these chromenone derivatives exhibited strong correlations with steric and electrostatic factors of the molecules (q2 =0.503, r_ncv^2 = 0.997, and r_bs^2 = 0.989 for CoMFA standard model and q2 =0.623, r_ncv^2 = 0.973, and r_bs^2 = 0.990 for CoMSIA model). 3D-QSAR field contour maps could be generated to identify the important structural features of chromenone analogues relevant to the IL-5 inhibitory activity.

Privileged Scaffolds for Potent and Specific Inhibitors of Mono-ADP-Ribosylating PARPs.

The identification of new targets to address unmet medical needs, better in a personalized way, is an urgent necessity. The introduction of PARP1 inhibitors into therapy, almost ten years ago, has represented a step forward this need being an innovate cancer treatment through a precision medicine approach. The PARP family consists of 17 members of which PARP1 that works by poly-ADP ribosylating the substrate is the sole enzyme so far exploited as therapeutic target. Most of the other members are mono-ADP-ribosylating (mono-ARTs) enzymes, and recent studies have deciphered their pathophysiological roles which appear to be very extensive with various potential therapeutic applications. In parallel, a handful of mono-ARTs inhibitors emerged that have been collected in a perspective on 2022. After that, additional very interesting compounds were identified highlighting the hot-topic nature of this research field and prompting an update. From the present review, where we have reported only mono-ARTs inhibitors endowed with the appropriate profile of pharmacological tools or drug candidate, four privileged scaffolds clearly stood out that constitute the basis for further drug discovery campaigns.

WNTinib is a multi-kinase inhibitor with specificity against β-catenin mutant hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. β-Catenin (CTNNB1)-mutated HCC represents 30% of cases of the disease with no precision therapeutics available. Using chemical libraries derived from clinical multi-kinase inhibitor (KI) scaffolds, we screened HCC organoids to identify WNTinib, a KI with exquisite selectivity in CTNNB1-mutated human and murine models, including patient samples. Multiomic and target engagement analyses, combined with rescue experiments and in vitro and in vivo efficacy studies, revealed that WNTinib is superior to clinical KIs and inhibits KIT/mitogen-activated protein kinase (MAPK) signaling at multiple nodes. Moreover, we demonstrate that reduced engagement on BRAF and p38α kinases by WNTinib relative to several multi-KIs is necessary to avoid compensatory feedback signaling-providing a durable and selective transcriptional repression of mutant β-catenin/Wnt targets through nuclear translocation of the EZH2 transcriptional repressor. Our studies uncover a previously unknown mechanism to harness the KIT/MAPK/EZH2 pathway to potently and selectively antagonize CTNNB1-mutant HCC with an unprecedented wide therapeutic index.

New Scaffolds of Proteasome Inhibitors: Boosting Anticancer Potential by Exploiting the Synergy of In Silico and In Vitro Methodologies.

Cancer is a complex multifactorial disease whose pathophysiology involves multiple metabolic pathways, including the ubiquitin-proteasome system, for which several proteasome inhibitors have already been approved for clinical use. However, the resistance to existing therapies and the occurrence of severe adverse effects is still a concern. The purpose of this study was the discovery of novel scaffolds of proteasome inhibitors with anticancer activity, aiming to overcome the limitations of the existing proteasome inhibitors. Thus, a structure-based virtual screening protocol was developed using the structure of the human 20S proteasome, and 246 compounds from virtual databases were selected for in vitro evaluation, namely proteasome inhibition assays and cell viability assays. Compound 4 (JHG58) was shortlisted as the best hit compound based on its potential in terms of proteasome inhibitory activity and its ability to induce cell death (both with IC50 values in the low micromolar range). Molecular docking studies revealed that compound 4 interacts with key residues, namely with the catalytic Thr1, Ala20, Thr21, Lys33, and Asp125 at the chymotrypsin-like catalytic active site. The hit compound is a good candidate for additional optimization through a hit-to-lead campaign.

Optimization of 4-amino-pyridazin-3(2H)-one as a valid core scaffold for FABP4 inhibitors.

Current clinical research suggests that fatty acid-binding protein 4 inhibitors (FABP4is), which are of biological and therapeutic interest, may show potential in treating cancer and other illnesses. We sought to uncover new structures through the optimization of the previously reported 4-amino and 4-ureido pyridazinone-based series of FABP4is as part of a larger research effort to create more potent FABP4 inhibitors. This led to the identification of 14e as the most potent analogwith IC50 = 1.57 μM, which is lower than the IC50 of the positive control. Advanced modeling investigations and in silico absorption, distribution, metabolism, and excretion - toxicity calculations suggested that 14e represents a potential candidate for in vivo studies such as FABP4i.

Synthesis, biological evaluation and docking studies of silicon incorporated diarylpyrroles as MmpL3 inhibitors: An effective strategy towards development of potent anti-tubercular agents

Growing global demand for new molecules to treat tuberculosis has created an urgent need to develop novel strategies to combat the menace. BM212 related compounds were found to be potent anti-TB agents and they inhibit mycolic acid transporter, MmpL3, a known potent drug target from Mycobacterium tuberculosis. In order to enhance their inhibitory potency, several silicon analogues of diarylpyrroles related to BM212 were designed, synthesized, and evaluated for anti-tubercular activities. In Alamar blue assay, most of the silicon-incorporated compounds were found to be more potent than the parent compound (BM212), against Mycobacterium tuberculosis (MIC = 1.7 μM, H37Rv). Docking results from the crystal structure of MmpL3 and silicon analogues as pharmacophore model also strongly correlate with the biological assays and suggest that the incorporation of silicon in the inhibitor scaffold could enhance their potency by stabilizing the hydrophobic residues at the binding pocket. The best docking hit, compound 12 showed an MIC of 0.1 μM against H37Rv with an acceptable in vitro ADME profile and excellent selectivity index. Overall, the present study indicates that, the designed silicon analogues, especially compound 12 could be a good inhibitor for an intrinsically flexible drug-binding pocket of MmpL3 and has potential for further development as anti-tubercular agents.

Discovery of Cycloalkyl[c]thiophenes as Novel Scaffolds for Hypoxia-Inducible Factor-2α Inhibitors.

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors induced in diverse pathophysiological settings. Inhibition of HIF-2α has become a strategy for cancer treatment since the discovery that small molecules, upon binding into a small cavity of the HIF-2α PAS B domain, can alter its conformation and disturb the activity of the HIF dimer complex. Herein, the design, synthesis, and systematic SAR exploration of cycloalkyl[c]thiophenes as novel HIF-2α inhibitors are described, providing the first chemotype featuring an alkoxy-aryl scaffold. X-ray data confirmed the ability of these inhibitors to induce perturbation of key amino acids by appropriately presenting key pharmacophoric elements in the hydrophobic cavity. Selected compounds showed inhibition of VEGF-A secretion in cancer cells and prevention of Arg1 expression and activity in IL4-stimulated macrophages. Moreover, in vivo target gene modulation was demonstrated with compound 35r. Thus, the disclosed HIF-2α inhibitors represent valuable tools for investigating selective HIF-2α inhibition and its effect on tumor biology.

Crystal structure of [1,2,4]triazolo[4,3-b]pyridazine derivatives as BRD4 bromodomain inhibitors and structure–activity relationship study

BRD4 contains two tandem bromodomains (BD1 and BD2) that recognize acetylated lysine for epigenetic reading, and these bromodomains are promising therapeutic targets for treating various diseases, including cancers. BRD4 is a well-studied target, and many chemical scaffolds for inhibitors have been developed. Research on the development of BRD4 inhibitors against various diseases is actively being conducted. Herein, we propose a series of [1,2,4]triazolo[4,3-b]pyridazine derivatives as bromodomain inhibitors with micromolar IC50 values. We characterized the binding modes by determining the crystal structures of BD1 in complex with four selected inhibitors. Compounds containing [1,2,4] triazolo[4,3-b]pyridazine derivatives offer promising starting molecules for designing potent BRD4 BD inhibitors.

In-silico approach to designing effective antiviral drugs against SARS-CoV-2 and SARS-CoV-1 from reported phytochemicals: a quality improvement study.

Computer-aided drug design by molecular docking, statistical analysis like multiple linear regression (MLR), principal component analysis (PCA), and molecular dynamics studies can emerge as an efficient approach to designing promising core scaffolds for coronavirus medication. The main protease [3-chymotrypsin-like protease (3CLpro)] of severe acute respiratory syndrome coronavirus (SARS-CoV)-1 and SARS-CoV-2 is one of the critical targets for designing and developing broad-spectrum antiviral therapeutic drugs. The main objective of this study was to investigate potential phytochemicals against SARS-CoV-1 and SARS-CoV-2 to ensure effective natural product-induced therapy. In this evaluation, we have selected 40 reported phytochemicals to design efficient core scaffolds that can act as potent inhibitors against the main proteases of SARS-CoV-2 and SARS-CoV-1. We categorized the selected phytochemicals into a more bioavailable and less bioavailable set, considering phytochemical drug likeliness properties. All the selected phytochemicals vigorously interacted with the catalytic dyads His41 and Cys145. Statistical analysis by MLR confirmed their contribution to structural features on binding affinities and PCA analysis for structural activity relationships for their structural pattern recognition to determine the core scaffold inhibitors. We confirmed that 4'-Hydroxyisolonchocarpin and BrussochalconeA were safe and exhibited excellent pharmacological properties. Because 4'-Hydroxyisolonchocarpin and BrussochalconeA are flavonoid derivatives, they exhibit the chalcone's ring. The presence of the reactive α,β-unsaturated system in the chalcone's rings showed different potential pharmacokinetics with an insignificant toxicological profile. Our comprehensive computational and statistical analysis reveals that these selected phytochemicals (4'-Hydroxyisolonchocarpin, BrussochalconeA) can be used to design potential broad antiviral inhibitors against SARS-CoV-2 and SARS-CoV-1.

5-Nitro-3-(2-(4-phenylthiazol-2-yl)hydrazineylidene)indolin-2-one derivatives inhibit HIV-1 replication by a multitarget mechanism of action.

In the effort to identify and develop new HIV-1 inhibitors endowed with innovative mechanisms, we focused our attention on the possibility to target more than one viral encoded enzymatic function with a single molecule. In this respect, we have previously identified by virtual screening a new indolinone-based scaffold for dual allosteric inhibitors targeting both reverse transcriptase-associated functions: polymerase and RNase H. Pursuing with the structural optimization of these dual inhibitors, we synthesized a series of 35 new 3-[2-(4-aryl-1,3-thiazol-2-ylidene)hydrazin-1-ylidene]1-indol-2-one and 3-[3-methyl-4-arylthiazol-2-ylidene)hydrazine-1-ylidene)indolin-2-one derivatives, which maintain their dual inhibitory activity in the low micromolar range. Interestingly, compounds 1a, 3a, 10a, and 9b are able to block HIV-1 replication with EC50 < 20 µM. Mechanism of action studies showed that such compounds could block HIV-1 integrase. In particular, compound 10a is the most promising for further multitarget compound development.

Discovery of Multitarget Inhibitors against Insect Chitinolytic Enzymes via Machine Learning-Based Virtual Screening.

Multitarget inhibitors of insect chitinolytic enzymes are promising sources of green insecticides. Machine learning (ML) is an emerging virtual screening method that can accelerate drug discovery and reduce costs. Taking advantage of the data from our previous high-throughput screening work, we established a strategy integrating ML and molecular docking to screen a large natural product library (17 600 compounds) to identify novel multitarget inhibitors of four chitinolytic enzymes from the insect Ostrinia furnacalis (OfChtI, OfChtII, OfChi-h, and OfHex1). 3,5-Di-O-caffeoylquinic acid and γ-mangostin were identified as inhibitors of all of these enzymes with Ki values at the μM level. Moreover, they showed significant biological activities against lepidopteran pests. Transcriptomic analysis of compound-treated insects suggested the physiological relationship between these compounds and chitinolytic enzymes. This study highlights the potential of ML for insecticide discovery and provides novel and low-cost scaffolds of multitarget inhibitors against insect chitinolytic enzymes as potential pesticide leads.

Photoswitchable Inhibitors to Optically Control Specific Kinase Activity.

Potent and selective small-molecule inhibitors are valuable tools to elucidate the functions of protein kinases within complex signaling networks. Incorporation of a photoswitchable moiety into the inhibitor scaffold offers the opportunity to steer inhibitor potency with temporal precision, while the challenge of selective inhibition can often be addressed by employing a chemical genetic approach, termed the analog-sensitive method. Here, we combine the perks of these two approaches and report photoswitchable azopyrazoles to target calcium-dependent protein kinase 1 (CDPK1) from Toxoplasma gondii, a kinase naturally susceptible to analog-sensitive kinase inhibitors due to its glycine gatekeeper residue. The most promising azopyrazoles display favorable photochemical properties, thermal stability, and a substantial difference in IC50 values between both photostationary states. Consequently, the CDPK1 kinase reaction can be controlled dynamically and reversibly by applying light of different wavelengths. Inhibition of CDPK1 by the azopyrazoles drastically relies on the nature of the gatekeeper residue as a successive increase in gatekeeper size causes a concurrent loss of inhibitory activity. Furthermore, two photoswitchable inhibitors exhibit activity against T. gondii and Cryptosporidium parvum infection in a cell culture model, making them a promising addition to the toolbox for dissecting the role of CDPK1 in the infectious cycle with high temporal control. Overall, this work merges the benefits of the analog-sensitive approach and photopharmacology without compromising inhibitory potency and thus holds great promise for application to other protein kinases in the future.

Design and Development of COX-II Inhibitors: Current Scenario and Future Perspective.

Innate inflammation beyond a threshold is a significant problem involved in cardiovascular diseases, cancer, and many other chronic conditions. Cyclooxygenase (COX) enzymes are key inflammatory markers as they catalyze prostaglandins production and are crucial for inflammation processes. While COX-I is constitutively expressed and is generally involved in "housekeeping" roles, the expression of the COX-II isoform is induced by the stimulation of different inflammatory cytokines and also promotes the further generation of pro-inflammatory cytokines and chemokines, which affect the prognosis of various diseases. Hence, COX-II is considered an important therapeutic target for drug development against inflammation-related illnesses. Several selective COX-II inhibitors with safe gastric safety profiles features that do not cause gastrointestinal complications associated with classic anti-inflammatory drugs have been developed. Nevertheless, there is mounting evidence of cardiovascular side effects from COX-II inhibitors that resulted in the withdrawal of market-approved anti-COX-II drugs. This necessitates the development of COX-II inhibitors that not only exhibit inhibit potency but also are free of side effects. Probing the scaffold diversity of known inhibitors is vital to achieving this goal. A systematic review and discussion on the scaffold diversity of COX inhibitors are still limited. To address this gap, herein we present an overview of chemical structures and inhibitory activity of different scaffolds of known COX-II inhibitors. The insights from this article could be helpful in seeding the development of next-generation COX-II inhibitors.

Exploration of natural compounds against the human mpox virus DNA-dependent RNA polymerase in silico

BackgroundLast year, the human monkeypox virus (hMPXV) emerged as an alarming threat to the community, with a detectable outbreak outside the African continent for the first time. According to The American Centers for Disease Control and Prevention (CDC), the virus is reported globally, with 86,746 confirmed cases (until April 08, 2023). DNA-dependent RNA polymerase (DdRp) is an essential protein for viral replication; hence it is a promising drug target for developing antiviral drugs against DNA viruses. Therefore, this study was conducted to search for natural compounds that could provide scaffolds for RNA polymerase inhibitors. MethodsIn this study, the DdRp structure of hMPXV was modeled and used to screen the natural compounds database (COCONUT). The virtual screening revealed 15 compounds able to tightly bind to the active site of the DdRp (binding energies less than −7.0 kcal/mol) compared to the physiological nucleotide, guanosine triphosphate (GTP). Molecular dynamics simulation was then performed on the top four hits and compared to GTP ResultsThe results revealed the potential of four compounds (comp289, comp295, comp441, and comp449) in binding the hMPXV DdRp active site with a comparable binding affinity (−17.06 ± 2.96, −11.6 ± 5.34, −14.85 ± 2.66, and −10.79 ± 4.49 kcal/mol) with GTP (−21.03 ± 7.55 kcal/mol) ConclusionThese findings may also pave the way for developing new hMPXV inhibitors based on natural product scaffolds. These results need further experimental validation but promising as it was validated by unbiased all-atom MD simulations and binding free energy calculations.

Sequence and Structural Motifs Controlling the Broad Substrate Specificity of the Mycobacterial Hormone-Sensitive Lipase LipN.

Mycobacterium tuberculosis has a complex life cycle transitioning between active and dormant growth states depending on environmental conditions. LipN (Rv2970c) is a conserved mycobacterial serine hydrolase with regulated catalytic activity at the interface between active and dormant growth conditions. LipN also catalyzes the xenobiotic degradation of a tertiary ester substrate and contains multiple conserved motifs connected with the ability to catalyze the hydrolysis of difficult tertiary ester substrates. Herein, we expanded a library of fluorogenic ester substrates to include more tertiary and constrained esters and screened 33 fluorogenic substrates for activation by LipN, identifying its unique substrate signature. LipN preferred short, unbranched ester substrates, but had its second highest activity against a heteroaromatic five-membered oxazole ester. Oxazole esters are present in multiple mycobacterial serine hydrolase inhibitors but have not been tested widely as ester substrates. Combined structural modeling, kinetic measurements, and substitutional analysis of LipN showcased a fairly rigid binding pocket preorganized for catalysis of short ester substrates. Substitution of diverse amino acids across the binding pocket significantly impacted the folded stability and catalytic activity of LipN with two conserved motifs (HGGGW and GDSAG) playing interconnected, multidimensional roles in regulating its substrate specificity. Together this detailed substrate specificity profile of LipN illustrates the complex interplay between structure and function in mycobacterial hormone-sensitive lipase homologues and indicates oxazole esters as promising inhibitor and substrate scaffolds for mycobacterial hydrolases.

Utilization of Supervised Machine Learning to Understand Kinase Inhibitor Toxophore Profiles.

There have been more than 70 FDA-approved drugs to target the ATP binding site of kinases, mainly in the field of oncology. These compounds are usually developed to target specific kinases, but in practice, most of these drugs are multi-kinase inhibitors that leverage the conserved nature of the ATP pocket across multiple kinases to increase their clinical efficacy. To utilize kinase inhibitors in targeted therapy and outside of oncology, a narrower kinome profile and an understanding of the toxicity profile is imperative. This is essential when considering treating chronic diseases with kinase targets, including neurodegeneration and inflammation. This will require the exploration of inhibitor chemical space and an in-depth understanding of off-target interactions. We have developed an early pipeline toxicity screening platform that uses supervised machine learning (ML) to classify test compounds' cell stress phenotypes relative to a training set of on-market and withdrawn drugs. Here, we apply it to better understand the toxophores of some literature kinase inhibitor scaffolds, looking specifically at a series of 4-anilinoquinoline and 4-anilinoquinazoline model libraries.