Discovery of Novel Diarylamide N-Containing Heterocyclic Derivatives as New Tubulin Polymerization Inhibitors with Anti-Cancer Activity

Tubulin has been regarded as an attractive and successful molecular target in cancer therapy and drug discovery. Vicinal diaryl is a simple scaffold found in many colchicine site tubulin inhibitors, which is also an important pharmacophoric point of tubulin binding and anti-cancer activity. As the continuation of our research work on colchicine binding site tubulin inhibitors, we designed and synthesized a series of diarylamide N-containing heterocyclic derivatives by the combination of vicinal diaryl core and N-containing heterocyclic skeletons into one hybrid though proper linkers. Among of these compounds, compound 15b containing a 5-methoxyindole group exhibited the most potent inhibitory activity against the tested three human cancer cell lines (MGC-803, PC-3 and EC-109) with IC₅₀ values of 1.56 μM, 3.56 μM and 14.5 μM, respectively. Besides, the SARs of these compounds were preliminarily studied and summarized. The most active compound 15b produced the inhibition of tubulin polymerization in a dose-dependent manner and caused microtubule network disruption in MGC-803 cells. Therefore, compound 15b was identified as a novel tubulin polymerization inhibitor targeting the colchicine binding site. In addition, the results of molecular docking also suggested compound 15b could tightly bind into the colchicine binding site of β-tubulin.     

Competitive mass spectrometry binding assay for characterization of three binding sites of tubulin

Tubulin is an attractive and established target for anticancer therapy. To date, the only method to determine the binding of inhibitor to tubulin has been competitive radioligand binding assays. We developed a non‐radioactive mass spectrometry (MS) binding assay to study the tubulin binding of colchicine, vinblastine and paclitaxel and to identify which of these three binding sites that a novel inhibitor binds. The method involves a very simple step of separating the unbound ligand from macromolecules using ultrafiltration. The unbound ligand in the filtrate can be accurately determined using highly sensitive and specific liquid chromatography tandem mass spectrometry (LC‐MS/MS) method using multiple reaction monitoring (MRM) mode. The assay was validated using podophyllotoxin, vincristine and docetaxel, drugs that compete to the colchicine‐, vinblastine‐ and paclitaxel‐binding sites in tubulin, respectively. This competitive binding assay allowed the reliable detection of interactions of these drugs with three binding sites on tubulin. This method was subsequently applied to determine the tubulin‐binding site of 4‐substituted methoxylbenzoyl‐aryl‐thiazoles (SMART‐H), a potent antitubulin agent developed in our laboratory. The results indicated that SMART‐H specifically and reversibly bound only to the colchicine‐binding site, but not to vinblastine‐ or paclitaxel sites. This new non‐radioligand binding method to determine the binding site on tubulin will function as a useful tool to study the binding sites of tubulin inhibitors. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis and biological evaluation of indole-2-carbohydrazides and thiazolidinyl-indole-2-carboxamides as potent tubulin polymerization inhibitors

A new series of N’-(substituted phenyl)-5-chloro/iodo-3-phenyl-1H-indole-2-carbohydrazide (5, 6) and N-[2-(substituted phenyl)-4-oxo-1,3-thiazolidin-3-yl]-5-iodo/chloro-3-phenyl-1H-indole-2-carboxamide (7, 8) derivatives were synthesized and evaluated for their anticancer properties. Compounds 5a and 6b, selected as prototypes by the National Cancer Institute for screening against the full panel of 60 human tumor cell lines at a minimum of five concentrations at 10-fold dilutions, demonstrated remarkable antiproliferative activity against leukemia, non-small cell lung cancer, colon cancer, central nervous system (CNS) cancer, melanoma, ovarian cancer, renal cancer, and breast cancer (MCF-7) cell lines with GI₅₀ values < 0.4 μM. A subset of the compounds was then tested for their potential to inhibit tubulin polymerization. Compounds 6f and 6g showed significant cytotoxicity at the nM level on MCF-7 cells and exhibited significant inhibitory activity on tubulin assembly and colchicine binding at about the same level as combretastatin A-4. Finally, docking calculations were performed to identify the binding mode of these compounds. Group 5 and 6 compounds interacted with the colchicine binding site through hydrophobic interactions similar to those of colchicine. These compounds with antiproliferative activity at high nanomolar concentration can serve as scaffolds for the design of novel microtubule targeting agents.     

In Vitro Anti-Tubulin Activity on MCF10A Cell Line and In Silico Rigid/Semiflexible-Residues Docking,of Two Lignans from Bursera Fagaroides var. Fagaroides

Podophyllotoxins are natural lignans with known cytotoxic activity on several cell lines. The structural basis for their actions is mainly by the aryltetralin-lignan skeleton. Authors have proposed a cytotoxic mechanism of podophyllotoxins through the topoisomerase-II inhibition activity; however, several studies have also suggested that podophyllotoxins can inhibit the microtubules polymerization. In this work, the two possible mechanisms of action of two previously isolated compounds from the stem bark of Bursera fagaroides var. fagaroides: acetylpodophyllotoxin (1) and 5’-desmethoxydeoxypodophyllotoxin (2), was analyzed. An in vitro anti-tubulin epifluorescence on the MCF10A cell line and enzymatic topoisomerase II assays were performed. The binding affinities of compounds 1 and 2 in the colchicine binding site of tubulin by using rigid- and semiflexible-residues were calculated and compared using in silico docking methods. The two lignans were active by the in vitro anti-tubulin assay but could not inhibit TOP2 activity. In the in silico analysis, the binding modes of compounds into both rigid- and semiflexible-residues of tubulin were predicted, and only for the semiflexible docking method, a linear correlation between the dissociation constant and IC₅₀ previously reported was found. Our results suggest that a simple semiflexible-residues modification in docking methods could provide an in vitro correlation when analyzing very structurally similar compounds.     

Uracil-coumarin based hybrid molecules as potent anti-cancer and anti-bacterial agents

In the current report, we have rationally designed a series of uracil-coumarin based bifunctional molecular hybrids roped by 1,2,3-triazole moiety. The designed compounds were synthesized and tested against a panel of six human cancer cell lines namely Colo-205, MCF-7, A-549, PA-1, PC-3 and Hela cells by Sulforhodamine B assay. The results indicated that the hybrid molecules can specifically inhibit the MCF-7 cancer cell proliferation amongst which A-2 was found to be most potent hybrid (GI₅₀ = 1.55 µM) with fluorine atom as R with two carbon chain length between triazole and coumarin moieties. Cell cycle analysis revealed that A-2 significantly arrest the G2/M phase to inhibit proliferation of MCF-7 cells. Due to its mitotic arrest, A-2 was further analyzed to predict its various binding interactions within the active site of tubulin, which revealed its best binding pattern within the vinblastine binding site. In addition to this, antibacterial potential of all the synthetics was also evaluated which resulted in two hit lead molecules A-2 (MIC = 11.7 μg/mL) and A-3 (MIC = 7.23 μg/mL) that can significantly inhibit the bacterial strain Staphylococcus aureus comparable to that of standard drug levofloxacin (MIC = 3.12 μg/mL). Binding interactions within the active site of dihydrofolate reductase (DHFR) were also streamlined by using molecular docking studies. Overall studies revealed some interesting features of synthetics to be active which stated that, the compounds with electronegative atom on R and compounds with two carbon chain length between triazole and coumarin showed best results.     

Tubulin is a molecular target of the Wnt-activating chemical probe

Background

In drug discovery research, cell-based phenotypic screening is an essential method for obtaining potential drug candidates. Revealing the mechanism of action is a key step on the path to drug discovery. However, elucidating the target molecules of hit compounds from phenotypic screening campaigns remains a difficult and troublesome process. Simple and efficient methods for identifying the target molecules are essential.

Results

2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) was identified as a senescence inducer from a phenotypic screening campaign. The compound is widely used as a Wnt agonist, although its target molecules remain to be clarified. To identify its target proteins, we compared a series of cellular assay results for the compound with our pathway profiling database. The database comprises the activities of compounds from simple assays of cellular reporter genes and cellular proliferations. In this database, compounds were classified on the basis of statistical analysis of their activities, which corresponded to a mechanism of action by the representative compounds. In addition, the mechanisms of action of the compounds of interest could be predicted using the database. Based on our database analysis, the compound was anticipated to be a tubulin disruptor, which was subsequently confirmed by its inhibitory activity of tubulin polymerization.

Conclusion

These results demonstrate that tubulin is identified for the first time as a target molecule of the Wnt-activating small molecule and that this might have misled the conclusions of some previous studies. Moreover, the present study also emphasizes that our pathway profiling database is a simple and potent tool for revealing the mechanisms of action of hit compounds obtained from phenotypic screenings and off targets of chemical probes.

     

Design,synthesis and biological evaluation of isoxazole-naphthalene derivatives as anti-tubulin agents

In this study, a novel series of isoxazole-naphthalene derivatives as tubulin polymerization inhibitors were designed, synthesized and evaluated for their anti-proliferative activities against human breast cancer cell line MCF-7. Most of the synthesized compounds exhibited moderate to potent antiproliferative activity (IC₅₀ < 10.0 μM), as compared to cisplatin (15.24 ± 1.27 μM). Among them, compound 5j containing 4-ethoxy substitution at phenyl ring was found to be the most active compound with IC₅₀ value of 1.23 ± 0.16 μM. Mechanistic studies revealed that compound 5j arrested cell cycle at G2/M phase and induces apoptosis. Furthermore, in vitro tubulin polymerization assay showed that compound 5j displayed better inhibition activity on tubulin polymerization (IC₅₀ = 3.4 μM) than colchicine (IC₅₀ = 7.5 μM). Molecular docking study also revealed that compound 5j binds to the colchicine binding site of tubulin.     

Identification of potential inhibitors against nuclear Dam1 complex subunit Ask1 of Candida albicans using virtual screening and MD simulations

Identification of hit compounds against specific target form the starting point for a drug discovery program. A consistent decline of new chemical entities (NCEs) in recent years prompted a challenge to explore newer approaches to discover potential hit compounds that in turn can be converted into leads, and ultimately drug with desired therapeutic efficacy. The vast amount of omics and activity data available in public databases offers an opportunity to identify novel targets and their potential inhibitors. State of the art in silico methods viz., clustering of compounds, virtual screening, molecular docking, MD simulations and MMPBSA calculations were employed in a pipeline to identify potential ‘hits’ against those targets as well whose structures, as of now, could only predict through threading approaches. In the present work, we have started from scratch, amino acid sequence of target and compounds retrieved from PubChem compound database, modeled it in such a way that led to the identification of possible inhibitors of Dam1 complex subunit Ask1 of Candida albicans. We also propose a ligand based binding site determination approach. We have identified potential inhibitors of Ask1 subunit of a Dam1 complex of C. albicans, which is required to prevent precocious spindle elongation in pre-mitotic phases. The proposed scheme may aid to find virtually potential inhibitors of other unique targets against candida.     

Chirality and Spectroscopic Changes Induced by the Recognition of Ethyl 5-Amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl Carbamate Analogs by Tubulin

The two chiral isomers of ethyl 5-amino-2-methyl-l,2-di-hydro-3-phenylpyrido[3,4-b]pyrazin-7-yl carbamate, NSC 613863 (R-isomer)-(+) and NSC 613862 (S-isomer)-(-) (CI980) and the three achiral analogs NSC 330770 (2-de-methylated analog A), NSC 337238 and C179 are potent microtubule inhibitors. These ligands interact with tubulin overlapping the colchicine binding site. This study addresses the effects of recognition by tubulin on the conformational properties of the ligands. The near-UV CD (circular dichroism) band of the R-isomer was suppressed, while that of the S-isomer displayed a more intense negative band when these compounds were bound to tubulin. Interestingly, the three other initially achiral compounds became optically active upon binding to tubulin; particularly, analog A exhibited a negative CD band on the order of magnitude of chiral compounds. The CD changes are reversible, highly specific and actually permit measuring the binding of the ligands by tubulin. These CD changes are compatible with the deformation of the bound ligands. Fluorescence emission is strongly enhanced and blue shifted upon binding to tubulin. Water among a solvent series had a specific solvent effect, except on the 1,2-dehydro analogs NSC 337238 and C179, suggesting hydrogen bonding to Nl. The emission of tubulin-bound R-isomer, S-isomer and analog A could be mimicked by solvent viscosity, supporting the notion that the intramolecular rotation between the pyridopyrazine and phenyl rings is frozen upon binding.     

Indole-derived chalcones as anti-dermatophyte agents: In vitro evaluation and in silico study

A series of indole-derived methoxylated chalcones were described as anti-dermatophyte agents. The in vitro antifungal susceptibility testing against different dermatophytes revealed that most of compounds had potent activity against the dermatophyte strains. In particular, the 4-ethoxy derivative 4d with MIC values of 0.25−2 μg/ml was the most potent compound against Trichophyton interdigitale, Trichophyton veruccosum and Microsporum fulvum. Moreover, the 4-butoxy analog 4i displaying MIC values in the range of 1−16 μg/ml had the highest inhibitory activity against Trichophyton mentagrophytes, Microsporum canis, and Arthroderma benhamiae. To predict whether the synthesized compounds interact with tubulin binding site of dermatophytes, the 3D-structure of target protein was modeled by homology modeling and then used for molecular docking and molecular dynamics (MD) simulation studies. Docking simulation revealed that the promising compound 4d can properly bind with tubulin. The molecular dynamics analysis showed that interactions of compound 4d with the active site of target protein have binding stability throughout MD simulation. The results of this study could utilize in the design of more effective antifungal drugs with tubulin inhibition mechanism against keratinophilic fungi.     

Design,synthesis and biological evaluation of novel 2-phenyl-4,5,6,7-tetrahydro-1H-​indole derivatives as potential anticancer agents and tubulin polymerization inhibitors

A new series of 2-phenyl-4,5,6,7-tetrahydro-1H-?indole derivatives as tubulin polymerization inhibitors were synthesized and evaluated for the anti-proliferative activities. All newly prepared compounds were tested for their antiproliferative activity in vitro on the human breast cancer cell line (MCF-7) and human lung adenocarcinoma cell line (A549). Among them, compound 7b with a 4-methoxyl substituent at the phenylhydrazone moiety exhibited the most potent anticancer activity against MCF-7 and A549 with IC₅₀ values of 1.77 ± 0.37 and 3.75 ± 0.11 μM, respectively. Interestingly, 7b displayed significant selectivity in inhibiting cancer cells over LO2 (normal human liver cells). Further mechanism studies revealed that 7b significantly arrested cell cycle at G2/M phase and induced apoptosis in a dose-dependent manner. Additionally, 7b effectively inhibited tubulin polymerization with an inhibitory manner similar to that of colchicine. Furthermore, molecular docking study suggested that 7b had high binding affinities for the colchicine binding pocket of tubulin. Hence, this study demonstrates for the first time that tetrahydroindole can be used as a functional group for the design and development of new tubulin polymerization inhibitors.     

Probing the interaction of HTI-286 with tubulin using a stilbene analogue

HTI-286 is a synthetic analogue of the natural product hemiasterlin. HTI-286 is a potent antitumor agent that induces tubulin oligomerization. To investigate the binding stoichiometry and the binding site during this ligand-induced tubulin association, we synthesized an analogue of HTI-286 containing the chromophore stilbene. Using the distinct absorbance of the stilbene analogue, we determined the amounts of inhibitors bound to different tubulin oligomers by analytical ultracentrifugation. Herein we describe our findings based on these experiments. At the ratio of inhibitor to protein equal to or greater than 1, the stilbene analogue induces oligomerization of tubulin to a ring structure. The binding stoichiometry in the ring is one inhibitor per tubulin monomer (defined as an alpha/beta-heterodimer). At the ratio of inhibitor to protein less than 1, tubulin forms multiple intermediates, with the binding stoichiometry less than one inhibitor per tubulin monomer for all intermediates. The stable complex between the inhibitor and tubulin monomer was not detected under our experimental conditions. The binding site of the stilbene analogue does not overlap with the classic tubulin-binding agent, colchicine.     

SYNTHESIS AND EVALUATION OF 2-DIAZO-3,3,3-TRIFLUOROPROPANOYL DERIVATIVES OF COLCHICINE AND PODOPHYLLOTOXIN AS PHOTOAFFINITY LABELS: REACTIVITY, PHOTOCHEMISTRY, AND TUBULIN BINDING

Derivatives of the tubulin polymerization inhibitors colchicine and podophyllotoxin bearing the photoreactive 2-diazo-3,3,3-trifluoropropanoyl (DTFP) group were synthesized for evaluation as potential photoaffinity labels of the tubulin binding site. All labels were assayed for their ability to inhibit tubulin polymerization, and N-DTFP-deacetylthiocolchicine was shown to competitively inhibit tubulin-colchicine binding with a Ki of 4-5 microM. The tubulin off-rate of this analog was similar to that of podophyllotoxin, rather than to the relatively irreversibly bound colchicine. Photochemical solvent insertion reactions of the labels were investigated. Radioactive samples of the two most active labels were prepared and used in initial protein-labeling experiments, during which the fractional occupancy of tubulin and extent of covalent incorporation were determined. A rearrangement of DTFP amides was encountered which is relevant to the utility of this moiety for use in synthesis of photoaffinity labels.     

Pharmacoinformatics exploration of polyphenol oxidases leading to novel inhibitors by virtual screening and molecular dynamic simulation study

Polyphenol oxidases (PPOs)/tyrosinases are metal-dependent enzymes and known as important targets for melanogenesis. Although considerable attempts have been conducted to control the melanin-associated diseases by using various inhibitors. However, the exploration of the best anti-melanin inhibitor without side effect still remains a challenge in drug discovery. In present study, protein structure prediction, ligand-based pharmacophore modeling, virtual screening, molecular docking and dynamic simulation study were used to screen the strong novel inhibitor to cure melanogenesis. The 3D structures of PPO1 and PPO2 were built through homology modeling, while the 3D crystal structures of PPO3 and PPO4 were retrieved from PDB. Pharmacophore modeling was performed using LigandScout 3.1 software and top five models were selected to screen the libraries (2601 of Aurora and 727, 842 of ZINC). Top 10 hit compounds (C1-10) were short-listed having strong binding affinities for PPO1-4. Drug and synthetic accessibility (SA) scores along with absorption, distribution, metabolism, excretion and toxicity (ADMET) assessment were employed to scrutinize the best lead hit. C4 (name) hit showed the best predicted SA score (5.75), ADMET properties and drug-likeness behavior among the short-listed compounds. Furthermore, docking simulations were performed to check the binding affinity of C1-C10 compounds against target proteins (PPOs). The binding affinity values of complex between C4 and PPOs were higher than those of other complexes (−11.70, −12.1, −9.90 and −11.20 kcal/mol with PPO1, PPO2, PPO3, or PPO4, respectively). From comparative docking energy and binding analyses, PPO2 may be considered as better target for melanogenesis than others. The potential binding modes of C4, C8 and C10 against PPO2 were explored using molecular dynamics simulations. The root mean square deviation and fluctuation (RMSD/RMSF) graphs results depict the significance of C4 over the other compounds. Overall, bioactivity and ligand efficiency profiles suggested that the proposed hit may be more effective inhibitors for melanogenesis.     

Free energy calculations on the binding of colchicine and its derivatives with the alpha/beta-tubulin isoforms

Tubulin is the target for numerous small molecule ligands which alter microtubule dynamics leading to cell cycle arrest and apoptosis. Many of these ligands are currently used clinically for the treatment of several types of cancer, and they bind to one of three distinct binding sites within beta-tubulin (paclitaxel, vinca, and colchicine), all of which have been identified crystallographically. Unfortunately, serious side effects always accompany chemotherapy since these drugs bind to tubulin indiscriminately, leading to the death of both cancerous and healthy cells. However, the existence and distribution of divergent tubulin isoforms provide a platform upon which we may build novel chemotherapeutic drugs that can differentiate between different cell types and therefore reduce undesirable side effects. We report results of computational analysis that aims at predicting differences between the binding energies of a family of colchicine derivatives against 10 human alpha/beta-tubulin isoforms. Free energy perturbation method has been used in our calculations and the results provide a proof of principle by indicating significant differences both among the derivatives and between tubulin isoforms.     

Identification of a nanomolar affinity α-synuclein fibril imaging probe by ultra-high throughput in silico screening

Small molecules that bind with high affinity and specificity to fibrils of the α-synuclein (αS) protein have the potential to serve as positron emission tomography (PET) imaging probes to aid in the diagnosis of Parkinson''s disease and related synucleinopathies. To identify such molecules, we employed an ultra-high throughput in silico screening strategy using idealized pseudo-ligands termed exemplars to identify compounds for experimental binding studies. For the top hit from this screen, we used photo-crosslinking to confirm its binding site and studied the structure–activity relationship of its analogs to develop multiple molecules with nanomolar affinity for αS fibrils and moderate specificity for αS over Aβ fibrils. Lastly, we demonstrated the potential of the lead analog as an imaging probe by measuring binding to αS-enriched homogenates from mouse brain tissue using a radiolabeled analog of the identified molecule. This study demonstrates the validity of our powerful new approach to the discovery of PET probes for challenging molecular targets.     

Design,synthesis, and biological evaluation of diarylpyrazole derivatives as antitumor agents targeting microtubules

A new series of novel diarylpyrazole derivatives as microtubule destabilizers were synthesized and evaluated for the anti-proliferative activities. Anti-proliferative assays were performed on the human cervix adenocarcinoma cell line (HeLa) and human gastric adenocarcinoma cell line (SGC-7901), and the compound 9s containing indole ring showed great anti-proliferative activity against HeLa cells with IC₅₀ value of 1.9 ± 0.11 μM. Further biological studies showed that 9s was able to inhibit tubulin polymerization, disrupt the cytoskeleton, block the cell cycle in the G2/M phase, and induce cell apoptosis in a concentration-dependent manner. In addition, the results of molecular docking studies showed that compound 9s could bind tightly to the colchicine binding site of tubulin through hydrogen bonding interaction. These preliminary results recommend that compound 9s is likely to be a microtubule destabilizer that deserves further investigation.     

In Silico Prediction,Molecular Docking and Dynamics Studies of Steroidal Alkaloids of Holarrhena pubescens Wall. ex G. Don to Guanylyl Cyclase C: Implications in Designing of Novel Antidiarrheal Therapeutic Strategies

The binding of heat stable enterotoxin (STa) secreted by enterotoxigenic Escherichia coli (ETEC) to the extracellular domain of guanylyl cyclase c (ECD_GC-C) causes activation of a signaling cascade, which ultimately results in watery diarrhea. We carried out this study with the objective of finding ligands that would interfere with the binding of STa on ECD_GC-C. With this view in mind, we tested the biological activity of a alkaloid rich fraction of Holarrhena pubescens against ETEC under in vitro conditions. Since this fraction showed significant antibacterial activity against ETEC, we decided to test the screen binding affinity of nine compounds of steroidal alkaloid type from Holarrhena pubescens against extracellular domain (ECD) by molecular docking and identified three compounds with significant binding energy. Molecular dynamics simulations were performed for all the three lead compounds to establish the stability of their interaction with the target protein. Pharmacokinetics and toxicity profiling of these leads demonstrated that they possessed good drug-like properties. Furthermore, the ability of these leads to inhibit the binding of STa to ECD was evaluated. This was first done by identifying amino acid residues of ECD_GC-C binding to STa by protein–protein docking. The results were matched with our molecular docking results. We report here that holadysenterine, one of the lead compounds that showed a strong affinity for the amino acid residues on ECD_GC-C, also binds to STa. This suggests that holadysenterine has the potential to inhibit binding of STa on ECD and can be considered for future study, involving its validation through in vitro assays and animal model studies.     

Virtual screening of B-Raf kinase inhibitors: A combination of pharmacophore modelling,molecular docking, 3D-QSAR model and binding free energy calculation studies

B-Raf kinase has been identified as an important target in recent cancer treatment. In order to discover structurally diverse and novel B-Raf inhibitors (BRIs), a virtual screening of BRIs against ZINC database was performed by using a combination of pharmacophore modelling, molecular docking, 3D-QSAR model and binding free energy (ΔG_bind) calculation studies in this work. After the virtual screening, six promising hit compounds were obtained, which were then tested for inhibitory activities of A375 cell lines. In the result, five hit compounds show good biological activities (IC₅₀ < 50 μM). The present method of virtual screening can be applied to find structurally diverse inhibitors, and the obtained five structurally diverse compounds are expected to develop novel BRIs.     

Virtual screening of dipeptidyl peptidase-4 inhibitors using quantitative structure–activity relationship-based artificial intelligence and molecular docking of hit compounds

Dipeptidyl peptidase-4 (DPP-4) inhibitors are becoming an essential drug in the treatment of type 2 diabetes mellitus; however, some classes of these drugs exert side effects, including joint pain and pancreatitis. Studies suggest that these side effects might be related to secondary inhibition of DPP-8 and DPP-9. In this study, we identified DPP-4-inhibitor hit compounds selective against DPP-8 and DPP-9. We built a virtual screening workflow using a quantitative structure–activity relationship (QSAR) strategy based on artificial intelligence to allow faster screening of millions of molecules for the DPP-4 target relative to other screening methods. Five regression machine learning algorithms and four classification machine learning algorithms were applied to build virtual screening workflows, with the QSAR model applied using support vector regression (R²_pred 0.78) and the classification QSAR model using the random forest algorithm with 92.2% accuracy. Virtual screening results of > 10 million molecules obtained 2 716 hits compounds with a pIC₅₀ value of > 7.5. Additionally, molecular docking results of several potential hit compounds for DPP-4, DPP-8, and DPP-9 identified CH0002 as showing high inhibitory potential against DPP-4 and low inhibitory potential for DPP-8 and DPP-9 enzymes. These results demonstrated the effectiveness of this technique for identifying DPP-4-inhibitor hit compounds selective for DPP-4 and against DPP-8 and DPP-9 and suggest its potential efficacy for applications to discover hit compounds of other targets.