A transcriptomics-based drug repositioning approach to identify drugs with similar activities for the treatment of muscle pathologies in spinal muscular atrophy (SMA) models.

Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder caused by the reduction of survival of motor neuron (SMN) protein levels. Although three SMN-augmentation therapies are clinically approved that significantly slow down disease progression, they are unfortunately not cures. Thus, complementary SMN-independent therapies that can target key SMA pathologies and that can support the clinically approved SMN-dependent drugs are the forefront of therapeutic development. We have previously demonstrated that prednisolone, a synthetic glucocorticoid (GC) improved muscle health and survival in severe Smn-/-;SMN2 and intermediate Smn2B/- SMA mice. However, long-term administration of prednisolone can promote myopathy. We thus wanted to identify genes and pathways targeted by prednisolone in skeletal muscle to discover clinically approved drugs that are predicted to emulate prednisolone's activities. Using an RNA-sequencing, bioinformatics, and drug repositioning pipeline on skeletal muscle from symptomatic prednisolone-treated and untreated Smn-/-; SMN2 SMA and Smn+/-; SMN2 healthy mice, we identified molecular targets linked to prednisolone's ameliorative effects and a list of 580 drug candidates with similar predicted activities. Two of these candidates, metformin and oxandrolone, were further investigated in SMA cellular and animal models, which highlighted that these compounds do not have the same ameliorative effects on SMA phenotypes as prednisolone; however, a number of other important drug targets remain. Overall, our work further supports the usefulness of prednisolone's potential as a second-generation therapy for SMA, identifies a list of potential SMA drug treatments and highlights improvements for future transcriptomic-based drug repositioning studies in SMA.

Effective α-glycosidase inhibitors based on polyphenolic benzothiazole heterocycles.

α-Glycosidase inhibition is one of the main approaches to treat Diabetes mellitus. Polyphenolic moieties are known to be responsible for yielding exhibit potent α-glycosidase inhibitory effects. In addition, compounds containing benzothiazole and Schiff base functionalities were previously reported to show α-glycosidase inhibition. In this paper, the synthesis of seven new phloroglucinol-containing benzothiazole Schiff base derivatives through the reaction of 6-substituted-2-aminobenzothiazole compounds with 2,4,6-trihydroxybenzaldehyde using acetic acid as a catalyst was reported. The synthesized compounds were characterized using spectroscopic methods such as FT-IR, 1H NMR, 13C NMR, and elemental analysis. The synthesized compounds were evaluated for their inhibitory effects on α-glycosidase, compounds 3f and 3g were found to show significant inhibitory properties when compared to the positive control. The IC50 values of 3f and 3g were calculated as 24.05 ± 2.28 and 18.51 ± 1.19 µM, respectively. Kinetic studies revealed that compounds 3f and 3g exhibited uncompetitive mode of inhibition against α-glycosidase. Molecular modeling predicted druglikeness for the title compounds and underpinned the importance of phloroglucinol hydroxyls for interacting with the key residues of α-glycosidase.

In vitro and in silico investigation of FDA-approved drugs to be repurposed against Alzheimer's disease.

Alzheimer's disease (AD), one of the main causes of dementia, is a neurodegenerative disorder. Cholinesterase inhibitors are used in the treatment of AD, but prolonged use of these drugs can lead to serious side effects. Drug repurposing is an approach that aims to reveal the effectiveness of drugs in different diseases beyond their clinical uses. In this work, we investigated in vitro and in silico inhibitory effects of 11 different drugs on cholinesterases. The results showed that trimebutine, theophylline, and levamisole had the highest acetylcholinesterase inhibitory actions among the tested drugs, and these drugs inhibited by 68.70 ± 0.46, 53.25 ± 3.40, and 44.03 ± 1.20%, respectively at 1000 µM. In addition, these drugs are bound to acetylcholinesterase via competitive manner. Molecular modeling predicted good fitness in acetylcholinesterase active site for these drugs and possible central nervous system action for trimebutine. All of these results demonstrated that trimebutine was determined to be the drug with the highest potential for use in AD.

The inhibitory effect of escitalopram on mouse detrusor contractility: The role of L-type calcium channels.

Selective serotonin reuptake inhibitors (SSRIs) are associated with urinary problems attributed to their central effects. ESC is a preferred SSRI and several case reports described that ESC is related to urinary retention. However, the direct effect of ESC on detrusor contractility is still not completely elucidated. Thus, we investigated the effect of ESC on detrusor contractility and mechanism(s) of its action in isolated mouse detrusor strips. Molecular docking and measurement of intracellular calcium were performed to determine the possible calcium channel blocking effect of ESC. The contractile responses to carbachol (CCh), KCl and electrical field stimulation of detrusor strips were significantly abolished by ESC (10 or 100 µM). ESC relaxed KCl-precontracted detrusor strips concentration-dependently, which was not affected by tetraethylammonium, glibenclamide, 4-aminopyridine, propranolol, L-NAME or methylene blue. ESC (10 or 100 µM) reduced both the CaCl2- and CCh-induced contractions under calcium-free conditions, indicating the role of calcium-involved mechanisms in ESC-mediated relaxation. Furthermore, ESC significantly decreased Bay K8644-induced contraction and the cytosolic calcium level in fura-2-loaded A7r5 cells. Molecular docking study also revealed the potential of ESC to bind L-type calcium (Cav1) channels. Our results demonstrate that ESC inhibits detrusor contractility via blocking Cav1 channels, which provides evidence for the direct effect of ESC on detrusor contractility and its mechanism.

Azole derivatives inhibit wildtype butyrylcholinesterase and its common mutants.

Azoles, which have been used for antifungal chemotherapy for decades, have recently been of interest for their efficacy against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). There is little known about the potential of azoles against BChE, however there is none regarding their inhibitory effects against mutants of BChE. In the current study, an azole library of 1-aryl-2-(1H-imidazol-1-yl)ethanol/ethanone oxime esters were tested against AChE and BChE, which yielded derivates more potent than the positive control, galantamine, against both isoforms. Kinetic analyses were performed for wildtype and mutant (A328F and A328Y) inhibition for the two most potent BChE inhibitors, pivalic and 3-bezoylpropanoic acid esters of 2-(1H-imidazol-1-yl)-1-(2-naphthyl)ethanol, which were found to have great affinity to the wildtype and mutant BChE types with Ki values as low as 0.173 ± 0.012 µM. The compounds were identified to show linear competitive or mixed type inhibition. Molecular modeling confirmed these kinetic data and provided further insights regarding molecular basis of BChE inhibition by the active derivatives. Thus, current study suggests new azole derivatives with promising cholinesterase inhibitory effects and reveals the first set of information to promote our understanding for the inhibitory behavior of this class against the mutant BChE forms.

Flavonoids as tyrosinase inhibitors in in silico and in vitro models: basic framework of SAR using a statistical modelling approach.

Flavonoids are widely distributed in plants and constitute the most common polyphenolic phytoconstituents in the human diet. In this study, the in vitro inhibitory activity of 44 different flavonoids (1-44) against mushroom tyrosinase was studied, and an in silico study and type of inhibition for the most active compounds were evaluated too. Tyrosinase inhibitors block melanogenesis and take part in melanin production or distribution leading to pigmentation diseases. The in vitro study showed that quercetin was a competitive inhibitor (IC50=44.38 ± 0.13 µM) and achieved higher antityrosinase activity than the control inhibitor kojic acid. The in silico results highlight the importance of the flavonoid core with a hydroxyl at C7 as a strong contributor of interference with tyrosinase activity. According to the developed statistical model, the activity of molecules depends on hydroxylation at C3 and methylation at C8, C7, and C3 in the benzo-γ-pyrane ring of the flavonoids.

In Vitro and in Silico Investigation of DNA Interaction, Topoisomerase I and II Inhibitory Properties of Polydatin.

Polydatin or piceid, is the 3-O-glucoside of resveratrol and is found abundantly in grapes, peanuts, wine, beer, and cacao products. Although anticancer activity of polydatin was reported before, and potential antiproliferative mechanisms of polydatin have been proposed, its direct effects on DNA and inhibitory potential against topoisomerase enzymes have remained unknown. In this study we aimed to reveal the link between polydatin's effects on DNA and DNA-topoisomerases and its antiproliferative promise. For this purpose, we evaluated the effects of polydatin on DNA and DNA topoisomerase using in vitro and in silico techniques. Polydatin was found to protect DNA against Fenton reaction-induced damage while not showing any hydrolytic nuclease effect. Further, polydatin inhibited topoisomerase II but not topoisomerase I. According to molecular docking studies, polydatin preferably showed minor groove binding to DNA where the stilbene moiety was important for binding to the DNA-topoisomerase II complex. As a result, topoisomerase II inhibition might be another anticancer mechanism of polydatin.

Bioactive Saponins of Primula vulgaris Huds. Promote Wound Healing through Inhibition of Collagenase and Elastase Enzymes: in Vivo, in Vitro and in Silico Evaluations.

Primula vulgaris Huds. leaves and roots were used to treat skin damage and inflammation in Anatolian Folk Medicine. This study aimed to assess the ethnopharmacological use of the plant using in vivo, in vitro, and in silico test models. Linear incision and circular excision wound models were used to determine the in vivo wound-healing potential of the plant extracts and fractions. In vitro assays including hyaluronidase, collagenase, and elastase inhibitory activities were carried out for the active compounds to discover their activity pathways. Structure-based molecular modeling was performed to understand inhibitory mechanisms regarding collagenase and elastase at the molecular level. The butanol fraction of the roots of P. vulgaris showed the highest wound-healing activity. Through activity-guided fractionation and isolation techniques, primulasaponin I (1) and primulasaponin I methyl ester (2) were stated as the major active compounds. These compounds exerted their activities through the inhibition of collagenase and elastase enzymes. Primulasaponin I methyl ester isolated from butanol fraction was found to be the strongest agent, especially with the values of 29.65 % on collagenase and 38.92 % on elastase inhibitory activity assays, as well as molecular docking studies. The present study supports scientific data for the traditional use of P. vulgaris and the wound healing properties of the plant can be referred to secondary metabolites as especially saponins found in the roots.

Antifungal Azole Derivatives Featuring Naphthalene Prove Potent and Competitive Cholinesterase Inhibitors with Potential CNS Penetration According to the in Vitro and in Silico Studies.

Cholinesterase inhibition is of great importance in the fight against neurodegenerative disorders such as Alzheimer's disease. Azole antifungals have come under the spotlight with recent discoveries that underline the efficacy and potential of miconazole and its derivatives against cholinesterase enzymes. In this study, we evaluated a library of azoles against acetylcholinesterase and butyrylcholinesterase using in vitro and in silico methods to identify potent inhibitors. Low micromolar IC50 values were obtained for imidazole derivatives, which were further tested and found potent competitive cholinesterase inhibitors via enzyme kinetics study. The active derivatives showed negligible toxicity in in vitro cytotoxicity tests. Molecular modeling studies predicted that these derivatives were druglike, could penetrate blood-brain barrier, and tightly bind to cholinesterase active site making key interactions via the imidazole moiety at protonated state. Thus, current study identifies potent and competitive cholinesterase inhibitor azoles with minor toxicity and potential to pass into the central nervous system.

Rutin increases alpha-tubulin acetylation via histone deacetylase 6 inhibition.

Microtubules are dynamic cytoskeletal filaments composed of alpha- (α) and beta (ß)-tubulin proteins. α-tubulin proteins are posttranslationally acetylated, and loss of acetylation is associated with axonal transport defects, a common alteration contributing to the pathomechanisms of several neurodegenerative diseases. Restoring α-tubulin acetylation by pharmacological inhibition of HDAC6, a primary α-tubulin deacetylase, can rescue impaired transport. Therefore, HDAC6 is considered a promising therapeutic target for neurodegenerative diseases, but currently, there is no clinically approved inhibitor for this purpose. In this study, using drug repurposing strategy, we aimed to identify compounds possessing HDAC6 inhibition activity and inducing α-tubulin acetylation. We systematically analyzed the FDA-approved library by utilizing virtual screening and consensus scoring approaches. Inhibition activities of promising compounds were tested using in vitro assays. Motor neuron-like NSC34 cells were treated with the candidate compounds, and α-tubulin acetylation levels were determined by Western blot. Our results demonstrated that rutin, a natural flavonoid, inhibits cellular HDAC6 activity without inducing any toxicity, and it significantly increases α-tubulin acetylation level in motor neuron-like cells.

Potential of nafimidone derivatives against co-morbidities of epilepsy: In vitro, in vivo, and in silico investigations.

Nafimidone is known for its clinical antiepileptic effects and alcohol derivatives of nafimidone were reported be potent anticonvulsants. These compounds are structurally similar to miconazole, which is known to inhibit cholinesterases, protect neurons, and ameliorate cognitive decline. Herein, we aimed to reveal the potential of three nafimidone alcohol esters (5 g, 5i, and 5 k), which were previously reported for their anticonvulsant effects, against co-morbidities of epilepsy such as inflammatory and neuropathic pain, cognitive and behavioral deficits, and neuron death, and understand their roles in related pathways such as γ-butyric acid type A (GABAA ) receptor and cholinesterases using in vitro, in vivo and in silico methods. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used for cytotoxicity evaluation, hippocampal slice culture assay for neuroprotection, formalin test for acute and inflammatory pain, sciatic ligation for neuropathic pain, Morris water maze and open field locomotor tasks for cognitive and behavioral deficits, radioligand binding for GABAA receptor affinity, spectrophotometric methods for cholinesterase inhibition in vitro, and molecular docking in silico. The compounds were non-toxic to fibroblast cells. 5 k was neuroprotective against kainic acid-induced neuron death. 5i reduced pain response of mice in both the acute and the inflammatory phases. 5i improved survival upon status epilepticus. The compounds showed no affinity to GABAA receptor but inhibited acetylcholinesterase, 5 k also inhibited butyrylcholinesterase. The compounds were predicted to interact mainly with the peripheric anionic site of cholinesterase enzymes. The title compounds showed neuroprotective, analgesic, and cholinesterase inhibitory effects, thus they bear promise against certain co-morbidities of epilepsy with neurological insults.

Inhibition of cholinesterases by safranin O: Integration of inhibition kinetics with molecular docking simulations.

In the present study, the inhibitory mechanisms and effects of a synthetic phenazine dye, safranin O (SO) on human plasma butyrylcholinesterase (BChE), human erythrocyte acetylcholinesterase (AChE) and recombinant BChE mutants were investigated. Kinetic studies showed the following information: SO leaded to linear competitive inhibition of human plasma BChE with Ki = 0.44 ± 0.085 µM; α = ∞. It acted as a hyperbolic noncompetitive inhibitor of human erythrocyte AChE with Ki = 0.69 ± 0.13; α = 1; ß = 0.08 ± 0.02. On the other hand, the inhibitory effects of SO on two BChE mutants, where A328 was modified to either F or Y, revealed differences in terms of inhibitory patterns and Ki values, compared to the obtained results with recombinant wild type BChE. SO was found to act as a linear competitive inhibitor of A328F and A328Y BChE mutants. Compared to recombinant wild type BChE, A328Y and A328F BChE mutants caused a 4- and 10-fold decrease in Ki value for SO, respectively. These findings were supported by molecular modelling studies. In conclusion, SO is a potent inhibitor of human cholinesterases and may be useful in the design and development of new drugs for the treatment of AD.

Azole derivatives with naphthalene showing potent antifungal effects against planktonic and biofilm forms of Candida spp.: an in vitro and in silico study.

Candida infections pose a serious public health threat due to increasing drug resistance. Azoles are first-line antifungal drugs for fungal infections. In this study, we tested an in-house azole collection incorporating naphthalene ring to find hits against planktonic and biofilm forms of resistant Candida spp. In the collection, potent derivatives were identified against the susceptible strains of Candida with minimum inhibitory concentration (MIC) values lower than those of the reference drug, fluconazole. MIC values of 0.125 µg/ml against C. albicans, 0.0625 µg/ml against C. parapsilosis, and 2 µg/ml against C. krusei, an intrinsically azole-resistant non-albicans Candida, were obtained. Some of the derivatives were highly active against fluconazole-resistant clinical isolate of C. tropicalis. Inhibition of C. albicans biofilms was also observed at 4 µg/ml similar as amphotericin B, the reference drug known for its antibiofilm activity. Through molecular docking studies, affinities and key interactions of the compounds with fungal lanosterol 14α-demethylase (CYP51), the target enzyme of azoles, were predicted. The interactions of imidazole with heme cofactor and of the naphthalene with Tyr118 were highlighted in line with the literature data. As a result, this study proves the importance of naphthalene for the antifungal activity of azoles against Candida spp. in both planktonic and biofilm forms.

Flavones as tyrosinase inhibitors: kinetic studies in vitro and in silico.

INTRODUCTION: Tyrosinase is a multifunctional copper-containing oxidase enzyme that catalyses the first steps in the formation of melanin pigments. Identification of tyrosinase inhibitors is of value for applications in cosmetics, medicine and agriculture. OBJECTIVE: To develop an analytical method that allows identification of drug-like natural products that can be further developed as tyrosinase inhibitors. Results of in vitro and in silico studies will be compared in order to gain a deeper insight into the mechanism of action of enzyme inhibition. METHOD: Using an in vitro assay we tested tyrosinase inhibitor effects of five structurally related flavones, i.e. luteolin (1), eupafolin (2), genkwanin (3), nobiletin (4), and chrysosplenetin (5). The strongest inhibitors were further investigated in silico, using enzyme docking simulations. RESULTS: All compounds tested showed modest tyrosinase inhibitory effect compared to the positive control, kojic acid. The polymethoxy flavones 4 and 5 exhibited the strongest tyrosinase inhibitory effect with the half maximal inhibitory concentration (IC50 ) values of 131.92 ± 1.75 µM and 99.87 ± 2.38 µM respectively. According to kinetic analysis 2, 4 and 5 were competitive inhibitors, whereas 1 and 3 were non-competitive inhibitors of tyrosinase. Docking studies indicated that methoxy groups on 4 and 5 caused steric hindrance which prevented alternative binding modes in the tyrosinase; the methoxy groups on the B-ring of these flavones faced the catalytic site in the enzyme. CONCLUSIONS: The docking simulations nicely complemented the in vitro kinetic studies, opening the way for the development of predictive models for use in drug design.

Antibacterial azole derivatives: Antibacterial activity, cytotoxicity, and in silico mechanistic studies.

Azole antifungal drugs are commonly used in antifungal chemotherapy. Antibacterial effects of some topical antifungals, such as miconazole and econazole, have lately been revealed, which suggests a promising venue in antimicrobial chemotherapy. In this study, we tested an in-house azole collection with antifungal properties for their antibacterial activity to identify dual-acting hits using the broth microdilution method. The in vitro screen yielded a number of potent derivatives against gram-positive bacteria, Enterococcus faecalis and Staphylococcus aureus. Compound 73's minimum inhibitory concentration (MIC) value less than 1 µg/ml against S. aureus; however, none of the compounds showed noteworthy activity against methicillin-resistant S. aureus (MRSA). All the active compounds were found safe at their MIC values against the healthy fibroblast cells in the in vitro cytotoxicity test. Molecular docking studies of the most active compounds using a set of docking programs with flavohemoglobin (flavoHb) structure, the proposed target of the azole antifungals with antibacterial activity, presented striking similarities regarding the binding modes and interactions between the tested compounds and the antifungal drugs with crystallographic data. In addition to being noncytotoxic, the library was predicted to be drug-like and free of pan-assay interference compounds (PAINS). As a result, the current study revealed several potential azole derivatives with both antifungal and antibacterial activities. Inhibition of bacterial flavoHb was suggested as a possible mechanism of action for the title compounds.

Tyrosinase inhibitory effects of Vinca major and its secondary metabolites: Enzyme kinetics and in silico inhibition model of the metabolites validated by pharmacophore modelling.

In the present study, we aimed to identify the tyrosinase enzyme inhibitory potential of Vinca major L. extract and its secondary metabolites. The extract possessed remarkable tyrosinase enzyme inhibitory effect with IC50 value of 20.39 ±â€¯0.44 µg/mL compared to the positive control, kojic acid (IC50 8.56 ±â€¯0.17 µg/mL). Compounds 1 and 5 were the most potent isolates with IC50 values of 32.41 ±â€¯0.99 and 31.34 ±â€¯0.75 µM, they were more potent than kojic acid (IC50: 60.25 ±â€¯0.54 µM). Compound 2 also exhibited remarkable tyrosinase inhibition with an IC50 value of 64.51 ±â€¯1.29 µM. An enzyme kinetics analysis revealed that 1 was a mixed-type, 2 and 5 were noncompetitive inhibitors. Using molecular docking, we predicted binding affinity and interactions of the compounds, which were in good alignment with a pharmacophore hypothesis generated out of a number of known tyrosinase inhibitors. The modelling studies underlined crucial interactions with the copper ions and residues around them such as Asn260, His263, and Met280.

Tyrosinase and α-glucosidase inhibitory potential of compounds isolated from Quercus coccifera bark: In vitro and in silico perspectives.

Bark of Quercus coccifera is widely used in folk medicine. We tested tyrosinase and α-glucosidase inhibitory effects of Q. coccifera bark extract and isolated compounds from it. The extract inhibited tyrosinase with an IC50 value of 75.13 ±â€¯0.44 µg/mL. Among the isolated compounds, polydatin (6) showed potent tyrosinase inhibition compared to the positive control, kojic acid, with an IC50 value of 4.05 ±â€¯0.30 µg/mL. The Q. coccifera extract also inhibited α-glucosidase significantly with an IC50 value of 3.26 ±â€¯0.08 µg/mL. (-)-8-Chlorocatechin (5) was the most potent isolate, also more potent than the positive control, acarbose, with an IC50 value of 43.60 ±â€¯0.67 µg/mL. According to the kinetic analysis, 6 was a noncompetitive and 5 was a competitive inhibitor of tyrosinase, and 5 was a noncompetitive α-glucosidase inhibitor. In the light of these findings, we performed in silico molecular docking studies for 5 and 6 with QM/MM optimizations to predict their tyrosinase inhibition mechanisms at molecular level and search for correlations with the in vitro results. We found that the ionized form of 5 (5i) showed higher affinity and more stable binding to tyrosinase catalytic site than its neutral form, while 6 bound to the predicted allosteric sites of the enzyme better than the catalytic site.

Synthesis, in vivo anticonvulsant testing, and molecular modeling studies of new nafimidone derivatives.

An estimated 50 million people suffer epilepsy worldwide and 30% of the cases do not respond to current antiepileptic drugs (AEDs). Here, we report synthesis and anticonvulsant screening of new derivatives of nafimidone, a well-known member of (arylakyl)azole anticonvulsants. The compounds showed promising protection against maximal electroshock (MES)-induced seizures in mice and rats when administered via intraperitoneal (ip) and oral route. Especially, 5b, 5c, and 5i displayed outstanding activity in rats in MES test when given ip (ED50 : 16.0, 15.8, and 11.8 mg/kg, respectively). Additionally, 5l was active against 6 Hz and corneal-kindled mice models. Behavioral toxicity of the compounds was very low and their therapeutic indexes were high compared to some currently available AEDs. A number of pharmaceutically relevant descriptors and properties were predicted for the compounds in silico in comparison with a set of known drugs. Favorable results were obtained such as good blood-brain barrier permeability and high oral absorption, as well as drug-likeness. 5l was found to show affinity to the benzodiazepine binding site of A-type GABA receptor via molecular docking simulations.

Synthesis, anticonvulsant screening, and molecular modeling studies of new arylalkylimidazole oxime ether derivatives.

In this study, 15 new oxime ether derivatives were synthesized and their anticonvulsant activities were screened in vivo. The compounds were synthesized by the reaction of various alkyl halides with 1-(2-naphthyl)-2-(1H-imidazol-1-yl)ethanone oxime. Their anticonvulsant activities were determined using acute (maximal electroshock, subcutaneous metrazol [SCM], and 6 Hz seizure test) and chronic (corneal-kindled mouse) seizure models, their neurotoxic effects were evaluated by models of behavioral toxicity according to the Epilepsy Therapy Screening Program protocol of the NIH. All our compounds were protective in at least one of the tests. Quantification studies were applied to some of the active compounds and the intraperitoneal ED50 values in mice were found between 25.48 and 99.56 mg/kg. Some pharmacokinetic properties of the compounds were predicted in silico and molecular docking studies were performed to provide insights into their possible anticonvulsant mechanism regarding their SCM activity.

Discovery and Characterisation of Dual Inhibitors of Tryptophan 2,3-Dioxygenase (TDO2) and Indoleamine 2,3-Dioxygenase 1 (IDO1) Using Virtual Screening.

Cancers express tryptophan catabolising enzymes indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2) to produce immunosuppressive tryptophan metabolites that undermine patients' immune systems, leading to poor disease outcomes. Both enzymes are validated targets for cancer immunotherapy but there is a paucity of potent TDO2 and dual IDO1/TDO2 inhibitors. To identify novel dual IDO1/TDO2 scaffolds, 3D shape similarity and pharmacophore in silico screening was conducted using TDO2 as a model for both systems. The obtained hits were tested in cancer cell lines expressing mainly IDO1 (SKOV3-ovarian), predominantly TDO2 (A172-brain), and both IDO1 and TDO2 (BT549-breast). Three virtual screening hits were confirmed as inhibitors (TD12, TD18 and TD34). Dose response experiments showed that TD34 is the most potent inhibitor capable of blocking both IDO1 and TDO2 activity, with the IC50 value for BT549 at 3.42 µM. This work identified new scaffolds able to inhibit both IDO1 and TDO2, thus enriching the collection of dual IDO1/TDO2 inhibitors and providing chemical matter for potential development into future anticancer drugs.