Synthesis, molecular docking study and biological evaluation of new pyrrole scaffolds as potential antitubercular agents for dual targeting of enoyl ACP reductase and dihydrofolate reductase.

In this study, new series of N'-(2-(substitutedphenoxy)acetyl)-4-(1H-pyrrol-1-yl)benzohydrazides (3a-j) 4-(2,5-dimethyl-1H-pyrrol-1-yl)-N'-(2-(substitutedphenoxy)acetyl)benzohydrazides (5a-j) were synthesized, characterized and assessed as inhibitors of enoyl ACP reductase and DHFR. Most of the compounds exhibited dual inhibition against the enzymes enoyl ACP reductase and DHFR. Several synthesized substances also demonstrated significant antibacterial and antitubercular properties. A molecular docking analysis was conducted in order to determine the potential mechanism of action of the synthesized compounds. The results indicated that there were binding interactions seen with the active sites of dihydrofolate reductase and enoyl ACP reductase. Additionally, important structural details were identified that play a critical role in sustaining the dual inhibitory activity. These findings were useful for the development of future dual inhibitors. Therefore, this study provided strong evidence that several synthesized molecules could exert their antitubercular properties at the cellular level through multi-target inhibition. By shedding light on the mechanisms through which these compounds exert their inhibitory effects, this research opens up promising avenues for the future development of dual inhibitors with enhanced antibacterial and antitubercular properties. The study's findings underscore the importance of multi-target approaches in drug design, providing a strong foundation for the design and optimization of novel compounds that can effectively target bacterial infections at the cellular level.

Deep mutational scanning of Pneumocystis jirovecii dihydrofolate reductase reveals allosteric mechanism of resistance to an antifolate.

Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro. One of the main drug targets in P. jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the baker's yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen.

Solution Ionic Strength Can Modulate Functional Loop Conformations in E. coli Dihydrofolate Reductase.

The observation of multiple conformations of a functional loop (termed M20) in the Escherichia coli dihydrofolate reductase (ecDHFR) enzyme triggered the proposition that large-scale motions of protein structural elements contribute to enzyme catalysis. The transition of the M20 loop from a closed conformation to an occluded conformation was thought to aid the rate-limiting release of the products. However, the influence of charged species in the solution environment on the observed M20 loop conformations, independent of charged ligands bound to the enzyme, had not been considered. Molecular dynamics simulations of ecDHFR in model CaCl2 solutions of varying molar ionic strengths IM reveal a substantial free energy barrier between occluded and closed M20 loop states at IM exceeding the E. coli threshold (∼0.24 M). This barrier may facilitate crystallization of ecDHFR in the occluded state, consistent with ecDHFR structures obtained at IM exceeding 0.3 M. At lower IM (≤0.15 M), the M20 loop can explore the occluded state, but prefers an open/partially closed conformation, again consistent with ecDHFR structures. Our findings caution against using ecDHFR structures obtained at nonphysiological ionic strengths in interpreting catalytic events or in structure-based drug design.

Construction and Docking Studies of Novel Pyrimido[4,5-b]quinolines as Antimicrobial Agents.

In order to develop novel antimicrobial agents, we prepared quinoline bearing pyrimidine analogues 2-7, 8 a-d and 9 a-d and their structures were elucidated by spectroscopic techniques. Furthermore, our second aim was to predict the interactions between the active compounds and enzymes (DNA gyrase and DHFR). In this work, fourteen pyrimido[4,5-b]quinoline derivatives were prepared and assessed for their antimicrobial potential by estimating zone of inhibition. All the screened candidates displayed antibacterial potential with zone of inhibition range of 9-24 mm compared with ampicillin (20-25 mm) as a reference drug. Moreover, the target derivatives 2 (ZI=16), 9 c (ZI=17 mm) and 9 d (ZI=16 mm) recorded higher antifungal activity against C. albicans to that exhibited by the antifungal drug amphotericin B (ZI=15 mm). Finally, the most potent pyrimidoquinoline compounds (2, 3, 8 c, 8 d, 9 c and 9 d) were docked inside DHFR and DNA gyrase active sites and they recorded excellent fitting within the active regions of DNA gyrase and DHFR. These outcomes revealed us that compounds (2, 3, 8 c, 8 d, 9 c and 9 d) could be lead compounds to discover novel antibacterial candidates.

Secondary Amine Catalysis in Enzyme Design: Broadening Protein Template Diversity through Genetic Code Expansion.

Secondary amines, due to their reactivity, can transform protein templates into catalytically active entities, accelerating the development of artificial enzymes. However, existing methods, predominantly reliant on modified ligands or N-terminal prolines, impose significant limitations on template selection. In this study, genetic code expansion was used to break this boundary, enabling secondary amines to be incorporated into alternative proteins and positions of choice. Pyrrolysine analogues carrying different secondary amines could be incorporated into superfolder green fluorescent protein (sfGFP), multidrug-binding LmrR and nucleotide-binding dihydrofolate reductase (DHFR). Notably, the analogue containing a D-proline moiety demonstrated both proteolytic stability and catalytic activity, conferring LmrR and DHFR with the desired transfer hydrogenation activity. While the LmrR variants were confined to the biomimetic 1-benzyl-1,4-dihydronicotinamide (BNAH) as the hydride source, the optimal DHFR variant favorably used the pro-R hydride from NADPH for stereoselective reactions (e.r. up to 92 : 8), highlighting that a switch of protein template could broaden the nucleophile option for catalysis. Owing to the cofactor compatibility, the DHFR-based secondary amine catalysis could be integrated into an enzymatic recycling scheme. This established method shows substantial potential in enzyme design, applicable from studies on enzyme evolution to the development of new biocatalysts.

Repurposing FDA-approved drugs to target malaria through inhibition of dihydrofolate reductase in the folate biosynthesis pathway: A prospective approach.

Dihydrofolate reductase (DHFR) is a ubiquitous enzyme that regulates the biosynthesis of tetrahydrofolate among various species of Plasmodium parasite. It is a validated target of the antifolate drug pyrimethamine (Pyr) in Plasmodium falciparum (Pf), but its clinical efficacy has been hampered due to the emergence of drug resistance. This has made the attempt to screen Food & Drug Administration-approved drugs against wild- and mutant PfDHFR by employing an in-silico pipeline to identify potent candidates. The current study has followed a virtual screening approach for identifying potential DHFR inhibitors from DrugBank database, based on a structure similarity search of candidates, followed by absorption, distribution, metabolism, and excretion estimation. The screened drugs were subjected to various parameters like docking, molecular mechanics with generalized born and surface area solvation calculations, and molecular simulations. We have thus identified two potential drug candidates, duloxetine and guanethidine, which can be repurposed to be tested for their efficacy against wild type and drug resistant falciparum malaria.

Oplodiol and nitidine as potential inhibitors of Plasmodium falciparum dihydrofolate reductase: insights from a computational study.

Many natural products have been shown to possess antiplasmodial activities, but their protein targets are unknown. This work employed molecular docking and molecular dynamics simulations to explore the inhibitory activity of some antiplasmodial natural products against wild-type and mutant strains of Plasmodium falciparum dihydrofolate reductase (PfDHFR). From the molecular docking study, 6 ligands preferentially bind at the active site of the DHFR domain with binding energies ranging from -6.4 to -9.5 kcal/mol. Interactions of compounds with MET55 and PHE58 were mostly observed in the molecular docking study. From the molecular dynamics study, the binding of 2 of the ligands-nitidine and oplodiol-was observed to be stable against all tested strains of PfDHFR. The average binding free energy of oplodiol in complex with the various PfDHFR strains was -93.701 kJ/mol whereas that of nitidine was -106.206 kJ/mol. The impressive in silico activities of the 2 compounds suggest they could be considered for development as potential antifolate agents.Communicated by Ramaswamy H. Sarma.

Investigating the anti-cancer potential of pyrimethamine analogues through a modern chemical biology lens.

Pharmacological inhibition of dihydrofolate reductase (DHFR) is an established approach for treating a variety of human diseases, including foreign infections and cancer. However, treatment with classic DHFR inhibitors, such as methotrexate (MTX), are associated with negative side-effects and resistance mechanisms that have prompted the search for alternatives. The DHFR inhibitor pyrimethamine (Pyr) has compelling anti-cancer activity in in vivo models, but lacks potency compared to MTX, thereby requiring higher concentrations to induce therapeutic responses. The purpose of this work was to investigate structural analogues of Pyr to improve its in vitro and cellular activity. A series of 36 Pyr analogues were synthesized and tested in a sequence of in vitro and cell-based assays to monitor their DHFR inhibitory activity, cellular target engagement, and impact on breast cancer cell viability. Ten top compounds were identified, two of which stood out as potential lead candidates, 32 and 34. These functionalized Pyr analogues potently engaged DHFR in cells, at concentrations as low as 1 nM and represent promising DHFR inhibitors that could be further explored as potential anti-cancer agents.

Epistasis and pleiotropy shape biophysical protein subspaces associated with drug resistance.

Protein space is a rich analogy for genotype-phenotype maps, where amino acid sequence is organized into a high-dimensional space that highlights the connectivity between protein variants. It is a useful abstraction for understanding the process of evolution, and for efforts to engineer proteins towards desirable phenotypes. Few mentions of protein space consider how protein phenotypes can be described in terms of their biophysical components, nor do they rigorously interrogate how forces like epistasis-describing the nonlinear interaction between mutations and their phenotypic consequences-manifest across these components. In this study, we deconstruct a low-dimensional protein space of a bacterial enzyme (dihydrofolate reductase; DHFR) into "subspaces" corresponding to a set of kinetic and thermodynamic traits [k_{cat}, K_{M}, K_{i}, and T_{m} (melting temperature)]. We then examine how combinations of three mutations (eight alleles in total) display pleiotropy, or unique effects on individual subspace traits. We examine protein spaces across three orthologous DHFR enzymes (Escherichia coli, Listeria grayi, and Chlamydia muridarum), adding a genotypic context dimension through which epistasis occurs across subspaces. In doing so, we reveal that protein space is a deceptively complex notion, and that future applications to bioengineering should consider how interactions between amino acid substitutions manifest across different phenotypic subspaces.

A rugged yet easily navigable fitness landscape.

Fitness landscape theory predicts that rugged landscapes with multiple peaks impair Darwinian evolution, but experimental evidence is limited. In this study, we used genome editing to map the fitness of >260,000 genotypes of the key metabolic enzyme dihydrofolate reductase in the presence of the antibiotic trimethoprim, which targets this enzyme. The resulting landscape is highly rugged and harbors 514 fitness peaks. However, its highest peaks are accessible to evolving populations via abundant fitness-increasing paths. Different peaks share large basins of attraction that render the outcome of adaptive evolution highly contingent on chance events. Our work shows that ruggedness need not be an obstacle to Darwinian evolution but can reduce its predictability. If true in general, the complexity of optimization problems on realistic landscapes may require reappraisal.

From a binding module to essential catalytic activity: how nature stumbled on a good thing.

Enzymes are complex macromolecules capable of catalyzing a wide variety of chemical reactions with high efficiency. Nonetheless, biological catalysis can be rudimentary. Here, we describe an enzyme that is built from a simple protein fold. This short protein sequence - almost a peptide - belongs to the ancient SH3 family of binding modules. Surprisingly, this binding module catalyzes the specific reduction of dihydrofolate using NADPH as a reducing cofactor, making this a dihydrofolate reductase. Too small to provide all the required binding and catalytic machinery on its own, it homotetramerizes, thus creating a large, central active site environment. Remarkably, none of the active site residues is essential to the catalytic function. Instead, backbone interactions juxtapose the reducing cofactor proximal to the target imine of the folate substrate, and a specific motion of the substrate promotes formation of the transition state. In this feature article, we describe the features that make this small protein a functional enzyme capable of catalyzing a metabolically essential reaction, highlighting the characteristics that make it a model for the evolution of primitive enzymes from binding modules.

Kinetic Barrier to Enzyme Inhibition Is Manipulated by Dynamical Local Interactions in E. coli DHFR.

Dihydrofolate reductase (DHFR) is an important drug target and a highly studied model protein for understanding enzyme dynamics. DHFR's crucial role in folate synthesis renders it an ideal candidate to understand protein function and protein evolution mechanisms. In this study, to understand how a newly proposed DHFR inhibitor, 4'-deoxy methyl trimethoprim (4'-DTMP), alters evolutionary trajectories, we studied interactions that lead to its superior performance over that of trimethoprim (TMP). To elucidate the inhibition mechanism of 4'-DTMP, we first confirmed, both computationally and experimentally, that the relative binding free energy cost for the mutation of TMP and 4'-DTMP is the same, pointing the origin of the characteristic differences to be kinetic rather than thermodynamic. We then employed an interaction-based analysis by focusing first on the active site and then on the whole enzyme. We confirmed that the polar modification in 4'-DTMP induces additional local interactions with the enzyme, particularly, the M20 loop. These changes are propagated to the whole enzyme as shifts in the hydrogen bond networks. To shed light on the allosteric interactions, we support our analysis with network-based community analysis and show that segmentation of the loop domain of inhibitor-bound DHFR must be avoided by a successful inhibitor.

Allosteric regulatory control in dihydrofolate reductase is revealed by dynamic asymmetry.

We investigated the relationship between mutations and dynamics in Escherichia coli dihydrofolate reductase (DHFR) using computational methods. Our study focused on the M20 and FG loops, which are known to be functionally important and affected by mutations distal to the loops. We used molecular dynamics simulations and developed position-specific metrics, including the dynamic flexibility index (DFI) and dynamic coupling index (DCI), to analyze the dynamics of wild-type DHFR and compared our results with existing deep mutational scanning data. Our analysis showed a statistically significant association between DFI and mutational tolerance of the DHFR positions, indicating that DFI can predict functionally beneficial or detrimental substitutions. We also applied an asymmetric version of our DCI metric (DCIasym ) to DHFR and found that certain distal residues control the dynamics of the M20 and FG loops, whereas others are controlled by them. Residues that are suggested to control the M20 and FG loops by our DCIasym metric are evolutionarily nonconserved; mutations at these sites can enhance enzyme activity. On the other hand, residues controlled by the loops are mostly deleterious to function when mutated and are also evolutionary conserved. Our results suggest that dynamics-based metrics can identify residues that explain the relationship between mutation and protein function or can be targeted to rationally engineer enzymes with enhanced activity.

In silico screening of inhibitors against human dihydrofolate reductase to identify potential anticancer compounds.

In all species, dihydrofolate reductase (DHFR) is an essential enzyme that regulates the cellular amount of tetrahydrofolate. Human DHFR (hDHFR) activity inhibition results in tetrahydrofolate depletion and cell death. This property has made hDHFR a therapeutic target for cancer. Methotrexate is a well-known hDHFR inhibitor, but its administration has shown some light to severe adverse effects. Therefore, we aimed to find new potential hDHFR inhibitors using structure-based virtual screening, ADMET prediction, molecular docking, and molecular dynamics simulations. Here, we used the PubChem database to find all compounds with at least 90% structural similarity to known natural DHFR inhibitors. To explore their interaction pattern and estimate their binding affinities, the screened compounds (2023) were subjected to structure-based molecular docking against hDHFR. The fifteen compounds that showed higher binding affinity to the hDHFR than the reference compound (methotrexate) displayed important molecular orientation and interactions with key residues in the enzyme's active site. These compounds were subjected to Lipinski and ADMET prediction. PubChem CIDs: 46886812 and 638190 were identified as putative inhibitors. In addition, molecular dynamics simulations revealed that the binding of compounds (CIDs: 46886812 and 63819) stabilized the hDHFR structure and caused minor conformational changes. Our findings suggest that two compounds (CIDs: 46886812 and 63819) could be promising potential inhibitors of hDHFR in cancer therapy.Communicated by Ramaswamy H. Sarma.

Phosphorylation of Thymidylate Synthase and Dihydrofolate Reductase in Cancer Cells and the Effect of CK2α Silencing.

Our previous research suggests an important regulatory role of CK2-mediated phosphorylation of enzymes involved in the thymidylate biosynthesis cycle, i.e., thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT). The aim of this study was to show whether silencing of the CK2α gene affects TS and DHFR expression in A-549 cells. Additionally, we attempted to identify the endogenous kinases that phosphorylate TS and DHFR in CCRF-CEM and A-549 cells. We used immunodetection, immunofluorescence/confocal analyses, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), in-gel kinase assay, and mass spectrometry analysis. Our results demonstrate that silencing of the CK2α gene in lung adenocarcinoma cells significantly increases both TS and DHFR expression and affects their cellular distribution. Additionally, we show for the first time that both TS and DHFR are very likely phosphorylated by endogenous CK2 in two types of cancer cells, i.e., acute lymphoblastic leukaemia and lung adenocarcinoma. Moreover, our studies indicate that DHFR is phosphorylated intracellularly by CK2 to a greater extent in leukaemia cells than in lung adenocarcinoma cells. Interestingly, in-gel kinase assay results indicate that the CK2α' isoform was more active than the CK2α subunit. Our results confirm the previous studies concerning the physiological relevance of CK2-mediated phosphorylation of TS and DHFR.

Unveiling the Efficacy of Sesquiterpenes from Marine Sponge Dactylospongia elegans in Inhibiting Dihydrofolate Reductase Using Docking and Molecular Dynamic Studies.

Dihydrofolate reductase (DHFR) is a crucial enzyme that maintains the levels of 5,6,7,8-tetrahydrofolate (THF) required for the biological synthesis of the building blocks of DNA, RNA, and proteins. Over-activation of DHFR results in the progression of multiple pathological conditions such as cancer, bacterial infection, and inflammation. Therefore, DHFR inhibition plays a major role in treating these illnesses. Sesquiterpenes of various types are prime metabolites derived from the marine sponge Dactylospongia elegans and have demonstrated antitumor, anti-inflammation, and antibacterial capacities. Here, we investigated the in silico potential inhibitory effects of 87 D. elegans metabolites on DHFR and predicted their ADMET properties. Compounds were prepared computationally for molecular docking into the selected crystal structure of DHFR (PDB: 1KMV). The docking scores of metabolites 34, 28, and 44 were the highest among this series (gscore values of -12.431, -11.502, and -10.62 kcal/mol, respectively), even above the co-crystallized inhibitor SRI-9662 score (-10.432 kcal/mol). The binding affinity and protein stability of these top three scored compounds were further estimated using molecular dynamic simulation. Compounds 34, 28, and 44 revealed high binding affinity to the enzyme and could be possible leads for DHFR inhibitors; however, further in vitro and in vivo investigations are required to validate their potential.

Exploring the potential and identifying Withania somnifera alkaloids as novel dihydrofolate reductase (DHFR) inhibitors by the AlteQ method.

There is an urgent need to discover and develop novel drugs to combat Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) in humans. Alkaloids have been shown to have wide-ranging therapeutic application and could be ideal candidates for drug development, and research is underway to develop new anti-tubercular drugs from natural sources. In this regard, the current research deals with finding novel lead compounds from the Withania somnifera (WS) plant. Broad health benefits of WS are due to the presence of diverse chemical constituents which include anaferine and anahygrine and which belong to the alkaloid family. In the present study, these two compounds have been theoretically studied to understand their electronic properties using the density functional theory (DFT) at the B3LYP/6-311 + G (d,p) level. HOMO and LUMO properties and molecular electrostatic potential (MEP) surface were calculated. Further, to understand the mechanism of action of these compounds and to identify their putative drug target, molecular docking and dynamics studies were employed against Mycobacterium tuberculosis dihydrofolate reductase (DHFR). It was determined that NADP+ affects stability of the complexes by reducing fluctuations of residues 14-23 and 117-126. It was also found that Ile5 and Gln28 play an important role in complexation. Electron density analysis (using the AlteQ method) of the intermolecular region, analyzing both the anaferin-NADP+ and anahygrin-NADP+ complexes showed that anaferin and anahygrin complexes are more stable in the presence of NADP+. It has been established that in most intermolecular contacts the contribution of the ligand to the electron density is greater than that of NADP+. The present study thus provides an excellent way to analyze the effect of anaferine and anahygrine in essential processes of M. tuberculosis.Communicated by Ramaswamy H. Sarma.

A conserved SH3-like fold in diverse putative proteins tetramerizes into an oxidoreductase providing an antimicrobial resistance phenotype.

We present a potential mechanism for emergence of catalytic activity that is essential for survival, from a non-catalytic protein fold. The type B dihydrofolate reductase (DfrB) family of enzymes were first identified in pathogenic bacteria because their dihydrofolate reductase activity is sufficient to provide trimethoprim (TMP) resistance. DfrB enzymes are described as poorly evolved as a result of their unusual structural and kinetic features. No characterized protein shares sequence homology with DfrB enzymes; how they evolved to emerge in the modern resistome is unknown. In this work, we identify DfrB homologues from a database of putative and uncharacterized proteins. These proteins include an SH3-like fold homologous to the DfrB enzymes, embedded in a variety of additional structural domains. By means of functional, structural and biophysical characterization, we demonstrate that these distant homologues and their extracted SH3-like fold can display dihydrofolate reductase activity and confer TMP resistance. We provide evidence of tetrameric assembly and catalytic mechanism analogous to that of DfrB enzymes. These results contribute, to our knowledge, the first insights into a potential evolutionary path taken by this SH3-like fold to emerge in the modern resistome following introduction of TMP. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Arylazopyrazole-Based Photoswitchable Inhibitors Selective for Escherichia coli Dihydrofolate Reductase.

Selective inhibitors of Escherichia coli dihydrofolate reductase (eDHFR) are crucial chemical biology tools that have widespread clinical applications. We developed a set of eDHFR-selective photoswitchable inhibitors by derivatizing the structure of our previously reported methotrexate (MTX) azolog, azoMTX. Substitution of the skeletal p-phenylene group of azoMTX with bulky bis-alkylated arylazopyrazole moieties significantly increased its selectivity toward eDHFR over human DHFR. Owing to the physical properties of arylazopyrazoles, the new ligands exhibited nearly complete Z-to-E photoconversion and high thermostability of Z-isomers. In addition, real-time photoreversible control of eDHFR activity was achieved by alternatively switching the illumination light wavelengths.

Screening of indole derivatives as the potent anticancer agents on dihydrofolate reductase: pharmaco-informatics and molecular dynamics simulation.

Dihydrofolate reductase (DHFR) is a ubiquitous cellular enzyme involved in the biosynthesis of nucleotide and protein precursors, thus, the inhibition of human DHFR can be a promising strategy in cancer treatment. The design of effective anticancer drugs is an urgent need today according to the high spread of cancer. The indole molecule with diverse mechanisms of action and anticancer properties is one of the efficient pharmacophores in drug design. Hence, a virtual library of indole derivatives as a scaffold was selected for designing safer and more effective anticancer drugs against DHFR in this work. All indole derivatives utilized in the library design were selected regarding appreciable tumor growth inhibition. Structure-activity relationship (SAR), docking energy, ADMET (absorption, distribution, metabolism, excretion, and toxicity) parameters, and effective non-covalent interactions were used to identify potential anticancer with indole scaffold. Results showed a higher number of indole moieties provide a strong attachment to the DHFR binding pocket and therefore more effective anticancer activity. The indole scaffold in combination with dichlorobenzene improves DHFR inhibition whereas barbituric acid weakens inhibition activity. In the following to validate the docking results, Molecular dynamics (MD) simulation and molecular mechanics generalized-Born surface area (MM-GBSA) indicated the permanent stability of the selected ligands into the DHFR binding pocket and the key amino acids. Therefore, promising pharmacophores based on indole-DHFR interactions were discovered, and the outcome could be useful in guiding future in vitro and in vivo drug discovery in cancer medicine.Communicated by Ramaswamy H. Sarma.