Facilitated diffusion of glucosamine-6-phosphate synthase inhibitors enhances their antifungal activity

N3-(4-Methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP) and 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP) are strong inhibitors of the essential fungal enzyme, glucosamine-6-phosphate synthase, but their antifungal activity is poor, due to slow penetration of these agents through the cytoplasmic membrane. In the present studies we have exploited the possibility of enhancement of ADGP and FMDP antifungal activity by improving their transport properties. It has been found that membrane-permeabilising polyene macrolides amphotericin B (AMB) and its N-methyl-N-fructosyl methyl ester derivative (MF-AME), at subinhibitory concentrations, facilitate diffusion of ADGP through the fungal cell membrane, thus allowing a decrease of its minimal inhibitory concentration (MIC). Synergistic effects have been observed for combinations of ADGP with AMB or MF-AME. Fractional inhibitory concentration (FIC) indexes, determined against a number of Candida spp., have been in the 0.18-0.81 range. Weak antifungal synergistic effects have been found for combinations of FMDP with AMB or MF-AME. ADGP can be easily encapsulated into unilamellar lipid vesicles. Liposomal preparations of ADGP demonstrated stronger antifungal activity against some fungal strains than free ADGP.     

Hydrophobic derivatives of 2-amino-2-deoxy-D-glucitol-6-phosphate: a new type of D-glucosamine-6-phosphate synthase inhibitors with antifungal action

Several N-acyl and ester derivatives of 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP) have been synthesised and tested as inhibitors of fungal enzymes involved in early steps of chitin biosynthesis and for antifungal activity. All the tested derivatives were found to be much poorer inhibitors of the enzyme, D-glucosamine-6-phosphate (GlcN-6-P) synthase, than the parent compound but some of them exhibited much better antifungal activity. MIC values for the investigated compounds ranged between 10 mg mL(-1), found for ADGP and 0.3 mg mL(-1) for the most active derivative, namely ADGP dimethyl ester. Increased affinity of ADGP derivatives to the artificial immobilised cell membrane was correlated with their enhanced ability to be taken up by fungal cells by free diffusion. It was found that some of the examined derivatives behaved as 'pro-drugs' and after internalisation were converted into ADGP in the cell-free extract. This conversion was relatively rapid for ADGP esters but very slow for N-acyl derivatives. Results of our studies demonstrate a possibility of design and preparation of GlcN-6-P synthase inhibitors exhibiting antifungal activity.     

Glucosamine-6-phosphate synthase, a novel target for antifungal agents. Molecular modelling studies in drug design

Fungal infections are a growing problem in contemporary medicine, yet only a few antifungal agents are used in clinical practice. In our laboratory we proposed the enzyme L-glutamine: D-fructose-6-phosphate amidotransferase (EC 2.6.1.16) as a new target for antifungals. The structure of this enzyme consists of two domains, N-terminal and C-terminal ones, catalysing glutamine hydrolysis and sugar-phosphate isomerisation, respectively. In our laboratory a series of potent selective inhibitors of GlcN-6-P synthase have been designed and synthesised. One group of these compounds, including the most studied N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid (FMDP), behave like glutamine analogs acting as active-site-directed inactivators, blocking the N-terminal, glutamine-binding domain of the enzyme. The second group of GlcN-6-P synthase inhibitors mimic the transition state of the reaction taking place in the C-terminal sugar isomerising domain. Surprisingly, in spite of the fact that glutamine is the source of nitrogen for a number of enzymes it turned out that the glutamine analogue FMDP and its derivatives are selective against GlcN-6-P synthase and they do not block other enzymes, even belonging to the same family of glutamine amidotransferases. Our molecular modelling studies of this phenomenon revealed that even within the family of related enzymes substantial differences may exist in the geometry of the active site. In the case of the glutamine amidotransferase family the glutamine binding site of GlcN-6-P synthase fits a different region of the glutamine conformational space than other amidotransferases. Detailed analysis of the interaction pattern for the best known, so far, inhibitor of the sugar isomerising domain, namely 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP), allowed us to suggest changes in the structure of the inhibitor that should improve the interaction pattern. The novel ligand was designed and synthesised. Biological experiments confirmed our predictions. The new compound named ADMP is a much better inhibitor of glucosamine-6-phosphate synthase than ADGP.     

Inhibitors of glucosamine-6-phosphate synthase as potential antimicrobials or antidiabetics – synthesis and properties

Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is known as a promising target for antimicrobial agents and antidiabetics. Several compounds of natural or synthetic origin have been identified as inhibitors of this enzyme. This set comprises highly selective l-glutamine, amino sugar phosphate or transition state intermediate cis-enolamine analogues. Relatively low antimicrobial activity of these inhibitors, poorly penetrating microbial cell membranes, has been improved using the pro-drug approach. On the other hand, a number of heterocyclic and polycyclic compounds demonstrating antimicrobial activity have been presented as putative inhibitors of the enzyme, based on the results of molecular docking to GlcN-6-P synthase matrix. The most active compounds of this group could be considered promising leads for development of novel antimicrobial drugs or antidiabetics, provided their selective toxicity is confirmed.     

Studies on substrate specificity and activity regulating factors of trehalose-6-phosphate synthase of Saccharomyces cerevisiae

Purified trehalose-6-phosphate synthase (TPS) of Saccharomyces cerevisiae was effective over a wide range of substrates, although differing with regard to their relative activity. Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity, particularly when a pyrimidine glucose nucleotide like UDPG was used, rather than a purine glucose nucleotide like GDPG. A high V_max and a low K_m value of UDPG show its greater affinity with TPS than GDPG or TDPG. Among the glucosyl acceptors TPS showed maximum activity with G-6-P which was followed by M-6-P and F-6-P. Effect of heparin was also extended to the purification of TPS activity, as it helped to retain both stability and activity of the final purified enzyme. Metal co-factors, specifically MnCl₂ and ZnCl₂ acted as stimulators, while enzyme inhibitors had very little effect on TPS activity. Metal chelators like CDTA, EGTA stimulated enzyme activity by chelation of metal inhibitors. Temperature and pH optima of the purified enzyme were determined to be 40 °C and pH 8.5 respectively. Enzyme activity was stable at 0–40 °C and at alkaline pH.     

Variability analyses of functional domains within glucosamine-6-phosphate synthase of mycosescausing fungi

The immunosuppressive individuals are highly prone to get afflicted with invasive opportunistic fungal infections such as Candidiasis, Aspergillosis, Histoplasmosis, Coccidioidomycosis, Blastomycosis, Penicilliosis, Cryptococcosis and Zygomycosis which are becoming a cause of concern to the mankind due to their high morbidity and mortality rates. The existing antifungal agents are not completely effective due to their severe side-effects and recurrent drug resistance in fungi. Hence, there is an urgent need to develop newer and better antifungal drugs. The enzyme Glucosamine-6-phosphate (G-6-P) synthase catalyzes the ratelimiting step of the fungal cell-wall biosynthetic pathway and targeting it can inhibit the growth of the fungus. The present study attempts to investigate the inherent variations in functional domain viz. Glutaminase (GATase II) and Sugar Isomerising (SIS) of Glucosamine-6-phosphate (G-6-P) synthase enzyme of mycoses-causing fungi. These domains may be identified as probable active site(s). Multiple sequence alignment performed using ClustalX2 and construction of phylogenetic tree of individual domains by MEGA v5.0 helped in the analyses of several variable amino acid sites within the domains suggesting their vital role in the pathogenesis of the fungi. Further, the online server ConSurf implied that mostly, the highly conserved residues of the domains were functional and exposed on the surface of the active site, making it an easy target for the drugs. Consequently, variable analysis of functional domains of target implicated the importance of target specific drug discovery for the treatment of invasive fungal infections or mycoses.     

Investigation of the inhibition pathway of glucosamine synthase by N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid by semiempirical quantum mechanical and molecular mechanics methods

Glucosamine synthase (E.C. 2.6.1.16) is a promising target in antifungal drug design. It has been reported that its potent inhibitor, N³-(4-methoxyfu-maroyl)-L-2,3-diaminopropanoic acid (FMDP), inactivates the enzyme by the Michael addition of the S-H group to the FMDP molecule followed by cyclisation reactions. In this study we have investigated, by means of semiempirical MNDO, PM3 and molecular mechanics methods, the energetics and kinetic possibility of the formation of various stereoisomers of the products of cyclisation of the Michael addition products detected experimentally. It was found that the substituted 1,4-thiazin-3-one can be formed in one step under alkaline conditions; the stereoisomers of this compound predicted to be the most stable on the basis of theoretical calculations are also the dominant ones in reality.Abbreviations FMDP N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid - MNDO Modified Neglect of Diatomic Overlap - AM1 Advanced Method 1 - PM3 (full abbreviation: MNDOPM3) Modified Neglect of Diatomic Overlap, Parametrised Method 3 Correspondence to: A. Liwo     

Rational design of N-alkyl derivatives of 2-amino-2-deoxy-d-glucitol-6P as antifungal agents

N-Alkyl and N,N-dialkyl derivatives of 2-amino-2-deoxy-d-glucitol-6P (ADGP) were synthesized and found to inhibit growth of human pathogenic fungi (MICs in the 0.08-0.625mgmL(-1) range for the most active compounds). It was thus shown that N-alkylation of ADGP provides novel inhibitors of a fungal enzyme, glucosamine-6P synthase, exhibiting higher antifungal activity than the parent compound, due to the increased lipophilicity and better uptake by fungal cells.     

Peptide dendrimers as antifungal agents and carriers for potential antifungal agent—N3‐(4‐methoxyfumaroyl)‐(S)‐2,3‐diaminopropanoic acid—synthesis and antimicrobial activity

A series of peptide dendrimers and their conjugates with antimicrobial agent FMDP (N³‐(4‐methoxyfumaroyl)‐(S)‐2,3‐diamino‐propanoic acid) were synthesized. The obtained compounds were tested for the antibacterial and antifungal activity. All novel dendrimers displayed much better activity against the tested strains than FMDP itself. Moreover, their conjugates with FMDP also exhibited antimicrobial activity. The most promising molecules were tested against a broad selection of fungal strains. The analysis of their antifungal properties indicates that the examined molecules are efficient growth inhibitors of fluconazole‐resistant hospital‐acquired strains. Moreover, an application of amphiphilic branched peptides such as FMDP carriers suggests that transport mechanism involves more likely the cell membrane perturbation than the mediation of the specific transport proteins. The activity of obtained compounds strongly depends on the specific structure of the molecule.     

Crystal structure of Bifidobacterium Longum phosphoketolase; key enzyme for glucose metabolism in Bifidobacterium

The crystal structure of Bifidobacterium longum phosphoketolase, a thiamine diphosphate (TPP) dependent enzyme, has been determined at 2.2 Å resolution. The enzyme is a dimer with the active sites located at the interface between the two identical subunits with molecular mass of 92.5 kDa. The bound TPP is almost completely shielded from solvent except for the catalytically important C2-carbon of the thiazolium ring, which can be accessed by a substrate sugar through a narrow funnel-shaped channel. In silico docking studies of B. longum phosphoketolase with its substrate enable us to propose a model for substrate binding.

Structured summary

MINT-7985878: PKT (uniprotkb:Q6R2Q7) and PKT (uniprotkb:Q6R2Q7) bind (MI:0407) by X-ray crystallography (MI:0114)     

Characterization of a glyphosate-insensitive 5-enolpyruvylshikimic acid-3-phosphate synthase

A glyphosate (N-[phosphonomethyl]glycine)-insensitive 5-enolpyruvylshikimic acid-3-phosphate (EPSP) synthase has been purified from a strain of Klebsiella pneumoniae which is resistant to this herbicide [(1984) Arch. Microbiol. 137, 121-123] and its properties compared with those of the glyphosate-sensitive EPSP synthase of the parent strain. The apparent K_m values of the insensitive enzyme for phosphoenolpyruvate (PEP) and shikimate 3-phosphate (S-3-P) were increased 15.6- and 4.3-fold, respectively, as compared to those of the sensitive enzyme, and significant differences were found for the optimal pH and temperature, as well as the isoelectric points of the two enzymes. While PEP protected both enzymes against inactivation by N-ethylmaleimide, 3-bromopyruvate, and phenylglyoxal, glyphosate protected only the sensitive enzyme.     

Design, synthesis, and evaluations of antifungal activity of novel phenyl(2H-tetrazol-5-yl)methanamine derivatives

Fungal infections pose a continuous and serious threat to human health and life. The intrinsic resistance has been observed in many genera of fungi. Many fungal infections are caused by opportunistic pathogens that may be endogenous (Candida infections) or acquired from the environment (Cryptococcus and Aspergillus infections). So, new therapeutic strategies are needed to combat various fungal infections. Fluconazole shows good antifungal activity with relatively low toxicity and is preferred as first line antifungal therapy, but it has suffered from severe drug resistance. So, there is a need to design novel analogues by modification of fluconazole-like structure. A novel series of phenyl(2H-tetrazol-5-yl)methanamine derivatives were synthesized by reaction of α-amino nitrile with sodium azide and ZnCl₂ in presence of isopropyl alcohol. They were evaluated for antifungal activity against Candida albicans and Aspergillus niger and subjected to docking study against 1EA1.     

Long range molecular dynamics study of regulation of eukaryotic glucosamine-6-phosphate synthase activity by UDP-GlcNAc

Glucosamine-6-phosphate (GlcN-6-P) synthase catalyses the first and practically irreversible step in hexosamine metabolism. The final product of this pathway, uridine 5’ diphospho N-acetyl-D-glucosamine (UDP-GlcNAc), is an essential substrate for assembly of bacterial and fungal cell walls. Moreover, the enzyme is involved in phenomenon of hexosamine induced insulin resistance in type II diabetes, which makes it a potential target for antifungal, antibacterial and antidiabetic therapy. The crystal structure of the isomerase domain of GlcN-6-P synthase from human pathogenic fungus Candida albicans, in complex with UDP-GlcNAc has been solved recently but it has not revealed the molecular mechanism of inhibition taking place under UDP-GlcNAc influence, the unique feature of the eukaryotic enzyme. UDP-GlcNAc is a physiological inhibitor of GlcN-6-P synthase, binding about 1 nm away from the active site of the enzyme. In the present work, comparative molecular dynamics simulations of the free and UDP-GlcNAc-bounded structures of GlcN-6-P synthase have been performed. The aim was to complete static X-ray structural data and detect possible changes in the dynamics of the two structures. Results of the simulation studies demonstrated higher mobility of the free structure when compared to the liganded one. Several amino acid residues were identified, flexibility of which is strongly affected upon UDP-GlcNAc binding. Importantly, the most fixed residues are those related to the inhibitor binding process and to the catalytic reaction. The obtained results constitute an important step toward understanding of mechanism of GlcN-6-P synthase inhibition by UDP-GlcNAc molecule.     

Modeling of human M1 aminopeptidases for in silico screening of potential Plasmodium falciparum alanine aminopeptidase (PfA-M1) specific inhibitors

Plasmodium falciparum alanine M1-aminopeptidase (PfA-M1) is a validated target for anti-malarial drug development. Presence of significant similarity between PfA-M1 and human M1-aminopeptidases, particularly within regions of enzyme active site leads to problem of non-specificity and off-target binding for known aminopeptidase inhibitors. Molecular docking based in silico screening approach for off-target binding has high potential but requires 3D-structure of all human M1-aminopeptidaes. Therefore, in the present study 3D structural models of seven human M1-aminopeptidases were developed. The robustness of docking parameters and quality of predicted human M1-aminopeptidases structural models was evaluated by stereochemical analysis and docking of their respective known inhibitors. The docking scores were in agreement with the inhibitory concentrations elucidated in enzyme assays of respective inhibitor enzyme combinations (r2≈0.70). Further docking analysis of fifteen potential PfA-M1 inhibitors (virtual screening identified) showed that three compounds had less docking affinity for human M1-aminopeptidases as compared to PfA-M1. These three identified potential lead compounds can be validated with enzyme assays and used as a scaffold for designing of new compounds with increased specificity towards PfA-M1.     

Structural analogues of reactive intermediates as inhibitors of glucosamine-6-phosphate synthase and phosphoglucose isomerase

The active centers of phosphoglucose isomerase (PGI) and the hexose phosphate isomerase domain (HPI) of glucosamine-6-P (GlcN-6-P) synthase demonstrate apparent similarity in spatial arrangement of critical amino acid residues, except Arg272 of the former and Lys603 and Lys485 of the latter. Ten derivatives of d-hexitol-6-P, 5-phosphoarabinoate, or 6-phosphogluconate, structural analogues of putative cis-enolamine or cis-enolate intermediates, were tested as inhibitors of fungal GlcN-6-P synthase and PGI. None of the investigated compounds demonstrated equally high inhibitory potential against both enzymes. 2-Amino-2-deoxy-D-mannitol 6-P was found to be the strongest GlcN-6-P synthase inhibitor in the series, with an inhibition constant equal to 9.0 (+/-1.0) x 10(-6)M. On the contrary, 5-phosphoarabinoate (5PA) exhibited specificity for PGI, with K(i)=2.2 (+/-0.1) x 10(-6) M. N-acetylation substantially lowered the GlcN-6-P synthase inhibitory potential of 2-amino-2-deoxy-D-glucitol-6-P but strongly enhanced inhibitory potential of this compound towards PGI. Molecular modeling studies revealed that interactions of the C1-C2 part of transition state analogue inhibitors with the respective areas demonstrating different distribution of molecular electrostatic potential (MEP) inside HPI and PGI active centers determined enzyme:ligand affinity. In Escherichia coli HPI, a patch of the negative potential created by Glu488 aided by Val399, supposed to stabilize a putative positively charged intermediate, especially attracts ligands containing 2-amino function. The Arg272, Lys210, and Gly271 peptide bond nitrogen system, present in the corresponding space of rabbit PGI, creates an area of positive MEP, stabilizing cis-enolate intermediate and attracting its structural mimics, such as 5PA.     

Antifungal properties of a peptide derived from the signal peptide of the HIV-1 regulatory protein, Rev

Antifungal effects of nuclear entry inhibitory signal peptide of HIV-1 Rev protein (Rev-NIS) were investigated. Rev-NIS contained potent antifungal activities without hemolytic effects. To understand the antifungal mechanism(s), in vivo and in vitro fluorescent studies were conducted. Flow cytometric analysis with bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] and calcein-leakage measurement from large unilamellar vesicles (LUVs) indicated that Rev-NIS depolarized and disrupted the fungal membranes. These results were further confirmed by using giant unilamellar vesicles (GUVs). The current study suggests that Rev-NIS exerts its antifungal activity with membrane-active mechanism(s).     

Arginine mediated purification of trehalose-6-phosphate synthase (TPS) from Candida utilis: Its characterization and regulation

Background

Trehalose is the most important multifunctional, non-reducing disaccharide found in nature. It is synthesized in yeast by an enzyme complex: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP).

Methods

In the present study TPS is purified using a new methodology from Candida utilis cells by inclusion of 100 mM l-arginine during cell lysis and in the mobile phase of high performance gel filtration liquid chromatography (HPGFLC).

Results

An electrophoretically homogenous TPS that was purified was a 60 kDa protein with 22.1 fold purification having a specific activity of 2.03 U/mg. Alignment of the N-terminal sequence with TPS from Saccharomyces cerevisiae confirmed the 60 kDa protein to be TPS. Optimum activity of TPS was observed at a protein concentration of 1 μg, at a temperature of 37 °C and pH 8.5. Aggregation mediated enzyme regulation was indicated. Metal cofactors, especially MnCl₂, MgCl₂ and ZnSO₄, acted as stimulators. Metal chelators like CDTA and EGTA stimulated enzyme activity. Among the four glucosyl donors, the highest V_max and lowest K_m values were calculated as 2.96 U/mg and 1.36 mM when adenosine di phosphate synthase (ADPG) was used as substrate. Among the glucosyl acceptors, glucose-6-phosphate (G-6-P) showed maximum activity followed by fructose-6-phosphate (F-6-P). Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity with different glucosyl donors.

General significance

Substrate specificity, V_max and K_m values provided an insight into an altered trehalose metabolic pathway in the C. utilis strain where ADPG is the preferred substrate rather than the usual substrate uridine diphosphaphate glucose (UDPG). The present work employs a new purification strategy as well as highlights an altered pathway in C. utilis.     

Effects of cadmium and zinc ions on purified lamb kidney cortex glucose-6-phosphate dehydrogenase activity

Glucose-6-phosphate dehydrogenase (G-6-PD) is the first enzyme in the pentose phosphate pathway. Cadmium is a toxic heavy metal that inhibits several enzymes. Zinc is an essential metal but overdoses of zinc have toxic effects on enzyme activities. In this study G-6-PD from lamb kidney cortex was competitively inhibited by zinc both with respect to glucose-6-phosphate (G-6-P) and NADP⁺ with Ki values of 1.066 ± 0.106 and 0.111 ± 0.007 mM respectively whereas cadmium was a non-competitive inhibitor with respect to both G-6-P and NADP⁺ Ki values of 2.028 ± 0.175 and 2.044 ± 0.289 mM respectively.     

Ordering of C-terminal loop and glutaminase domains of glucosamine-6-phosphate synthase promotes sugar ring opening and formation of the ammonia channel

Glucosamine-6-phosphate synthase (GlmS) channels ammonia from glutamine at the glutaminase site to fructose 6-phosphate (Fru6P) at the synthase site. Escherichia coli GlmS is composed of two C-terminal synthase domains that form the dimer interface and two N-terminal glutaminase domains at its periphery. We report the crystal structures of GlmS alone and in complex with the glucosamine-6-phosphate product at 2.95 Å and 2.9 Å resolution, respectively. Surprisingly, although the whole protein is present in this crystal form, no electron density for the glutaminase domain was observed, indicating its mobility. Comparison of the two structures with that of the previously reported GlmS-Fru6P complex shows that, upon sugar binding, the C-terminal loop, which forms the major part of the channel walls, becomes ordered and covers the synthase site. The ordering of the glutaminase domains likely follows Fru6P binding by the anchoring of Trp74, which acts as the gate of the channel, on the closed C-terminal loop. This is accompanied by a major conformational change of the side chain of Lys503^# of the neighboring synthase domain that strengthens the interactions of the synthase domain with the C-terminal loop and completely shields the synthase site. The concomitant conformational change of the Lys503^#-Gly505^# tripeptide places catalytic His504^# in the proper position to open the sugar and buries the linear sugar, which is now in the vicinity of the catalytic groups involved in the sugar isomerization reaction. Together with the previously reported structures of GlmS in complex with Fru6P or glucose 6-phosphate and a glutamine analogue, the new structures reveal the structural changes occurring during the whole catalytic cycle.     

Identification of Macrolepiota procera extract as a novel G6PD inhibitor for the treatment of lung cancer

Tumor metabolism, an emerging hallmark of cancer, is characterized by aberrant expression of enzymes from various metabolic pathways including glycolysis and PPP (pentose phosphate pathway). Glucose 6 phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD), oxidative carboxylases of PPP, have been reported to accomplish different biosynthetic and energy requirements of cancer cells. G6PD and 6PGD have been proposed as potential therapeutic targets for cancer therapy during recent years due to their overexpression in various cancers. Here, we have employed enzymatic assay based screening using in-house G6PD and 6PGD assay protocols for the identification of mushroom extracts which could inhibit G6PD or 6PGD enzymatic activity for implications in cancer therapy. For the fulfillment of the objectives of present study, nine edible mushrooms were subjected to green extraction for preparation of ethanolic extracts. 6xhis-G6PD and pET-28a-h6PGD plasmids were expressed in BL21-DE3 E. coli cells for the expression and purification of protein of interests. Using purified proteins, in house enzymatic assay protocols were established. The preliminary screening identified two extracts (Macrolepiota procera and Terfezia boudieri) as potent and selective G6PD inhibitors, while no extract was found highly active against 6PGD. Further, evaluation of anticancer potential of mushroom extracts against lung cancer cells revealed Macrolepiota procera as potential inhibitor of cancer cell proliferation with IC₅₀ value of 6.18 μg/ml. Finally, screening of M. procera-derived compounds against G6PD via molecular docking has identified paraben, quercetin and syringic acid as virtual hit compounds possessing good binding affinity with G6PD. The result of present study provides novel findings for possible mechanism of action of M. procera extract against A549 via G6PD inhibition suggesting that M. procera might be of therapeutic interest for lung cancer treatment.