ABSTRACT
Wnts are lipid-modified glycoproteins that play key roles in both embryonic development and adult homeostasis. Wnt signaling is dysregulated in many cancers and preclinical data shows that targeting Wnt biosynthesis and secretion can be effective in Wnt-addicted cancers. An integral membrane protein known as Wntless (WLS/Evi) is essential for Wnt secretion. However, WLS remains undrugged thus far. The cryo-EM structure of WLS in complex with WNT8A shows that WLS has a druggable G-protein coupled receptor (GPCR) domain. Using Active Learning/Glide, we performed an ultra-large scale virtual screening from Enamine's REAL 350/3 Lead-Like library containing nearly 500 million compounds. 68 hits were examined after on-demand synthesis in cell-based Wnt reporter and other functional assays. ETC-451 emerged as a potential first-in-class WLS inhibitor. ETC-451 blocked WLS-WNT3A interaction and decreased Wnt-addicted pancreatic cancer cell line proliferation. The current hit provides a starting chemical scaffold for further structure or ligand-based drug discovery targeting WLS.
ABSTRACT
UBE2T is an attractive target for drug development due to its linkage with several types of cancers. However, the druggability of ubiquitin-conjugating E2 (UBE2T) is low because of the lack of a deep and hydrophobic pocket capable of forming strong binding interactions with drug-like small molecules. Here, we performed fragment screening using 19 F-nuclear magnetic resonance (NMR) and validated the hits with 1 H-15 N-heteronuclear single quantum coherence (HSQC) experiment and X-ray crystallographic studies. The cocrystal structures obtained revealed the binding modes of the hit fragments and allowed for the characterization of the fragment-binding sites. Further screening of structural analogues resulted in the identification of a compound series with inhibitory effect on UBE2T activity. Our current study has identified two new binding pockets in UBE2T, which will be useful for the development of small molecules to regulate the function of this protein. In addition, the compounds identified in this study can serve as chemical starting points for the development of UBE2T modulators.
Subject(s)
Ubiquitin-Conjugating Enzymes , Ubiquitin , Ubiquitin-Conjugating Enzymes/metabolism , Binding SitesABSTRACT
UBE2T is an E2 ubiquitin ligase critical for ubiquitination of substrate and plays important roles in many diseases. Despite the important function, UBE2T is considered as an undruggable target due to lack of a pocket for binding to small molecules with satisfied properties for clinical applications. To develop potent and specific UBE2T inhibitors, we adopted a high-throughput screening assay and two compounds-ETC-6152 and ETC-9004 containing a sulfone tetrazole scaffold were identified. Solution NMR study demonstrated the direct interactions between UBE2T and compounds in solution. Further co-crystal structures reveal the binding modes of these compounds. Both compound hydrolysation and formation of a hydrogen bond with the thiol group of the catalytic cysteine were observed. The formation of covalent complex was confirmed with mass spectrometry. As these two compounds inhibit ubiquitin transfer, our study provides a strategy to develop potent inhibitors of UBE2T.
Subject(s)
Cysteine , Ubiquitin , Ubiquitin/metabolism , Cysteine/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitination , High-Throughput Screening AssaysABSTRACT
The atypical protein kinase C-iota (PKC-ι) enzyme is implicated in various cancers and has been put forward as an attractive target for developing anticancer therapy. A high concentration biochemical screen identified pyridine fragment weakly inhibiting PKC-ι with IC50 = 424 µM. Driven by structure-activity relationships and guided by docking hypothesis, the weakly bound fragment was eventually optimized into a potent inhibitor of PKC-ι (IC50= 270 nM). Through the course of the optimization, an intermediate compound was crystallized with the protein, and careful analysis of the X-ray crystal structure revealed a unique binding mode involving the post-kinase domain (C-terminal tail) of PKC-ι.
ABSTRACT
Even though many GyrB and ParE inhibitors have been reported in the literature, few possess activity against Gram-negative bacteria. This is primarily due to limited permeability across Gram-negative bacterial membrane as well as bacterial efflux mechanisms. Permeability of compounds across Gram-negative bacterial membranes depends on many factors including physicochemical properties of the inhibitors. Herein, we show the optimization of pyridylureas leading to compounds with potent activity against Gram-negative bacterial species such as P.aeruginosa, E.coli and A.baumannii.
Subject(s)
Anti-Bacterial Agents/pharmacology , DNA Gyrase/metabolism , DNA Topoisomerase IV/antagonists & inhibitors , Drug Discovery , Escherichia coli/drug effects , Topoisomerase Inhibitors/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , DNA Topoisomerase IV/metabolism , Dose-Response Relationship, Drug , Escherichia coli/enzymology , Microbial Sensitivity Tests , Molecular Structure , Streptococcus pneumoniae/drug effects , Streptococcus pneumoniae/enzymology , Structure-Activity Relationship , Topoisomerase Inhibitors/chemical synthesis , Topoisomerase Inhibitors/chemistryABSTRACT
Protein kinase C iota (PKC-ι) is an atypical kinase implicated in the promotion of different cancer types. A biochemical screen of a fragment library has identified several hits from which an azaindole-based scaffold was chosen for optimization. Driven by a structure-activity relationship and supported by molecular modeling, a weakly bound fragment was systematically grown into a potent and selective inhibitor against PKC-ι.
Subject(s)
Carcinoma, Hepatocellular/drug therapy , Cell Proliferation/drug effects , Isoenzymes/antagonists & inhibitors , Liver Neoplasms/drug therapy , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Carcinoma, Hepatocellular/pathology , Humans , Liver Neoplasms/pathology , Models, Molecular , Molecular Structure , Protein Conformation , Tumor Cells, CulturedABSTRACT
Overexpression of the eukaryotic initiation factor 4E (eIF4E) is linked to a variety of cancers. Both mitogen-activated protein kinases-interacting kinases 1 and 2 (Mnk1/2) activate the oncogene eIF4E through posttranslational modification (phosphorylating it at the conserved Ser209). Inhibition of Mnk prevents eIF4E phosphorylation, making the Mnk-eIF4E axis a potential therapeutic target for oncology. Recently, the design and synthesis of a series of novel potent compounds inhibiting the Mnk1/2 kinases were carried out in-house. Here, we describe computational models of the interactions between Mnk1/2 kinases and these inhibitors. Molecular modeling combined with free energy calculations show that these compounds bind to the inactive forms of the kinases. All compounds adopt similar conformations in the catalytic sites of both kinases, stabilized by hydrogen bonds with the hinge regions and with the catalytic Lys78 (Mnk1) and Lys113 (Mnk2). These hydrogen bond interactions clearly play a critical role in determining the conformational stability and potency of the compounds. We also find that van der Waals interactions with an allosteric pocket are key to their binding and potency. Two distinct hydration sites that appear to further stabilize the ligand binding/interactions were observed. Critically, the inclusion of explicit water molecules in the calculations results in improving the agreement between calculated and experimental binding free energies.
ABSTRACT
Bacterial topoisomerases are attractive antibacterial drug targets because of their importance in bacterial growth and low homology with other human topoisomerases. Structure-based drug design has been a proven approach of efficiently developing new antibiotics against these targets. Past studies have focused on developing lead compounds against the ATP binding pockets of both DNA gyrase and topoisomerase IV. A detailed understanding of the interactions between ligand and target in a solution state will provide valuable information for further developing drugs against topoisomerase IV targets. Here we describe a detailed characterization of a known potent inhibitor containing a 9H-pyrimido[4,5-b]indole scaffold against the N-terminal domain of the topoisomerase IV E subunit from Escherichia coli (eParE). Using a series of biophysical and biochemical experiments, it has been demonstrated that this inhibitor forms a tight complex with eParE. NMR studies revealed the exact protein residues responsible for inhibitor binding. Through comparative studies of two inhibitors of markedly varied potencies, it is hypothesized that gaining molecular interactions with residues in the α4 and residues close to the loop of ß1-α2 and residues in the loop of ß3-ß4 might improve the inhibitor potency.
Subject(s)
DNA Topoisomerase IV/antagonists & inhibitors , DNA Topoisomerase IV/chemistry , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Topoisomerase Inhibitors/chemistry , Humans , Indoles/chemistry , Nuclear Magnetic Resonance, Biomolecular , Protein Domains , Protein Structure, SecondaryABSTRACT
Clinically used BCR-ABL1 inhibitors for the treatment of chronic myeloid leukemia do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 and 2 inhibitors prevent phosphorylation of eIF4E and eliminate the self-renewal capacity of LSCs. Herein, we describe the identification of novel dual MNK1 and 2 and BCR-ABL1 inhibitors, starting from the known kinase inhibitor 2. Initial structure-activity relationship studies resulted in compound 27 with loss of BCR-ABL1 inhibition. Further modification led to orally bioavailable dual MNK1 and 2 and BCR-ABL1 inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level of phosphorylated eukaryotic translation initiation factor 4E in the tumor tissues. Kinase selectivity of these compounds is also presented.
Subject(s)
Fusion Proteins, bcr-abl/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Administration, Oral , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Biological Availability , Chemistry Techniques, Synthetic , Dose-Response Relationship, Drug , Eukaryotic Initiation Factor-4E/metabolism , Female , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Mice, SCID , Molecular Targeted Therapy/methods , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Structure-Activity Relationship , Xenograft Model Antitumor Assays/methodsABSTRACT
Bacterial DNA topoisomerases are essential for bacterial growth and are attractive, important targets for developing antibacterial drugs. Consequently, different potent inhibitors that target bacterial topoisomerases have been developed. However, the development of potent broad-spectrum inhibitors against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria has proven challenging. In this study, we carried out biophysical studies to better understand the molecular interactions between a potent bis-pyridylurea inhibitor and the active domains of the E-subunits of topoisomerase IV (ParE) from a G(+) strain (Streptococcus pneumoniae (sParE)) and a G(-) strain (Pseudomonas aeruginosa (pParE)). NMR results demonstrated that the inhibitor forms a tight complex with ParEs and the resulting complexes adopt structural conformations similar to those observed for free ParEs in solution. Further chemical-shift perturbation experiments and NOE analyses indicated that there are four regions in ParE that are important for inhibitor binding, namely, α2, the loop between ß2 and α3, and the ß2 and ß6 strands. Surface plasmon resonance showed that this inhibitor binds to sParE with a higher KD than pParE. Point mutations in α2 of ParE, such as A52S (sParE), affected its binding affinity with the inhibitor. Taken together, these results provide a better understanding of the development of broad-spectrum antibacterial agents.
Subject(s)
DNA Topoisomerase IV/chemistry , Amino Acid Sequence , DNA Topoisomerase IV/antagonists & inhibitors , DNA Topoisomerase IV/genetics , Models, Molecular , Molecular Sequence Data , Mutation , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Secondary , Pseudomonas aeruginosa , Solutions , Streptococcus pneumoniae , Surface Plasmon Resonance , TemperatureABSTRACT
Bacterial topoisomerase IV (ParE) is essential for DNA replication and serves as an attractive target for antibacterial drug development. The X-ray structure of the N-terminal 24 kDa ParE, responsible for ATP binding has been solved. Due to the accessibility of structural information of ParE, many potent ParE inhibitors have been discovered. In this study, a pyridylurea lead molecule against ParE of Escherichia coli (eParE) was characterized with a series of biochemical and biophysical techniques. More importantly, solution NMR analysis of compound binding to eParE provides better understanding of the molecular interactions between the inhibitor and eParE.
Subject(s)
Adenosine Triphosphate/metabolism , DNA Topoisomerase IV/metabolism , DNA Topoisomerase IV/pharmacology , Escherichia coli/enzymology , Adenosine Triphosphate/antagonists & inhibitors , Amino Acid Sequence , Anti-Bacterial Agents/pharmacology , Binding, Competitive , DNA Topoisomerase IV/antagonists & inhibitors , DNA Topoisomerase IV/chemistry , Drug Design , Molecular Sequence Data , Nuclear Magnetic Resonance, BiomolecularABSTRACT
The N-terminal ATP binding domain of the DNA gyrase B subunit is a validated drug target for antibacterial drug discovery. Structural information for this domain (pGyrB) from Pseudomonas aeruginosa is still missing. In this study, the interaction between pGyrB and a bis-pyridylurea inhibitor was characterized using several biophysical methods. We further carried out structural analysis of pGyrB using NMR spectroscopy. The secondary structures of free and inhibitor bound pGyrB were obtained based on backbone chemical shift assignment. Chemical shift perturbation and NOE experiments demonstrated that the inhibitor binds to the ATP binding pocket. The results of this study will be helpful for drug development targeting P. aeruginosa.
Subject(s)
Catalytic Domain , DNA Gyrase/chemistry , DNA Gyrase/metabolism , Pseudomonas aeruginosa/enzymology , Topoisomerase II Inhibitors/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Topoisomerase II Inhibitors/chemistry , Topoisomerase II Inhibitors/pharmacology , Urea/chemistry , Urea/metabolism , Urea/pharmacologyABSTRACT
PA-824 is a bicyclic 4-nitroimidazole, currently in phase II clinical trials for the treatment of tuberculosis. Dose fractionation pharmacokinetic-pharmacodynamic studies in mice indicated that the driver of PA-824 in vivo efficacy is the time during which the free drug concentrations in plasma are above the MIC (fT>MIC). In this study, a panel of closely related potent bicyclic 4-nitroimidazoles was profiled in both in vivo PK and efficacy studies. In an established murine TB model, the efficacy of diverse nitroimidazole analogs ranged between 0.5 and 2.3 log CFU reduction compared to untreated controls. Further, a retrospective analysis was performed for a set of seven nitroimidazole analogs to identify the PK parameters that correlate with in vivo efficacy. Our findings show that the in vivo efficacy of bicyclic 4-nitroimidazoles correlated better with lung PK than with plasma PK. Further, nitroimidazole analogs with moderate-to-high volume of distribution and Lung to plasma ratios of >2 showed good efficacy. Among all the PK-PD indices, total lung T>MIC correlated the best with in vivo efficacy (rsâ=â0.88) followed by lung Cmax/MIC and AUC/MIC. Thus, lung drug distribution studies could potentially be exploited to guide the selection of compounds for efficacy studies, thereby accelerating the drug discovery efforts in finding new nitroimidazole analogs.
Subject(s)
Nitroimidazoles/pharmacology , Nitroimidazoles/pharmacokinetics , Tuberculosis/drug therapy , Animals , Caco-2 Cells , Cell Line, Tumor , Disease Models, Animal , Female , Humans , Mice , Microbial Sensitivity Tests , Mycobacterium tuberculosis/drug effects , Retrospective StudiesABSTRACT
Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.
Subject(s)
Adenosine Triphosphate/antagonists & inhibitors , Antitubercular Agents/pharmacology , High-Throughput Screening Assays/methods , Homeostasis/drug effects , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/growth & development , Adenosine Triphosphate/physiology , Animals , Antitubercular Agents/chemistry , CHO Cells , Cell Survival/drug effects , Cell Survival/physiology , Cricetinae , Cricetulus , HeLa Cells , Homeostasis/physiology , Humans , Mycobacterium bovis/drug effects , Mycobacterium bovis/growth & developmentABSTRACT
Tuberculosis continues to be a global health threat, making bicyclic nitroimidazoles an important new class of therapeutics. A deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis catalyzes the reduction of nitroimidazoles such as PA-824, resulting in intracellular release of lethal reactive nitrogen species. The N-terminal 30 residues of Ddn are functionally important but are flexible or access multiple conformations, preventing structural characterization of the full-length, enzymatically active enzyme. Several structures were determined of a truncated, inactive Ddn protein core with and without bound F(420) deazaflavin coenzyme as well as of a catalytically competent homolog from Nocardia farcinica. Mutagenesis studies based on these structures identified residues important for binding of F(420) and PA-824. The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data.
Subject(s)
Models, Molecular , Mycobacterium tuberculosis/enzymology , Nitroreductases/chemistry , Nitroreductases/metabolism , Protein Conformation , Amino Acid Sequence , Flavins/metabolism , Molecular Sequence Data , Molecular Structure , Mutagenesis , Nitroimidazoles/metabolism , Nitroreductases/genetics , Protein Binding , Reactive Nitrogen Species/metabolismABSTRACT
The bicyclic 4-nitroimidazoles PA-824 and OPC-67683 represent a promising novel class of therapeutics for tuberculosis and are currently in phase II clinical development. Both compounds are pro-drugs that are reductively activated by a deazaflavin (F(420)) dependent nitroreductase (Ddn). Herein we describe the biochemical properties of Ddn including the optimal enzymatic turnover conditions and substrate specificity. The preference of the enzyme for the (S) isomer of PA-824 over the (R) isomer is directed by the presence of a long hydrophobic tail. Nitroimidazo-oxazoles bearing only short alkyl substituents at the C-7 position of the oxazole were reduced by Ddn without any stereochemical preference. However, with bulkier substitutions on the tail of the oxazole, Ddn displayed stereospecificity. Ddn mediated metabolism of PA-824 results in the release of reactive nitrogen species. We have employed a direct chemiluminescence based nitric oxide (NO) detection assay to measure the kinetics of NO production by Ddn. Binding affinity of PA-824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating a turnover-independent assessment of affinity. Our results indicate that (R)-PA-824, despite not being turned over by Ddn, binds to the enzyme with the same affinity as the active (S) isomer. This result, in combination with docking studies in the active site, suggests that the (R) isomer probably has a different binding mode than the (S) with the C-3 of the imidazole ring orienting in a non-productive position with respect to the incoming hydride from F(420). The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding.
Subject(s)
Antitubercular Agents/pharmacology , Flavins/metabolism , Mycobacterium tuberculosis/enzymology , Nitroimidazoles/pharmacology , Nitroreductases/metabolism , Oxazoles/pharmacology , Cloning, Molecular , Kinetics , Nitric Oxide/metabolism , Nitroreductases/genetics , Nitroreductases/isolation & purification , Protein Conformation , Stereoisomerism , Structure-Activity Relationship , Substrate SpecificityABSTRACT
The (S)-2-nitro-6-(4-(trifluoromethoxy)benzyloxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine named PA-824 (1) has demonstrated antitubercular activity in vitro and in animal models and is currently in clinical trials. We synthesized derivatives at three positions of the 4-(trifluoromethoxy)benzylamino tail, and these were tested for whole-cell activity against both replicating and nonreplicating Mycobacterium tuberculosis (Mtb). In addition, we determined their kinetic parameters as substrates of the deazaflavin-dependent nitroreductase (Ddn) from Mtb that reductively activates these pro-drugs. These studies yielded multiple compounds with 40 nM aerobic whole cell activity and 1.6 µM anaerobic whole cell activity: 10-fold improvements over both characteristics from the parent molecule. Some of these compounds exhibited enhanced solubility with acceptable stability to microsomal and in vivo metabolism. Analysis of the conformational preferences of these analogues using quantum chemistry suggests a preference for a pseudoequatorial orientation of the linker and lipophilic tail.
Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/metabolism , Mycobacterium tuberculosis/drug effects , Nitroimidazoles/pharmacology , Nitroreductases/metabolism , Prodrugs/pharmacology , Animals , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacokinetics , Drug Evaluation, Preclinical/methods , Humans , Kinetics , Metabolic Clearance Rate , Mice , Microbial Sensitivity Tests , Microsomes, Liver/metabolism , Molecular Structure , Mycobacterium tuberculosis/enzymology , Nitroimidazoles/chemistry , Nitroimidazoles/pharmacokinetics , Prodrugs/chemistry , Prodrugs/metabolism , Structure-Activity Relationship , Substrate Specificity , Tuberculosis/drug therapy , Tuberculosis/microbiologyABSTRACT
Antibiotics are typically more effective against replicating rather than nonreplicating bacteria. However, a major need in global health is to eradicate persistent or nonreplicating subpopulations of bacteria such as Mycobacterium tuberculosis (Mtb). Hence, identifying chemical inhibitors that selectively kill bacteria that are not replicating is of practical importance. To address this, we screened for inhibitors of dihydrolipoamide acyltransferase (DlaT), an enzyme required by Mtb to cause tuberculosis in guinea pigs and used by the bacterium to resist nitric oxide-derived reactive nitrogen intermediates, a stress encountered in the host. Chemical screening for inhibitors of Mtb DlaT identified select rhodanines as compounds that almost exclusively kill nonreplicating mycobacteria in synergy with products of host immunity, such as nitric oxide and hypoxia, and are effective on bacteria within macrophages, a cellular reservoir for latent Mtb. Compounds that kill nonreplicating pathogens in cooperation with host immunity could complement the conventional chemotherapy of infectious disease.
Subject(s)
Acyltransferases/antagonists & inhibitors , Antitubercular Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Microbial Viability/drug effects , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/immunology , Rhodanine/pharmacology , Acyltransferases/genetics , Animals , Bacterial Proteins/genetics , Cells, Cultured , Colony Count, Microbial , Enzyme Inhibitors/pharmacology , Gene Deletion , Genetic Complementation Test , Guinea Pigs , Hypoxia/immunology , Lung/microbiology , Macrophages/drug effects , Macrophages/microbiology , Molecular Structure , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/pathogenicity , Nitric Oxide/immunology , Rhodanine/chemistry , Rhodanine/toxicity , Tuberculosis/immunology , Tuberculosis/microbiology , Virulence , Virulence Factors/antagonists & inhibitors , Virulence Factors/geneticsABSTRACT
OBJECTIVE: To derive population norms on the Malayalam adaptation of Addenbrooke's Cognitive Examination (M-ACE) and the inclusive Malayalam mini mental state examination (M-MMSE). MATERIALS AND METHODS: Education-stratified norms were obtained on randomly selected cognitively unimpaired community elders (n = 519). RESULTS: Valid data on norms was available on 488 subjects (age 68.5 +/- 7.1 and education 7.9 +/- 5.4). Education and age, but not gender had a significant effect on both M-ACE and M-MMSE. When compared to the effect of age, the effect of education was sevenfold more on the M-ACE and ninefold more on the M-MMSE. The mean composite score on the M-ACE (and the M-MMSE) was 42.8 +/- 9.8 (14.9 +/- 3.1) for those with 0 (n = 72), 55.9 +/- 12.5 (19.7 +/- 4.1) with 1-4 (n = 96), 62.6 +/- 11.4 (21.9 +/- 3.7) with 5-8 (n = 81), 77 +/- 10.2 (25.7 +/- 2.4) with 9-12 (n = 136) and 83.4 +/- 7.2 (26.7 +/- 1.6) with > 12 (n = 103) years of formal education. CONCLUSIONS: Education has the most potent effect on performance on both M-ACE and M-MMSE in the Indian cohort. Education-stratified scores on the M-ACE and the M-MMSE, will provide a more appropriate means of establishing the cognitive status of patients. It is also our feeling that these cut-off scores will be useful across India.