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Protein tyrosine phosphatase 1B inhibitors from the fungus Malbranchea albolutea
Abstract
From solid rice-based cultures of Malbranchea albolutea, three undescribed ardeemins and sartoryglabrins analogs were discovered and named alboluteins A-C. 1H-Indole-3-carbaldehyde, and anthranilic acid were also isolated. 1D and 2D-NMR techniques, as well as DFT-calculated chemical shifts, allowed characterizing alboluteins A-C. Testing these compounds against PTP1B indicated their inhibitory activity with IC50's ranging from 19 to 129 μM (ursolic acid IC50 = 29.8 μM, positive control). Kinetic analysis revealed that albolutein C behaved as a non-competitive inhibitor. Docking studies of alboluteins A-C into the crystal structure of PTP1B (PDB ID: 1T49) predicted that all compounds prefer to bind at the allosteric site of the enzyme, with Ki values of 2.02 × 10⁻⁴, 1.31 × 10⁻⁴, and 2.67 × 10⁻⁴ mM, respectively. Molecular dynamic studies indicated that the active compounds remained tied to the enzyme with good binding energy.
... In the search for new antibacterial secondary metabolites from marine fungi, we identified a deep-sea sediment-derived fungus Malbranchea circinata SDU050 through activityguided screening. The genus Malbranchea is known for producing structurally diverse and biologically active secondary metabolites, including pyrrole alkaloids with anticancer properties, indole terpenoid alkaloids with vasorelaxant and calmodulin inhibitor activities, and polyketides with antidiabetic potential [17][18][19]. After the scale-up fermentation of M. circinata SDU050 in potato dextrose broth (PDB), we isolated and identified six compounds, including three isocoumarins (1-3), a butyrolactone derivative (4), a dipeptide (5), and a hydroxybenzoic acid derivative (6). ...
... To explore the biosynthetic potential of this strain, a metabolic blockade strategy was employed to activate or enhance the production of additional polyketides by inactivating the core PKS gene responsible for the biosynthesis of sclerin (1), the predominant metabolite produced by the strain. This approach successfully led to the isolation and identification of three new cytochalasins (7)(8)(9), along with four usnic acid derivatives (10)(11)(12)(13), four glycosylated anthraquinones (14)(15)(16)(17), two xanthones (18)(19), and three acetophenone derivatives (20)(21)(22). Compounds 1 and 10-13 were evaluated for their antibacterial activity and all isolates were evaluated for cytotoxic activity. ...
... Instead, a series of previously unobserved peaks was detected, indicating that the metabolic blockade strategy successfully activated or increased the production of previously undetected secondary metabolites (Figure 1a). A subsequent 20 L fermentation of the ∆sclA mutant strain resulted in the isolation and identification of 16 polyketides, classified into five PKS families: cytochalasins (7-9), usnic acids (10-13), glycosylated anthraquinones (14)(15)(16)(17), xanthones (18)(19), and acetophenone derivatives (20)(21)(22). Notably, three cytochalasins (7-9) were identified for the first time ( Figure 1d). ...
Malbranchea circinata SDU050, a fungus derived from deep-sea sediment, is a prolific producer of diverse secondary metabolites. Genome sequencing revealed the presence of at least 69 biosynthetic gene clusters (BGCs), including 30 encoding type I polyketide synthases (PKSs). This study reports the isolation and identification of four classes of secondary metabolites from wild-type M. circinata SDU050, alongside five additional metabolite classes, including three novel cytochalasins (7–9), obtained from a mutant strain through the metabolic blockade strategy. Furthermore, bioinformatic analysis of the BGC associated with the isocoumarin sclerin (1) enabled the deduction of its biosynthetic pathway based on gene function predictions. Bioactivity assays demonstrated that sclerin (1) and (−)-mycousnine (10) exhibited weak antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Bacillus subtilis. These findings underscore the chemical diversity and biosynthetic potential of M. circinata SDU050 and highlight an effective strategy for exploring marine fungal metabolites.
... In recent years, some metabolites isolated from Malbranchea spp., including glycosylated anthraquinones, anthranilic acid peptides, diketopiperazines, quinazolinone type alkaloids, and polyketides have demonstrated inhibitory activity against the enzymes AGs and protein tyrosine phosphatase 1B (PTP1B), both important enzymes involved in the metabolism of glucose. [7][8][9][10] According to the chemotaxonomic criteria, the present work aims to investigate Malbranchea flocciformis Sigler & J. W. Carmichael (Malbrancheaceae) as a new source of potential antidiabetic compounds. ...
... Thus, the compounds manually annotated found in M. flocciformis extract have also been isolated in some other Malbranchea spp.: Rosigenin (5) was previously isolated from M. flavorosea; malfilanol B (6), malfilamentosides A and B (7 and 8), malsterosides A and C (9 and 10), gotjawalide (11), gymnascolide A (1), and N-6-isopentenyladenosine (12; prenylated derivative of 2) from M. filamentosa; [8,[11][12][13][14] compound 1 was also found in M. dendritica; [9] and albolutein C (13) in M. albolutea. [7] Finally, the fungal metabolites cyclo D-Trp-L-Pro (14) and mucorisocoumarin (15) were also annotated by GNPS. Previous studies [9] have shown gymnoascolide A (1) as a promising bioactive compound due to its good antidiabetic properties. ...
... [15] The procedures for DNA extraction, PCR, Sanger sequencing, and molecular phylogenetic analysis using IQ-Tree and maximum Likelihood were as recently described. [38,7,9] The ITS sequences obtained (n = 2) were BLAST searched in the NCBI GenBank to verify identity and sequence homology with other similar ITS sequences using the Type database. [38] Based on a BLAST search in GenBank nucleotide database, the closest hit using the ITS sequence was M. flocciformis IFM 41298 ITS region; from TYPE material (GenBank NR_155357.1; ...
Compound 3, a trimeric anthranilic acid peptide, and another three metabolites were isolated from an organic extract from the culture medium of Malbranchea flocciformis ATCC 34530. The chemical structure proposed previously for 3 was unequivocally assigned via synthesis and X‐ray diffraction analysis. Tripeptide 3 showed insulinotropic properties by decreasing the postprandial peak in healthy and hyperglycemic mice. It also increased glucose‐induced insulin secretion in INS‐1E at 5 μM, specifically at higher glucose concentrations. These results revealed that 3 might act as an insulin sensitizer and a non‐classical insulin secretagogue. Altogether, these findings are in harmony with the in vivo oral glucose tolerance test and acute oral hypoglycemic assay. Finally, the chemical composition of the extract was established by the Global Natural Products Social Molecular Network platform. Phylogenetic analysis using the internal transcribed spacer region revealed that M. flocciformis ATCC 34530 is related to the Malbrancheaceae.
... 7 Finally, Malbranchea albolutea Sigler & Carmichael biosynthesizes ardeemin derivatives with potent PTP-1B inhibitory effects. 8 Malbranchea dendritica Sigler & Carmichael is a fungus isolated from soil worldwide. 9 From the chemical and pharmacological point of view, this species has not been investigated. ...
... 52 To place the strain ATCC 34527 in a phylogenetic framework, we downloaded all types and other sequences from various closely related Malbranchea and Auxarthron spp 53−55 and performed a Maximum Likelihood analysis using methods detailed recently. 8 ModelFinder 56 predicted TIM3e+I+G4 as the best fitting substitution model according to the Akaike Information Criterion. 57 The trimmed nucleotide alignment after removing ambiguous nucleotide positions with GBlocks 58,59 was then used to run the Maximum Likelihood analysis using IQ-Tree implemented using the program PhyloSuite with 5000 Ultrafast bootstrapping. ...
... A recombinant human protein tyrosine phosphatase 1B (PTP-1B) was used. 8 The fungal extract, isolated compounds, and positive control (ursolic acid) were dissolved in DMSO or tris buffer solution (100 mM pH = 6.8). Aliquots of 0−10 μL of testing materials (triplicated) were incubated for 5 min with 20 μL of 1.56 mg/mL PTP-1B enzyme solution. ...
An extract from a PDB static culture of Malbranchea dendritica exhibited α-glucosidase and PTP-1B inhibitory activities. Fractionation of the active extract led to the isolation of gymnoascolide A (1), a γ-butenolide, and xanthones sydowinin A (2), sydowinin B (3), and AGI-B4 (4), as well as orcinol (5). Compound 1 exhibited important inhibitory activity against yeast α-glucosidase (IC50 = 0.556 ± 0.009 mM) in comparison to acarbose (IC50 = 0.403 ± 0.010 mM). Kinetic analysis revealed that 1 is a mixed-type inhibitor. Furthermore, compound 1 significantly reduced the postprandial peak in mice during a sucrose tolerance test at the doses of 5.16 and 10 mg/kg. Compound 1 was reduced with Pd/C to yield a mixture of enantiomers 1a and 1b; the mixture showed similar activity against α-glucosidase (IC50 = 0.396 ± 0.003 mM) and kinetic behavior as the parent compound but might possess better drug-likeness properties according to SwissADME and Osiris Property Explorer tools. Docking analysis with yeast α-glucosidase (pdb: 3A4A) and the C-terminal subunit of human maltase-glucoamylase (pdb: 3TOP) predicted that 1, 1a, and 1b bind to an allosteric site of the enzymes. Compounds 1–5 were evaluated against PTP-1B, but only xanthone 3 moderately inhibited in a noncompetitive fashion the enzyme with an IC50 of 0.081 ± 0.004 mM. This result was consistent with that of docking analysis, which revealed that 3 might bind to an allosteric site of the enzyme. From the inactive barley-based semisolid culture of M. dendritica, the natural pigment erythroglaucin (6) and the nucleosides deoxyadenosine (7), adenosine (8), thymidine (9), and uridine (10) were also isolated and identified.
... Compounds 1a-8a, resulting from the docking studies at the PTP1B's catalytic, were selected to determine its inhibitory activity against PTP1B by using a spectrophotometric assay. [39,40] The compounds to be assessed included estradiol (7a), which was also found to target the allosteric site. Other estradiol derivatives, such as 3b, 4b, and 5b, also showed good in silico binding energies on the allosteric site; however, since all of them are structural analogues, only the evaluation of estradiol was considered because it was the most representative. ...
... The spectrophotometric method previously described was used. [39] The compounds and the positive control were dissolved in dimethylsulfoxide (DMSO). Aliquots of 0-10 μL solution of the compounds to be tested (in triplicate) were incubated with 85 μL of the enzyme (66 nM) in Tris [50 mM; Tris-HCI Buffer, pH 6.8] and 5 microliters of the substrate (pNPP, 10 mM), for 15 min at 37°C. ...
Current treatments for type 2 diabetes (T2D) mainly rely on exercise, dietary control, and anti‐diabetic drugs to enhance insulin secretion and improve insulin sensitivity. However, there is a need for more therapeutic options, as approved drugs targeting different pharmacological objectives are still unavailable. One potential target that has attracted attention is the protein tyrosine phosphatase 1B (PTP1B), which negatively regulates the insulin signaling pathway. In this work, a comprehensive computational screening was carried out using cheminformatics and molecular docking on PTP1B, employing a rigorous repurposing approach. The screening involved approved drugs and compounds under research as anti‐diabetics that bind to targets such as peroxisome proliferator‐activated receptor gamma (PPAR‐γ) and α‐glucosidase. Several computational hits were then meticulously tested in vitro against PTP1B, with 13‐cis‐retinoic acid (3a) showing an IC50 of 0.044 mM and competitive inhibition. Molecular dynamics studies further confirmed that 3a can bind to the catalytic binding site of PTP1B. Finally, 3a is the first time it has been reported as an inhibitor of PTP1B, making it a potentially valuable candidate for further studies in D2T treatment.
... Nevertheless, the inhibition type of FC-122 remained unknown to date. Therefore, we performed an enzyme kinetic assay for PTP1B1-400 [59][60][61] using p-nitrophenyl phosphate (pNPP) as substrate at different concentrations as well as the inhibitor. Figure 2C depicts the double reciprocal plot of Lineweaver-Burk, showing the effect of FC-122 on the PTP1B activity. ...
... Nevertheless, the inhibition type of FC-122 remained unknown to date. Therefore, we performed an enzyme kinetic assay for PTP1B 1-400 [59][60][61] using p-nitrophenyl phosphate (pNPP) as substrate at different concentrations as well as the inhibitor. Figure 2C depicts the double reciprocal plot of Lineweaver-Burk, showing the effect of FC-122 on the PTP1B activity. ...
Type 2 diabetes (T2D) is one of the most common diseases and the 8th leading cause of death worldwide. Individuals with T2D are at risk for several health complications that reduce their life expectancy and quality of life. Although several drugs for treating T2D are currently available, many of them have reported side effects ranging from mild to severe. In this work, we present the synthesis in a gram-scale as well as the in silico and in vitro activity of two semisynthetic glycyrrhetinic acid (GA) derivatives (namely FC-114 and FC-122) against Protein Tyrosine Phosphatase 1B (PTP1B) and α-glucosidase enzymes. Furthermore, the in vitro cytotoxicity assay on Human Foreskin fibroblast and the in vivo acute oral toxicity was also conducted. The anti-diabetic activity was determined in streptozotocin-induced diabetic rats after oral administration with FC-114 or FC-122. Results showed that both GA derivatives have potent PTP1B inhibitory activity being FC-122, a dual PTP1B/α-glucosidase inhibitor that could increase insulin sensitivity and reduce intestinal glucose absorption. Molecular docking, molecular dynamics, and enzymatic kinetics studies revealed the inhibition mechanism of FC-122 against α-glucosidase. Both GA derivatives were safe and showed better anti-diabetic activity in vivo than the reference drug acarbose. Moreover, FC-114 improves insulin levels while decreasing LDL and total cholesterol levels without decreasing HDL cholesterol.
... inhibitors from natural sources (Rebollar-Ramos et al., 2021;Díaz-Rojas et al., 2021;Rangel-Grimaldo et al., 2020), herein we report the enzymatic inhibition of the components from an infusion prepared from the aerial parts of J. spicigera. ...
... The conditions for preparation of reagents (enzyme and substrate), incubation and analysis of results was the same as those described in the previous sections (Rangel-Grimaldo et al., 2020;Jiménez-Arreola et al., 2020;Díaz-Rojas et al., 2021). Additional steps in the methodology are described below. ...
An infusion from the aerial parts of Justicia spicigera Schltdl., an herb commonly used to treat diabetes, inhibited the activity of protein tyrosine phosphatase 1B (PTP1B). Two undescribed compounds, 2-N-(p-coumaroyl)-3H-phenoxazin-3-one, and 3″-O-acetyl-kaempferitrin, along with kaempferitrin, kaempferol 7-O-α-L-rhamnopyranoside, perisbivalvine B and 2,5-dimethoxy-p-benzoquinone were isolated from the active extract. Their structures were elucidated by a combination of spectroscopic and spectrometric methods. The isolates were evaluated for their inhibitory activity against PTP1B; the most active compounds were 2-N-(p-coumaroyl)-3H-phenoxazin-3-one, and perisbivalvine B with IC50 values of 159.1 ± 0.02 μM and 106.6 ± 0.01 μM, respectively. However, perisbivalvine B was unstable. Kinetic analysis of 2-N-(p-coumaroyl)-3H-phenoxazin-3-one and 2,5-dimethoxy-p-benzoquinone (obtained in good amounts) indicated that both compounds behaved as parabolic competitive inhibitors and bind to the enzyme forming complexes with 1:1 and 1:2 stoichiometry. Docking of 2-N-(p-coumaroyl)-3H-phenoxazin-3-one and 2,5-dimethoxy-p-benzoquinone to PTP1B1-400 predicted a good affinity of these compounds for PTP1B catalytic site and demonstrated that the binding of a second ligand is sterically possible. The 1:2 complex was also supported by the second docking analysis, which predicted an important contribution of π-stacking interactions to the stability of these 1:2 complexes. Finally, an UHPLC-MS method was developed and validated to quantify the content of kaempferitrin in the infusion of the plant.
... For these purposes, a first report of a great interest were received on M. albolutea for the ability to produce alboluteins A-C (1-3), ardeemins and sartoryglabrins analogs; these compounds are involved in the PTP1B inhibitions, a negative modulator of insulin and leptin signaling, highly validated pharmacological target against insulin resistance and obesity (Kerru et al. 2018). For these reasons, the importance to future deep study and comparison among several M. albolutea strains, on the possible effects that alboluteins A-C have antitumoral potential since PTP1B is like a positive regulator of tumor progression (Díaz-Rojas et al. 2021). ...
Background The barn owl Tyto alba is a medium-sized nocturnal raptor, predator of small mammals, birds, reptiles, and insects, distributed all over the world. Environmentally uncontaminated wild barn owl pellets are hard and rare to find and provide a natural matrix for biological investigations. Results Different solutions for collecting wild barn owl pellets at low environmental contamination are proposed. These solutions are based on a daily sanitized surface for sample retrieval, followed by rapid analysis. As an example of the possible investigations that can be carried out on these matrices, with the aim to highlight the relevance of the obtained results also from a biotechnological perspective. Two rare pellet samples from wild barn owl T. alba, appropriately collected in a rural area of central Italy, were analyzed using culture-dependent, molecular (Next-Generation Sequencing), and enzyme profile analysis techniques. The osteological observations of the bone remains provided useful information for identifying the prey, mainly rodents (Apodemus sp.). Under our experimental conditions, the results revealed both a wide heterogeneity between the pellet microbiota and a great percentage of uncultured bacteria not classified at the species level. Furthermore, microbial cultures of Malbranchea albolutea, Debaryomyces hansenii, and Lactobacillus sp. were isolated and studied. Finally, we quantified the environmental impact of our experimental work in terms of CO 2 equivalent release. To compensate for the release of 300 kg/CO 2 eq., three Taxus. baccata L. were planted. Conclusions This work provides a starting point for developing an effective strategy to study and characterize wild barn owl pellets at low environmental contamination. It presents a simple and easy technique/protocol for collecting the pellets. The microbiota heterogeneity found in the two analyzed samples suggests that barn owl pellets can represent a potential natural reservoir for the discovery of new microorganisms to be used in various biotechnological applications. This could open the way for further large-scale studies on a greater number of samples and populations.
... The conditions for preparing reagents (enzyme and substrate), incubation, and analysis of results were the same as that described in Section 2.1 (Jiménez-Arreola et al., 2020;Díaz-Rojas et al., 2021). Additional steps in the methodology are described below. ...
This work aimed to discover protein tyrosine phosphatase 1B (PTP1B) inhibitors from a small molecule library of natural products (NPs) derived from selected Mexican medicinal plants and fungi to find new hits for developing antidiabetic drugs. The products showing similar IC50 values to ursolic acid (UA) (positive control, IC50 = 26.5) were considered hits. These compounds were canophyllol (1), 5-O-(β-D-glucopyranosyl)-7-methoxy-3′,4′-dihydroxy-4-phenylcoumarin (2), 3,4-dimethoxy-2,5-phenanthrenediol (3), masticadienonic acid (4), 4′,5,6-trihydroxy-3′,7-dimethoxyflavone (5), E/Z vermelhotin (6), tajixanthone hydrate (7), quercetin-3-O-(6″-benzoyl)-β-D-galactoside (8), lichexanthone (9), melianodiol (10), and confusarin (11). According to the double-reciprocal plots, 1 was a non-competitive inhibitor, 3 a mixed-type, and 6 competitive. The chemical space analysis of the hits (IC50 < 100 μM) and compounds possessing activity (IC50 in the range of 100–1,000 μM) with the BIOFACQUIM library indicated that the active molecules are chemically diverse, covering most of the known Mexican NPs’ chemical space. Finally, a structure–activity similarity (SAS) map was built using the Tanimoto similarity index and PTP1B absolute inhibitory activity, which allows the identification of seven scaffold hops, namely, compounds 3, 5, 6, 7, 8, 9, and 11. Canophyllol (1), on the other hand, is a true analog of UA since it is an SAR continuous zone of the SAS map.
... The genus Arthrographis is still related to Malbranchea (SJA10) due to the presence of arthroconidia, resembling Oidiodendron, but the conidiophores of Arthrographis do not have characteristic pigmentation (Sainaghi et al. 2015;Hernandez-Restrepo et al. 2020). Three analogs of ardeemin and sartoryglabrins, namely albolutein A-C, isolated from Malbranchea albolutea, function as protein tyrosine phosphate inhibitors (Díaz-Rojas et al. 2021). ...
‘Aini K, Elfita, Widjajanti H, Setiawan A. 2022. Diversity and antibacterial activity of endophytic fungi isolated from the medicinal plant of Syzygium jambos. Biodiversitas 23: 2981-2989. Currently, antimicrobial resistance is one of the most important threats to global public health. This is due to the various mechanisms of antimicrobial resistance. Thus, the need for new sources of bioactive compounds outside conventional antibiotics is a top priority. This investigation evaluated the diversity and antibacterial activities of endophytic fungi isolated from the leaves and root bark of jambu mawar (Syzygium jambos (L.) Alston). The endophytic fungi were grown on PDA media, and their antibacterial activities were tested using the Kirby-Bauer method on two Gram-positive and two Gram-negative bacteria. A total of 10 (SJD1-SJD10) and 11 isolates (SJA1-SJA11) of endophytic fungi were identified from the leaves and root bark of S. jambos, respectively. Among them, SJA8 isolate exhibited strong antibacterial activity. Based on morphological characterization and phylogenetic tree analysis, SJA8 was identified as Pleiocarpon livistonae. Isolation and identification of pure compounds from the fungi may reveal potential candidates for new antibiotic substances.
... Docking studies of alboluteins A-C (68-70) into the crystal structure of PTP1B (PDB ID: 1T49) predicted that all compounds prefer to bind at the allosteric site of the enzyme, with Ki values of 2.02 × 10 -4 , 1.31 × 10 -4 , and 2.67 × 10 -4 mM, respectively. Molecular dynamic studies indicated that the active compounds remain tied to the enzyme with good binding energy [40]. ...
Diabetes is a growing health concern worldwide because it affects people of all age groups and increases the risk of other diseases such as renal impairment and neural and cardiovascular disorders. Oral hypoglycemic drugs mainly control diabetes; however, their associated side effects limit their use in patients with other complications. PTP1B is a viable drug target to explore new antidiabetic drugs. PTP1B acts as a negative regulator of the insulin-signaling pathway, and therefore, PTP1B inhibitors display antihyperglycemic activity. Several classes of compounds from natural and synthetic sources act as PTP1B inhibitors. Fungi are comprehensive in their diversity and recognized as a valuable source for therapeutically active molecules. In recent years, researchers have reported diverse classes of fungal secondary metabolites as potent PTP1B inhibitors. Some metabolites such as 6-O-methylalaternin, fumosorinone A, nordivaricatic acid, and the divarinyl divarate showed good activity and can be taken forward as a lead to develop novel PTP1B inhibitors and antidiabetic drugs. Therefore, the present review focuses on the fungal metabolites identified in the last five years possessing PTP1B inhibitory activity. A total of 128 metabolites are reviewed. Their fungal species and source, chemical structure, and activity in terms of IC50 are highlighted.
... To test the inhibitory activity of the compounds against PTP1B, a spectrocolorimetric method previously described was employed [59,60]. The IC 50 was calculated by regression analysis using Equation: ...
Regulating insulin and leptin levels using a protein tyrosine phosphatase 1B (PTP1B) inhibitor is an attractive strategy to treat diabetes and obesity. Glycyrrhetinic acid (GA), a triterpenoid, may weakly inhibit this enzyme. Nonetheless, semisynthetic derivatives of GA have not been developed as PTP1B inhibitors to date. Herein we describe the synthesis and evaluation of two series of indole- and N-phenylpyrazole-GA derivatives (4a–f and 5a–f). We measured their inhibitory activity and enzyme kinetics against PTP1B using p-nitrophenylphosphate (pNPP) assay. GA derivatives bearing substituted indoles or N-phenylpyrazoles fused to their A-ring showed a 50% inhibitory concentration for PTP1B in a range from 2.5 to 10.1 µM. The trifluoromethyl derivative of indole-GA (4f) exhibited non-competitive inhibition of PTP1B as well as higher potency (IC50 = 2.5 µM) than that of positive controls ursolic acid (IC50 = 5.6 µM), claramine (IC50 = 13.7 µM) and suramin (IC50 = 4.1 µM). Finally, docking and molecular dynamics simulations provided the theoretical basis for the favorable activity of the designed compounds.
Allosteric modulation is a direct and effective method for regulating the function of biological macromolecules, which play vital roles in various cellular activities. Unlike orthosteric modulators, allosteric modulators bind to sites distant from the protein's orthosteric/active site and can have specific effects on the protein's function or activity without competing with endogenous ligands. Compared to traditional orthosteric modulators, allosteric modulators offer several advantages, including reduced side effects, greater specificity, and lower toxicity, making them a promising strategy for developing novel drugs. Indole-fused architectures are widely distributed in natural products and bioactive drug leads, displaying diverse biological activities that attract the interest of both chemists and biologists in drug discovery. Currently, an increasing number of indole-fused compounds have exhibited potent activities in allosteric modulation. In this review, we provide a brief summary of examples of allosteric modulators based on the indole-fused complex architecture, highlighting the strategies for drug design/discovery and the structure-activity relationships of allosteric modulators from the perspective of medicinal chemistry.
The glucosinolate pathway, which is present in the order Brassicales, is one of the most researched defensive natural product biosynthesis pathways. Its core molecules, the glucosinolates are broken down upon pathogen challenge or tissue damage to yield an array of natural products that may help plants defend against the stressor. Though the most widely known glucosinolate decomposition products are the antimicrobial isothiocyanates, there is a wide range of other volatile and non-volatile natural products that arise from this biosynthetic pathway.
This review summarizes our current knowledge on the interaction of these much less examined, non-isothiocyanate products with fungi. It deals with compounds including (1) glucosinolates and their biosynthesis precursors; (2) glucosinolate-derived nitriles (e.g. derivatives of 1H-indole-3-acetonitrile), thiocyanates, epithionitriles and oxazolidine-2-thiones; (3) putative isothiocyanate downstream products such as raphanusamic acid, 1H-indole-3-methanol (= indole-3-carbinol) and its oligomers, 1H-indol-3-ylmethanamine and ascorbigen; (4) 1H-indole-3-acetonitrile downstream products such as 1H-indole-3-carbaldehyde (indole-3-carboxaldehyde), 1H-indole-3-carboxylic acid and their derivatives; and (5) indole phytoalexins including brassinin, cyclobrassinin and brassilexin.
Herein, a literature review on the following aspects is provided: their direct antifungal activity and the proposed mechanisms of antifungal action, increased biosynthesis after fungal challenge, as well as data on their biotransformation/detoxification by fungi, including but not limited to fungal myrosinase activity.
The aim of this review was to discuss an overview of type 2 diabetes; biology of PTP1B; role of PTP1B in metabolic disorders; and recent updates in the development of PTP1B inhibitors reported in literature since 1994. In this study, extensive literature search was carried out on PTP1B inhibitors of natural as well as synthetic origin in various scientific databases and research articles related to discovery of PTP1B inhibitors were selected for this study. Protein tyrosine phosphatase 1B (PTP1B) is an important therapeutic target for several human diseases including type 2 diabetes, obesity and cancer because of its seminal part as a negative modulator in both insulin and leptin signaling pathways. A large number of molecules of broad chemical diversity were reported as potent and selective PTP1B inhibitors over other protein tyrosine phosphatases. Several of these molecules have shown their potential in the treatment of various human diseases including type 2 diabetes, obesity, inflammation and cancer in various animal models. But only a very limited number of PTP1B inhibitors (including ertiprotafib, trodusquemine and JTT-551) has entered clinical trials and are finally withdrawn owing to their unsatisfactory effectiveness and undesirable adverse effects. Consequently, it is still highly imperative and of great importance to develop potent, highly selective and safe PTP1B inhibitors.
Protein tyrosine phosphatase 1B (PTP1B) is an active target for developing drugs to treat type II diabetes, obesity, and cancer. However, in the past, research programs targeting this enzyme focused on discovering inhibitors of truncated models (hPTP1B1-282, hPTP1B1-298, or hPTP1B1-321), losing valuable information about the ligands' mechanism of inhibition and selectivity. Nevertheless, finding an allosteric site in hPTP1B1-321, and the full-length (hPTP1B1-400) protein expression, have shifted the strategies to discover new PTP1B inhibitors. Accordingly, as part of a research program directed at finding non-competitive inhibitors of hPTP1B1-400 from Pezizomycotina, the extract of Penicillium sp. (IQ-429) was chemically investigated. This study led to xanthoepocin (1) isolation, which was elucidated by means of spectroscopic and spectrometric data. The absolute configuration of 1 was determined to be 7R8S9R7'R8'S9'R by comparing the theoretical and experimental ECD spectra and by GIAO-NMR DP4+ statistical analysis. Xanthoepocin (1) inhibited the phosphatase activity of hPTP1B1-400 (IC50 value of 8.8 ± 1.0 µM) in a mixed type fashion with ki and αki values of 5.5 µM of 6.6, respectively. Docking xanthoepocin (1) with a homologated model of hPTP1B1-400 indicated that it binds in a pocket different from the catalytic triad at the interface of the N and C-terminal domains. Molecular dynamics (MD) simulations showed that 1 locks the WPD loop of hPTP1B1-400 in a closed conformation, avoiding substrate binding, products release, and catalysis, suggesting an allosteric modulation triggered by large-scale conformational and dynamics changes. Intrinsic quenching fluorescence experiments indicated that 1 behaves like a static quencher of hPTP1B1-400 (KSV = 1.1 ×10⁵ M⁻¹), and corroborated that it binds to the enzyme with an affinity constant (ka) of 3.7 × 10⁵ M⁻¹. Finally, the drug-likeness and medicinal chemistry friendliness of 1 were predicted with SwissADME.
Molecular dynamics (MD) simulations have become increasingly popular in studying the motions and functions of biomolecules. The accuracy of the simulation, however, is highly determined by the molecular mechanics (MM) force field (FF), a set of functions with adjustable parameters to compute the potential energies from atomic positions. However, the overall quality of the FF, such as our previously published ff99SB and ff14SB, can be limited by assumptions that were made years ago. In the updated model presented here (ff19SB), we have significantly improved the backbone profiles for all 20 amino acids. We fit coupled ϕ/ψ parameters using 2D ϕ/ψ conformational scans for multiple amino acids, using as reference data the entire 2D quantum mechanics (QM) energy surface. We address the polarization inconsistency during dihedral parameter fitting by using both QM and MM in solution. Finally, we examine possible dependency of the backbone fitting on side chain rotamer. To extensively validate ff19SB parameters, we have performed a total of ~5 milliseconds MD simulations in explicit solvent. Our results show that after amino-acid specific training against QM data with solvent polarization, ff19SB not only reproduces the differences in amino acid specific Protein Data Bank (PDB) Ramachandran maps better, but also shows significantly improved capability to differentiate amino acid dependent properties such as helical propensities. We also conclude that an inherent underestimation of helicity is present in ff14SB, which is (inexactly) compensated by an increase in helical content driven by the TIP3P bias toward overly compact structures. In summary, ff19SB, when combined with a more accurate water model such as OPC, should have better predictive power for modeling sequence-specific behavior, protein mutations, and also rational protein design.
Multigene and genomic data sets have become commonplace in the field of phylogenetics, but many existing tools are not designed for such data sets, which often makes the analysis time‐consuming and tedious. Here, we present phylosuite, a (cross‐platform, open‐source, stand‐alone Python graphical user interface) user‐friendly workflow desktop platform dedicated to streamlining molecular sequence data management and evolutionary phylogenetics studies. It uses a plugin‐based system that integrates several phylogenetic and bioinformatic tools, thereby streamlining the entire procedure, from data acquisition to phylogenetic tree annotation (in combination with iTOL). It has the following features: (a) point‐and‐click and drag‐and‐drop graphical user interface; (b) a workplace to manage and organize molecular sequence data and results of analyses; (c) GenBank entry extraction and comparative statistics; and (d) a phylogenetic workflow with batch processing capability, comprising sequence alignment (mafft and macse), alignment optimization (trimAl, HmmCleaner and Gblocks), data set concatenation, best partitioning scheme and best evolutionary model selection (partitionfinder and modelfinder), and phylogenetic inference (mrbayes and iq‐tree). phylosuite is designed for both beginners and experienced researchers, allowing the former to quick‐start their way into phylogenetic analysis, and the latter to conduct, store and manage their work in a streamlined way, and spend more time investigating scientific questions instead of wasting it on transferring files from one software program to another.
Diabetes mellitus is a medical condition characterized by the body's loss of control over blood sugar. The frequency of diagnosed cases and consequential increases in medical costs makes it a rapidly growing chronic disease that threatens human health worldwide. In addition, its unnerving statistical projections are perilous to both the economy of the nation and man's life expectancy. Type-I and type-II diabetes are the two clinical forms of diabetes mellitus. Type-II diabetes mellitus (T2DM) is illustrated by the abnormality of glucose homeostasis in the body, resulting in hyperglycemia. Although significant research attention has been devoted to the development of diabetes regimens, which demonstrates success in lowering blood glucose levels, their efficacies are unsustainable due to undesirable side effects such as weight gain and hypoglycemia. Over the years, heterocyclic scaffolds have been the basis of anti-diabetic chemotherapies; hence, in this review we consolidate the use of bioactive scaffolds, which have been evaluated for their biological response as inhibitors against their respective anti-diabetic molecular targets over the past five years (2012-2017). Our investigation reveals a diverse target set which includes; protein tyrosine phosphatase 1 B (PTP1B), dipeptidly peptidase-4 (DPP-4), free fatty acid receptors 1 (FFAR1), G protein-coupled receptors (GPCR), peroxisome proliferator activated receptor-γ (PPARγ), sodium glucose co-transporter-2 (SGLT2), α-glucosidase, aldose reductase, glycogen phosphorylase (GP), fructose-1,6-bisphosphatase (FBPase), glucagon receptor (GCGr) and phosphoenolpyruvate carboxykinase (PEPCK). This review offers a medium on which future drug design and development toward diabetes management may be modelled (i.e. optimization via structural derivatization), as many of the drug candidates highlighted show promise as an effective anti-diabetic chemotherapy.
To be effective as a drug, a potent molecule must reach its target in the body in sufficient concentration, and stay there in a bioactive form long enough for the expected biologic events to occur. Drug development involves assessment of absorption, distribution, metabolism and excretion (ADME) increasingly earlier in the discovery process, at a stage when considered compounds are numerous but access to the physical samples is limited. In that context, computer models constitute valid alternatives to experiments. Here, we present the new SwissADME web tool that gives free access to a pool of fast yet robust predictive models for physicochemical properties, pharmacokinetics, drug-likeness and medicinal chemistry friendliness, among which in-house proficient methods such as the BOILED-Egg, iLOGP and Bioavailability Radar. Easy efficient input and interpretation are ensured thanks to a user-friendly interface through the login-free website http://www.swissadme.ch. Specialists, but also nonexpert in cheminformatics or computational chemistry can predict rapidly key parameters for a collection of molecules to support their drug discovery endeavours.
Fungi are morphologically, ecologically, metabolically, and phylogenetically diverse. They are known to produce numerous bioactive molecules, which makes them very useful for natural products researchers in their pursuit of discovering new chemical diversity with agricultural, industrial, and pharmaceutical applications. Despite their importance in natural products chemistry, identification of fungi remains a daunting task for chemists, especially those who do not work with a trained mycologist. The purpose of this review is to update natural products researchers about the tools available for molecular identification of fungi. In particular, we discuss (1) problems of using morphology alone in the identification of fungi to the species level; (2) the three nuclear ribosomal genes most commonly used in fungal identification and the potential advantages and limitations of the ITS region, which is the official DNA barcoding marker for species-level identification of fungi; (3) how to use NCBI-BLAST search for DNA barcoding, with a cautionary note regarding its limitations; (4) the numerous curated molecular databases containing fungal sequences; (5) the various protein-coding genes used to augment or supplant ITS in species-level identification of certain fungal groups; and (6) methods used in the construction of phylogenetic trees from DNA sequences to facilitate fungal species identification. We recommend that, whenever possible, both morphology and molecular data be used for fungal identification. Our goal is that this review will provide a set of standardized procedures for the molecular identification of fungi that can be utilized by the natural products research community.
From an extract prepared from the grain-based culture of Malbranchea flavorosea two new polyketides, namely, 8-chloroxylarinol A (1) and flavoroseoside (2), along with the known compounds xylarinol A (3), xylarinol B (4), massarigenins B and C (5 and 6), and clavatol (7), were isolated. The structures of 1 and 2 were elucidated using spectroscopic methods and corroborated by single-crystal X-ray diffraction analysis. In the case of compound 2 the absolute configuration at the stereogenic centers was established according to the method of Flack. In addition, the X-ray structure of compound 6 is reported for the first time. Compounds 3, 4, and 6 significantly inhibited yeast α-glucosidase. Compound 6 also inhibited the postprandial peak during an oral sucrose tolerance assay when tested in vivo, using normal and NA/STZ-induced hyperglycemic mice.
Malbranchea filamentosa and M. albolutea were reexamined and connected to teleomorphs in the genus Auxarthron. DNA sequences were obtained from the small subunit (SSU) and internal transcribed spacer (ITS) regions of the nuclear ribosomal rRNA gene to evaluate conspecificity of the M. filamentosa isolates and to evaluate relationships among taxa within the Auxarthron clade. Five of eight isolates putatively identified as M. filamentosa produced fertile ascomata in matings and with F1 progeny. Auxarthron filamentosum sp. nov. is described for the teleomorph. Three nonmating isolates appeared conspecific based on morphology, but one was excluded based on sequence divergence. An Auxarthron teleomorph was described for M. albolutea in 1976, but not named because of uncertainty about its distinction from A. thaxteri and A. umbrinum. Auxarthron alboluteum sp. nov. is shown to be phylogenetically distinct. Phylogenetic analysis based on newly derived sequences showed that members of the genus Auxarthron and Malbranchea dendritica formed a strongly supported monophyletic group. In the ITS analysis, most species were placed into two well supported clades that correlated with the shape of the ascospores; the position of the type species A. californiense, was not clearly resolved. Differences were found between newly derived SSU sequences and those on deposit in GenBank for the same strains. After re-evaluation of the strains, the following sequences are considered to be incorrect: M. albolutea L28063 (UAMH 2846), A. zuffianum L28062 (UAMH 4098), M. dendritica L28064 (UAMH 2731) and M. filamentosa L28065 (UAMH 4097). The sequence for U29389 is correct for Malbranchea albolutea not M. dendritica as stated in the original GenBank record. The problems with these sequences were not uncovered in prior published analyses because insufficient representatives of Auxarthron species were sampled.
In a survey of coprophilous fungi in Italy, two interesting ascomycetes were recovered from hedgehog dung. These fungi showed a sexual morph characterized by gymnothecia, globose reticulate ascospores, a Malbranchea asexual state and a keratinolytic ability, and were identified morphologically and molecularly as Auxarthron umbrinum and A. concentricum of the Onygenaceae. The history, ecology, and morphology of the genus Auxarthron as a whole, and of A.umbrinum and A. concentricum in particular, are revised. A preliminary evaluation of the antagonistic properties of Auxarthron umbrinum and A. concentricum against phytopathogenic fungi has been performed in dual cultures. Growth inhibition of some plant pathogenic fungi was recorded, and the effects were growth medium dependent. When Solid State Fermentation (SSF) substrate of both Auxarthron isolates has been submitted to extraction, both the organic extract residues (n-hexane and CH2Cl2) and the lyophilized aqueous phases were used for the antibiotic test against plant pathogenic fungi. Significant antifungal activity was obtained by organic fractions and aqueous residue of A. concentricum against Alternaria brassicicola and Botrytis cinerea whereas A. umbrinum appeared to be less effective. Analysis of ITS nrDNA sequences suggests that an extensive phylogenetic revision of the genus Auxarthron is necessary.
Large phylogenomics data sets require fast tree inference methods, especially for maximum-likelihood (ML) phylogenies. Fast
programs exist, but due to inherent heuristics to find optimal trees, it is not clear whether the best tree is found. Thus,
there is need for additional approaches that employ different search strategies to find ML trees and that are at the same
time as fast as currently available ML programs. We show that a combination of hill-climbing approaches and a stochastic perturbation
method can be time-efficiently implemented. If we allow the same CPU time as RAxML and PhyML, then our software IQ-TREE found
higher likelihoods between 62.2% and 87.1% of the studied alignments, thus efficiently exploring the tree-space. If we use
the IQ-TREE stopping rule, RAxML and PhyML are faster in 75.7% and 47.1% of the DNA alignments and 42.2% and 100% of the protein
alignments, respectively. However, the range of obtaining higher likelihoods with IQ-TREE improves to 73.3–97.1%. IQ-TREE
is freely available at http://www.cibiv.at/software/iqtree.
Four ardeemin derivatives, 5-N-acetylardeemin (1), 5-N-acetyl-15bβ-hydroxyardeemin (2), 5-N-acetyl-15b-didehydroardeemin (3), and 5-N-acetyl-16α-hydroxyardeemin (4), were isolated from the fermentation broth of an endophytic Aspergillus fumigatus SPS-02 associated with Artemisia annua L. The structures of these metabolites were elucidated by a combination of spectroscopic data, including 1D-, 2D-NMR and MS. In vitro chemosensitization assay indicated that these ardeemins had different activities of reversing the multidrug-resistant (MDR) phenotype in three cancer cell lines, leukemia doxorubicin resistant cell K562/DOX, human lung adenocarcinoma cis-platin-resistant cell A549/DDP, and ovarian cancer cisplatin-resistant cell SK-OV-S/DDP. Compound 4 exhibited the strongest MDR reversing effect at 5 μM concentration in K562/DOX and A549/DDP cell lines 5.2±0.18-fold, 8.2±0.23-fold, respectively, while compound 2 had the highest reversal capacity in SK-OV-S/DDP cell line with 10.8±0.28 fold. Preliminary investigation of their structureactivity relationship suggested that a OH group at C(15b) or C(16) in ardeemin plays a key role in reversing the MDR effect. It is the first report on ardeemin analogs from endophytic A. fumigatus with reversal effects on MDR cancer cell lines K562/DOX, A549/DDP and SK-OV-S/DDP.
Bioassay-directed fractionation of an ethyl acetate extract (mycelia and broth) of the fungus Malbranchea aurantiaca led to the isolation of the novel phytotoxic alkaloid (5aS,12aS,13aS)-8,9-dichloro-12,12-dimethyl-2,3,11,12,12a,13-hexahydro-1H,6H-5a,13a (epiminomethano)indolizino[7,6b]carbazol-14-one (1) of the brevianamide series. The phytotoxin was given the trivial name of malbrancheamide (1). The structure of 1 was unequivocally established by UV, NMR, MS and X-ray studies. The absolute configuration was established by X-ray analysis according to the method of Flack. According to the conformational studies using molecular mechanics analyses, 1 exists in one preferred conformation, which was optimized by DFT calculations. Compound 1 caused moderate inhibition of radicle growth of Amaranthus hypochondriacus (IC50=0.37 μM) and inhibited the activation of the calmodulin-dependent enzyme PDE1 (IC50=3.65±0.74 μM). This effect was comparable to that of chlorpromazine (IC50=2.75±0.87 μM) a well characterized CaM antagonist. The inhibition mechanism of 1 was competitive with respect to CaM according to a kinetic analysis.
Auxarthron is a genus within the Onygenales encompassing keratinophilic species with typical ascomata (gymnothecia) consisting of anastomosing network of thick-walled hyphae and small globose or oblate ascospores. No association of this genus with clinically relevant cases of human or animal infection has been reported. This paper describes the isolation of an undescribed Auxarthron species as an agent of proven onychomycosis affecting almost all fi ngernails in a man with psoriasis. The causality of the isolated fungus was verifi ed by repeated sampling and direct microscopy revealing irregular septate hyphae. Based on micro- and macromorphological features and unique sequence data (ITS region, benA and RPB2 gene), the isolated fungus is proposed as the new species A. ostraviense . The sibling species of A. ostraviense , A. umbrinum , was isolated from three patients with suspected onychomycosis and a detailed clinical history is provided for
one of these patients. All four isolates were tested for susceptibility to selected antifungal agents. Terbinafi ne and clotrimazole appear to be effective in vitro . The morphological identifi cation of Auxarthron spp. is non-trivial, time-consuming and requires cultivation media other than Sabouraud glucose agar which is routinely used in dermatomycology.
A historical perspective on the application of molecular dynamics (MD) to biological macromolecules is presented. Recent developments combining state-of-the-art force fields with continuum solvation calculations have allowed us to reach the fourth era of MD applications in which one can often derive both accurate structure and accurate relative free energies from molecular dynamics trajectories. We illustrate such applications on nucleic acid duplexes, RNA hairpins, protein folding trajectories, and protein−ligand, protein−protein, and protein−nucleic acid interactions.
Some fungi with pleomorphic life-cycles still bear two names despite more than 20 years of molecular phylogenetics that have shown how to merge the two systems of classification, the asexual "Deuteromycota" and the sexual "Eumycota". Mycologists have begun to flout nomenclatorial regulations and use just one name for one fungus. The International Code of Botanical Nomenclature (ICBN) must change to accommodate current practice or become irrelevant. The fundamental difference in the size of fungi and plants had a role in the origin of dual nomenclature and continues to hinder the development of an ICBN that fully accommodates microscopic fungi. A nomenclatorial crisis also looms due to environmental sequencing, which suggests that most fungi will have to be named without a physical specimen. Mycology may need to break from the ICBN and create a MycoCode to account for fungi known only from environmental nucleic acid sequence (i.e. ENAS fungi).
The Amsterdam Declaration on Fungal Nomenclature was agreed at an international symposium convened in Amsterdam on 19–20 April 2011 under the auspices of the International Commission on the Taxonomy of Fungi (ICTF). The purpose of the symposium was to address the issue of whether or how the current system of naming pleomorphic fungi should be maintained or changed now that molecular data are routinely available. The issue is urgent as mycologists currently follow different practices, and no consensus was achieved by a Special Committee appointed in 2005 by the International Botanical Congress to advise on the problem. The Declaration recognizes the need for an orderly transitition to a single-name nomenclatural system for all fungi, and to provide mechanisms to protect names that otherwise then become endangered. That is, meaning that priority should be given to the first described name, except where that is a younger name in general use when the first author to select a name of a pleomorphic monophyletic genus is to be followed, and suggests controversial cases are referred to a body, such as the ICTF, which will report to the Committee for Fungi. If appropriate, the ICTF could be mandated to promote the implementation of the Declaration. In addition, but not forming part of the Declaration, are reports of discussions held during the symposium on the governance of the nomenclature of fungi, and the naming of fungi known only from an environmental nucleic acid sequence in particular. Possible amendments to the Draft BioCode (2011) to allow for the needs of mycologists are suggested for further consideration, and a possible example of how a fungus only known from the environment might be described is presented.
Chemical investigation of a collection of the fungus Neosartorya glabra from Thailand furnished sartoryglabins A-C (1a, 1b and 2) which are analogs of the reverse prenylated indole alkaloids known as (-) ardeemins. Structures of these compounds were established by NMR spectrometry and an X-ray analysis. Sartoryglabins A-C were evaluated for their in vitro growth inhibitory activity on three human tumor cell lines: MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A375-C5 (melanoma). All the compounds exhibited strong to moderate activity against the MCF-7 cell line but weak or no activity against the NCI-H460 and A375-C5 cell lines. Sartoryglabin B was found to exhibit selectivity towards the MCF-7 cell line.
antifungal activity, cytotoxic activity, Malbranchea filamentosa, malfilanol, sesquiterpene
Seven new merosesquiterpenoids, trichothecrotocins D-J (1-7), two new trichothecene sesquiterpenoids, trichothecrotocins K (12) and L (13), and six known compounds (8-11, 14, and 15), were isolated from a potato-associated fungus, Trichothecium crotocinigenum. Compounds 5 and 6 were racemates which were further separated as pure enantiomers. Structures together with absolute configurations were established by extensive spectroscopic analysis, as well as quantum chemistry calculations on ECD and optical rotations. Compounds 1-4 are rare meroterpenoids featuring a seco-phenyl group, while 1 and 2 possessed a novel 6-6/5 fused ring system. Compounds 1-4, 8, 11, and 12 showed antifungal activity against four plant pathogens with MIC values of 8-128 μg/mL. It is suggested that the meroterpenoids produced by T. crotocinigenum may play an important role in the antifungal property of the fungus, thereby protecting the host plant, i.e., potato.
ConspectusStructural elucidation is an important and challenging stage in the discovery of new organic molecules. Single-crystal X-ray analysis provides the most unquestionable results, though in practice the availability of suitable crystals limits its broad use. On the other hand, NMR spectroscopy has become the leading and universal technique to accomplish the task. Despite continuous advances in the field, the misinterpretation of NMR data is commonplace, evidenced by the large number of erroneous structures being published in top journals. Quantum calculations of NMR chemical shifts and scalar coupling constants emerged as ideal complements to facilitate the elucidation process when experimental NMR data is inconclusive. Since seminal reports demonstrated that affordable DFT methods provide NMR predictions accurate enough to differentiate among closely related isomers, the discipline has experienced substantial growth. The impact has been felt in different areas, and nowadays the results of such calculations are routinely seen in high impact literature.This Account describes our investigations in the field of quantum NMR calculations, focusing on the development of tools for structural elucidation and practical applications. We pioneered the use of artificial intelligence methods in the development of novel strategies of structural validation. Our first generation of trained artificial neural networks (ANNs) showed excellent ability to identify mistakes at the atom connectivity level, whereas the use of multidimensional pattern recognition pushed the performance to the stereochemical limit. In a conceptually different approach, we developed DP4+, an updated version of the DP4 probability used to determine the most likely structure among two or more candidates when one set of experimental data is available. Increasing the level of theory in NMR calculations and including unscaled data in the formalism improved the performance of the method, further validated to settle the configuration of challenging motifs such as spiroepoxides or Mosher's derivatives. One of the limitations of DP4+ is related to the relatively large computational cost involved in obtaining DFT-optimized geometries, which led to the development of a fast variant including the valuable information provided by coupling constants (J-DP4 method).These tools were explored to suggest the most probable structure of controversial natural or unnatural products originally misassigned, with some predictions further validated by synthesis (as in the case of pseudorubriflordilactone B). The possibility of predicting the structure of a natural product without requiring authentic sample was investigated in collaboration with Prof. Pilli (UNICAMP, Brazil) in the computer-guided total synthesis and stereochemical revisions of several natural products. Despite these advances, there remain considerable challenges, such as the case of configurational assessment of polar systems featuring multiple intramolecular hydrogen bonding interactions because of the poor energy predictions provided by most DFT methods. In our latest work, we tackle this problem by averaging the results provided by randomly generated ensembles, paving the way for a new paradigm in quantum NMR-assisted structural elucidation.
A critical biological event that contributes to the appearance and progress of cancer and diabetes is the reversible phosphorylation of proteins, a process controlled by protein tyrosine-kinases (PTKs) and protein tyrosine-phosphatases (PTPs). Within the PTPs, PTP1B has gained significant interest since it is a validated target in drug discovery. Indeed, several PTP1B inhibitors have been developed, from both, synthesis and natural products. However, none have been approved by the FDA, due to their poor selectivity and/or pharmacokinetic properties. One of the most significant challenges to the discovery of PTP1B inhibitors (in vitro or in silico) is the use of truncated structures (PTP1B1-300), missing valuable information about the mechanisms of inhibition, and selectivity of ligands. The present study describes the biochemical characterization of a full-length PTP1B (hPTP1B1-400), as well as the description of phenalenones 1-4 and ursolic acid (5) as allosteric modulators. Compounds 1-5 showed inhibitory potential on hPTP1B1-400, with IC50 values ranging from 12.7 to 82.1 µM. Kinetic studies showed that 1 and 5 behave as mixed and non-competitive inhibitors, respectively. Circular dichroism experiments confirmed that 1 and 5 induced conformational changes to hPTP1B1-400. Further insights into the structure of hPTP1B1-400 were obtained from a homology model, which pointed out that the C-terminus (residues 301-400) is highly disordered. Molecular docking with the homologated model suggested that compounds 1 and 3-5 bind to the C-terminal domain, likely inducing conformational changes on the protein. Docking positions of compounds 1, 4, and 5 were refined with molecular dynamics simulations. Importantly, these simulations confirmed the high flexibility of the C-terminus of hPTP1B1-400, as well as the changes to its rigidity when bound to 1, 4, and 5.
During a search for new α-glucosidase and protein tyrosine phosphatase 1B inhibitors from fungal sources, eight new secondary metabolites, including two anthranilic acid-derived peptides (1 and 2), four glycosylated anthraquinones (3-6), 4-isoprenylravenelin (7), and a dimer of 5,8-dihydroxy-4-methoxy-α-tetralone (8), along with four known compounds (9-12), were isolated from solid rice-based cultures of Malbranchea circinata. The structural elucidation of these metabolites was performed using 1D and 2D NMR techniques and DFT-calculated chemical shifts. Compounds 1-3, 9, and 10 showed inhibitory activity to yeast α-glucosidase (αGHY), with IC50 values ranging from 57.4 to 261.3 μM (IC50 acarbose = 585.8 μM). The effect of 10 (10.0 mg/kg) was corroborated in vivo using a sucrose tolerance test in normoglucemic mice. The most active compounds against PTP-1B were 8-10, with IC50 values from 10.9 to 15.3 μM (IC50 ursolic acid = 27.8 μM). Docking analysis of the active compounds into the crystal structures of αGHY and PTP-1B predicted that all compounds bind to the catalytic domains of the enzymes. Together, these results showed that M. circinata is a potential source of antidiabetic drug leads.
Background
Protein tyrosine phosphatases are enzymes which help in signal transduction in diabetes, obesity, cancer, liver diseases and neurodegenerative diseases. PTP1B is the main member of this enzyme from the protein extract of human placenta. In phosphate inhibitors development significant progress has been made over the last 10 years, few compounds has reached in early-stage clinical trials whereas later stage trials or registration yet none have progressed. Many researchers are investigating different ways to improve the pharmacological properties of PTP1B inhibitors.
Objective
In the present review, authors have summarized various aspects related to involvement of PTP1B in various types of signal transduction mechanisms and its prominent role in various diseases like cancer, liver diseases and diabetes mellitus.
Conclusion
There are still certain challenges for selection of PTP1B as a drug target. Therefore continuous future efforts are needed to explore this target for development of PTP inhibitors to treat the prevailing diseases associated with it.
G protein-coupled receptor (GPCR) kinases (GRKs) are responsible for initiating desensitization of activated GPCRs. GRK5 is potently inhibited by the calcium-sensing protein calmodulin (CaM), which leads to nuclear translocation of GRK5 and promotion of cardiac hypertrophy. Herein, we report the architecture of the Ca ²⁺ ·CaM–GRK5 complex determined by small-angle X-ray scattering and negative-stain electron microscopy. Ca ²⁺ ·CaM binds primarily to the small lobe of the kinase domain of GRK5 near elements critical for receptor interaction and membrane association, thereby inhibiting receptor phosphorylation while activating the kinase for phosphorylation of soluble substrates. To define the role of each lobe of Ca ²⁺ ·CaM, we utilized the natural product malbrancheamide as a chemical probe to show that the C-terminal lobe of Ca ²⁺ ·CaM regulates membrane binding while the N-terminal lobe regulates receptor phosphorylation and kinase domain activation. In cells, malbrancheamide attenuated GRK5 nuclear translocation and effectively blocked the hypertrophic response, demonstrating the utility of this natural product and its derivatives in probing Ca ²⁺ ·CaM-dependent hypertrophy.
From the rice-based culture of Malbranchea flavorosea, three new compounds namely flavoroseoside B (5-desoxy-5-chloro-flavoroseoside) (2), 4-hydroxy-2-O-α-ribofuranosyl-5-methylacetophenone (3), and (S)-3,4-dihydro-3-(1H-indol-3-ylmethyl)-4-methyl-1H-1,4-benzodiazepine-2,5-dione (4), along with three known compounds, rosigenin (5), massarilactone B (6), and riboxylarinol B (7) were obtained. The structures were determined by spectroscopic methods. Compound 4 and its synthetic analog 3,4-dihydro-3-(1H-indol-3-ylmethyl)-1-methyl-1H-1,4-benzodiazepine-2,5-dione (9) inhibited the activity of Ruminococus obeum α-glucosidase enzyme. Molecular docking and dynamic studies revealed that compounds 4 and 9 might bind to this α-glucosidase at the catalytic center. Phylogenetic analysis using internal transcribed spacer region revealed that Malbranchea flavorosea ATCC 34529 is related to Myxotrichum spp.
Since PTP1B enzyme was discovered in 1988, it has captured the research community’s attention. This landmark discovery has stimulated numerous research studies on a variety of human diseases, including cancer, inflammation, and diabetes. Tremendous progress has been made in finding PTP1B inhibitors and exploring PTP1B regulatory mechanisms. This review investigates for the natural PTP1B inhibitors, and focuses on the common characteristics of the discovered structures and structure–activity relationships. To facilitate understanding, all the natural compounds are here divided into five different classes (fatty acids, phenolics, terpenoids, steroids, and alkaloids), according to their skeletons. These PTP1B inhibitors of scaffold structures could serve as a theoretical basis for new concept drug discovery and design.
Malbrancheamide is a dichlorinated fungal indole alkaloid isolated from Malbranchea aurantiaca that belongs to a family of natural products containing a characteristic bicyclo[2.2.2]diazaoctane core. The introduction of chlorine atoms on the indole ring of malbrancheamide differentiates it from other members of this family and contributes significantly to its biological activity. In this study, we characterized the flavin-dependent halogenase involved in the late-stage halogenation of malbrancheamide. MalA catalyzes the iterative dichlorination and monobromination of the free substrate premalbrancheamide as the final steps in the malbrancheamide biosynthetic pathway. Two unnatural bromo-chloro-malbrancheamide analogs were generated through MalA-mediated chemoenzymatic synthesis. Structural analysis and computational studies of MalA in complex with three substrates revealed that the enzyme represents a new class of zinc-binding flavin-dependent halogenases, and provides new insights into a potentially unique reaction mechanism.
Model-based molecular phylogenetics plays an important role in comparisons of genomic data, and model selection is a key step in all such analyses. We present ModelFinder, a fast model-selection method that greatly improves the accuracy of phylogenetic estimates by incorporating a model of rate heterogeneity across sites not previously considered in this context and by allowing concurrent searches of model space and tree space.
Diabetes mellitus and obesity are one of the most common health issues spread throughout world and raised the medical attention to find the new effective agents to treat these disease state. Occurrence of the drug resistance to the insulin and leptin receptor is also challenging major issues. The molecules that can overcome this resistance problem could be effective for the treatment of both type II diabetes and obesity. Protein Tyrosine Phosphatase (PTP) has emerged as new promising targets for therapeutic purpose in recent years. Protein Tyrosine Phosphatase 1B (PTP 1B) act as a negative regulator of insulin and leptin receptor signalling pathways. Several approaches have been successfully applied to find out potent and selective inhibitors. This article reviews PTP 1B inhibitors; natural, synthetic and semi-synthetic that showed inhibition towards enzyme as a major target for the management of type II diabetes. These studies could be contributing the future development of PTP 1B inhibitors as drugs.
In this study, the fungus Aspergillus felis (strain UFMGCB 8030) was isolated from rocks in the Atacama Desert, Chile. Its CH2Cl2 extract exhibited antifungal activity (MIC of 1.9 μg/mL) against Paracoccidioides brasiliensis (Pb18), which is the etiological agent for paracoccidioidomycosis. The crude extract was purified, and a new cytochalasin derivative, cytochalasin Z15E (1), and one ardeemin derivative, 5-N-acetyl-8-β-isopropyl-ardeemin (4), in addition to five known secondary metabolites, rosellichalasin (2), cytochalasin E (3), gancidin (5), pseurotin A1 (6), and 2,4-dihydroxyacetophenone (7), were isolated. The structures of these compounds were confirmed by 1D and 2D NMR spectroscopy and HR-ESI–MS. Cytochalasin E was the most active compound with MIC an value of 3.6 μM, while the other isolated compounds exhibited weak antifungal activities (MIC values >100.0 μM).
Experimental and computational approaches to estimate solubility and permeability in discovery and development settings are described. In the discovery setting 'the rule of 5' predicts that poor absorption or permeation is more likely when there are more than 5 H-bond donors, 10 H-bond acceptors, the molecular weight (MWT) is greater than 500 and the calculated Log P (CLogP) is greater than 5 (or MlogP>4.15). Computational methodology for the rule-based Moriguchi Log P (MLogP) calculation is described. Turbidimetric solubility measurement is described and applied to known drugs. High throughput screening (FITS) leads tend to have higher MWT and Log P and lower turbidimetric solubility than leads in the pre-HTS era. In the development setting, solubility calculations focus on exact value prediction and are difficult because of polymorphism. Recent work on linear free energy relationships and Log P approaches are critically reviewed. Useful predictions are possible in closely related analog series when coupled with experimental thermodynamic solubility measurements. (C) 2012 Published by Elsevier B.V.
The DP4 probability is one of the most sophisticated and reliable approaches for the stereochemical assignment of organic molecules using GIAO NMR chemical shift calculations when only one set of experimental data is available. In order to improve the performance of the method, we have developed a modified probability (DP4+), whose main differences from the original DP4 are the inclusion of unscaled data and the use of higher levels of theory for the NMR calculation procedure. With these modifications, a significant improvement in the overall performance was achieved, providing accurate and confident results in establishing the stereochemistry of 48 challenging isomeric compounds.
Objectives
This study was conducted to evaluate the vasorelaxant effect of the fungal alkaloids malbrancheamides on pre-contracted rat aorta rings. Also, we explored the probable mode of action using experimental and theoretical docking studies.Methods
The vasorelaxant effect was assessed on rat aorta rings pre-contracted with noradrenaline (0.1 μm). The mechanism of action was evaluated using different inhibitors of the pathways involved in the vasorelaxation process, such as l-NAME, indomethacin, tetraethylammonium and atropine. The docking analyses were carried out with AutoDock 4.2 software using the crystallized structure of the cyclooxygenase domain of eNOS.Key findingsMalbrancheamides (1–3) induced a significant vasorelaxant activity in a concentration- and endothelium-intact model in rat aorta rings, and a lesser effect in an endothelium-denuded model. Malbrancheamide-induced vasorelaxation was significantly weakened by pretreatment of endothelium-intact aortic rings with L-NAME (10 μm), indicating a nitrergic relaxant mechanism. Docking analysis predicted that 1–3 could activate eNOS throughout an allosteric fashion at C1 and C2 pockets.Conclusions
Experimental evidence revealed that malbrancheamides induced both endothelium-independent and endothelium-dependent relaxant effects. According to theoretical studies, it is feasible that the endothelium-independent relaxation exerted by malbrancheamide could be mediated by its calmodulin inhibitory properties throughout an interference with myosin light chain phosphorylation and a positive modulation of eNOS.
We present an implementation of explicit solvent all atom classical molecular dynamics (MD) within the AMBER program package that runs entirely on CUDA-enabled GPUs. First released publicly in April 2010 as part of version 11 of the AMBER MD package and further improved and optimized over the last two years, this implementation supports the three most widely used statistical mechanical ensembles (NVE, NVT, and NPT), uses particle mesh Ewald (PME) for the long-range electrostatics, and runs entirely on CUDA-enabled NVIDIA graphics processing units (GPUs), providing results that are statistically indistinguishable from the traditional CPU version of the software and with performance that exceeds that achievable by the CPU version of AMBER software running on all conventional CPU-based clusters and supercomputers. We briefly discuss three different precision models developed specifically for this work (SPDP, SPFP, and DPDP) and highlight the technical details of the approach as it extends beyond previously reported work [Götz et al., J. Chem. Theory Comput. 2012, DOI: 10.1021/ct200909j; Le Grand et al., Comp. Phys. Comm. 2013, DOI: 10.1016/j.cpc.2012.09.022].We highlight the substantial improvements in performance that are seen over traditional CPU-only machines and provide validation of our implementation and precision models. We also provide evidence supporting our decision to deprecate the previously described fully single precision (SPSP) model from the latest release of the AMBER software package.
Malbranchea species belong to the family Onygenaceae and are taxonomically close to human and animal pathogenic fungi.¹ The fact prompted us to investigate the chemical constituents of Malbranchea fungi. We already have reported the isolation and structural characterization of 4-benzyl-3-phenyl-5H-furan-2-one as a vasodilator, malfilanols A and B as antifungal and cytotoxic sesquiterpenes, malbrancheosides A–D as triterpene glycosides and malfilamentosides A and B as furanone glycosides, from the fungus Malbranchea filamentosa IFM41300. Further purification of extracts of rice cultivated by the above fungus allowed us to isolate three new cytotoxic anthrasteroid glycosides, designated malsterosides A (1), B (2) and C (3) (Figure 1).
We describe PTRAJ and its successor CPPTRAJ, two complementary, portable, and freely available computer programs for the analysis and processing of time series of three-dimensional atomic positions (i.e., coordinate trajectories) and the data therein derived. Common tools include the ability to manipulate the data to convert among trajectory formats, process groups of trajectories generated with ensemble methods (e.g., replica exchange molecular dynamics), image with periodic boundary conditions, create average structures, strip subsets of the system, and perform calculations such as RMS fitting, measuring distances, B-factors, radii of gyration, radial distribution functions, and time correlations, among other actions and analyses. Both the PTRAJ and CPPTRAJ programs and source code are freely available under the GNU General Public License version 3 and are currently distributed within the AmberTools 12 suite of support programs that make up part of the Amber package of computer programs (see http://ambermd.org). This overview describes the general design, features, and history of these two programs, as well as algorithmic improvements and new features available in CPPTRAJ.
In the course of searching for new biologically active metabolites in Malbranchea filamentosa, four new triterpene glycosides, malbrancheosides A (1) - D (4), were isolated. The structures of 1-4 were confirmed by the chemical and spectroscopic investigation. Malbrancheosides A (1) -D (4) are the first example of triterpenoidal glycosides having D-glucosamine derivatives from the fungal sources.
Three new aromatic butenolides, gymnoascolides A-C (1-3), have been isolated from the Australian soil ascomycete Gymnoascus reessii and assigned structures on the basis of detailed spectroscopic analysis. The absolute configurations of gymnoascolides B (2) and C (3) at C-5 were solved using a combination of chemical derivatization and quantum chemical simulations.
Prenylated indole alkaloids are a diverse group of fungal secondary metabolites and represent an important biosynthetic class. In this study we have identified new halogenated prenyl-indole alkaloids from an invertebrate-derived Malbranchea graminicola strain. Using direct analysis in real time (DART) mass spectrometry, these compounds were initially detected from hyphae of the fungus grown on agar plates, without the need for any organic extraction. Subsequently, the metabolites were isolated from liquid culture in artificial seawater. The structures of two novel chlorinated metabolites, named (-)-spiromalbramide and (+)-isomalbrancheamide B, provide additional insights into the assembly of the malbrancheamide compound family. Remarkably, two new brominated analogues, (+)-malbrancheamide C and (+)-isomalbrancheamide C, were produced by enriching the growth medium with bromine salts.
The Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) and the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) methods calculate binding free energies for macromolecules by combining molecular mechanics calculations and continuum solvation models. To systematically evaluate the performance of these methods, we report here an extensive study of 59 ligands interacting with six different proteins. First, we explored the effects of the length of the molecular dynamics (MD) simulation, ranging from 400 to 4800 ps, and the solute dielectric constant (1, 2, or 4) on the binding free energies predicted by MM/PBSA. The following three important conclusions could be observed: (1) MD simulation length has an obvious impact on the predictions, and longer MD simulation is not always necessary to achieve better predictions. (2) The predictions are quite sensitive to the solute dielectric constant, and this parameter should be carefully determined according to the characteristics of the protein/ligand binding interface. (3) Conformational entropy often show large fluctuations in MD trajectories, and a large number of snapshots are necessary to achieve stable predictions. Next, we evaluated the accuracy of the binding free energies calculated by three Generalized Born (GB) models. We found that the GB model developed by Onufriev and Case was the most successful model in ranking the binding affinities of the studied inhibitors. Finally, we evaluated the performance of MM/GBSA and MM/PBSA in predicting binding free energies. Our results showed that MM/PBSA performed better in calculating absolute, but not necessarily relative, binding free energies than MM/GBSA. Considering its computational efficiency, MM/GBSA can serve as a powerful tool in drug design, where correct ranking of inhibitors is often emphasized.
A new malbrancheamide analogue, isomalbrancheamide B (3), along with three known compounds, malbrancheamide (1), isomalbrancheamide (2), and premalbrancheamide (4), were isolated in higher yields from the alkaloid fraction of the fungus Malbranchea aurantiaca. The interaction of the alkaloids 1-4 with calmodulin (CaM) was analyzed using different enzymatic, fluorescence, spectroscopic, nuclear magnetic resonance (NMR), and molecular modelling techniques. On the basis of the enzymatic and fluorescence experiments, malbrancheamides 1-3 are classical CaM inhibitors. Compound 4, however, did not quench the extrinsic fluorescence of the CaM biosensor indicating that it could be a functional inhibitor. Circular dichroism, NMR, and molecular modelling studies revealed that 1 binds to CaM in the same hydrophobic pocket than the chlorpromazine and trifluoperazine, two classical CaM inhibitors. Thus, malbrancheamide and related monochlorinated analogues are compounds with a high potential for the development of new therapeutic agents, involving CaM as their molecular target.
This report describes a number of substructural features which can help to identify compounds that appear as frequent hitters (promiscuous compounds) in many biochemical high throughput screens. The compounds identified by such substructural features are not recognized by filters commonly used to identify reactive compounds. Even though these substructural features were identified using only one assay detection technology, such compounds have been reported to be active from many different assays. In fact, these compounds are increasingly prevalent in the literature as potential starting points for further exploration, whereas they may not be.
Six photosensitive polyketides, malbranpyrroles A-F, were discovered from the thermophilic fungus Malbranchea sulfurea by using intact-cell desorption/ionization on silicon mass (ICD-MS) and LC-SPE-NMR. These two strategies facilitate the searching and structural determination of unstable natural products. The ICD-MS indicated that only brown hyphae of M. sulfurea can produce malbranpyrroles. The biosynthetic pathway of malbranpyrroles was evidenced by 13C isotope precursors and amino acid feeding experiments. The cytotoxicity data revealed that the conformation of the conjugated system in malbranpyrroles does not affect cytotoxic potency against cancer cell lines. In addition, the chlorine atom was shown to be the pharmacophore for cytotoxicity.
An advanced metabolite, named premalbrancheamide, involved in the biosynthesis of malbrancheamide (1) and malbrancheamide B (2) has been synthesized in double (13)C-labeled form and was incorporated into the indole alkaloid 2 by Malbranchea aurantiaca. In addition, premalbrancheamide has been detected as a natural metabolite in cultures of M. aurantiaca. The biosynthetic implications of these experiments are discussed.
Literature data on compounds both well- and poorly-absorbed in humans were used to build a statistical pattern recognition model of passive intestinal absorption. Robust outlier detection was utilized to analyze the well-absorbed compounds, some of which were intermingled with the poorly-absorbed compounds in the model space. Outliers were identified as being actively transported. The descriptors chosen for inclusion in the model were PSA and AlogP98, based on consideration of the physical processes involved in membrane permeability and the interrelationships and redundancies between available descriptors. These descriptors are quite straightforward for a medicinal chemist to interpret, enhancing the utility of the model. Molecular weight, while often used in passive absorption models, was shown to be superfluous, as it is already a component of both PSA and AlogP98. Extensive validation of the model on hundreds of known orally delivered drugs, "drug-like" molecules, and Pharmacopeia, Inc. compounds, which had been assayed for Caco-2 cell permeability, demonstrated a good rate of successful predictions (74-92%, depending on the dataset and exact criterion used).
A simple pharmacophore point filter has been developed that discriminates between drug-like and nondrug-like chemical matter. It is based on the observation that nondrugs are often underfunctionalized. Therefore, a minimum count of well-defined pharmacophore points is required to pass the filter. The application of the filter results in 66-69% of subsets of the MDDR database to be classified as drug-like. Furthermore, 61-68% of subsets of the CMC database are classified as drug-like. In contrast, only 36% of the ACD are found to be drug-like. While these results are not quite as good as those obtained with recently described neural net approaches, the method used here has clear advantages. In contrast to a neural net approach and also in contrast to decision tree methods described recently, the pharmacophore filter has been developed by using "chemical wisdom" that is unbiased from fitting the structural content of specific drug databases to prediction models. Similar to decision tree methods, the pharmacophore point filter provides a detailed structural reason for the classification of each molecule as drug or nondrug. The pharmacophore point filter results are compared to neural net filter results. A statistically significant overlap between compounds recognized as drug-like validates both approaches. The pharmacophore point filter complements neural net approaches as well as property profiling approaches used as drug-likeness filters in compound library analysis and design.
Oral bioavailability measurements in rats for over 1100 drug candidates studied at SmithKline Beecham Pharmaceuticals (now GlaxoSmithKline) have allowed us to analyze the relative importance of molecular properties considered to influence that drug property. Reduced molecular flexibility, as measured by the number of rotatable bonds, and low polar surface area or total hydrogen bond count (sum of donors and acceptors) are found to be important predictors of good oral bioavailability, independent of molecular weight. That on average both the number of rotatable bonds and polar surface area or hydrogen bond count tend to increase with molecular weight may in part explain the success of the molecular weight parameter in predicting oral bioavailability. The commonly applied molecular weight cutoff at 500 does not itself significantly separate compounds with poor oral bioavailability from those with acceptable values in this extensive data set. Our observations suggest that compounds which meet only the two criteria of (1) 10 or fewer rotatable bonds and (2) polar surface area equal to or less than 140 A(2) (or 12 or fewer H-bond donors and acceptors) will have a high probability of good oral bioavailability in the rat. Data sets for the artificial membrane permeation rate and for clearance in the rat were also examined. Reduced polar surface area correlates better with increased permeation rate than does lipophilicity (C log P), and increased rotatable bond count has a negative effect on the permeation rate. A threshold permeation rate is a prerequisite of oral bioavailability. The rotatable bond count does not correlate with the data examined here for the in vivo clearance rate in the rat.