Unknown Biological Activity Research Articles

In silico methods to select biologically active anti-protozoan natural products

Neglected diseases (ND) caused by tropical protozoan parasites threaten millions of people in the world. Despite the efforts from academia, government authorities and pharma companies, the available marketed drugs are insufficient. Natural products (NP) comprise a rich source of compounds that may lead to new drugs against ND. In 2011, the Research Network Natural Products against Neglected Diseases (ResNet NPND)1 has been established as an international research initiative to make efforts against this global threat. In this work, we describe the use of in silico methods to find NP hits against protozoan parasites. A ResNet NPND data bank with ca. 1,000 chemical structures of anti-parasitic NP and their biological data has been created2. 2D/3D structure descriptors were calculated and non-relevant variables were eliminated by correlation matrix followed by linear forward selection. The in vitro activity data of 513 chemical structures was grouped into four different classes against the following parasites: Plasmodium falciparum (A; 291 structures), Trypanosoma brucei (B; 63), T. cruzi (C; 98) and Leishmania donovani (D; 61). Random Forest (RF) and k-Nearest Neighbors (k-NN), machine-learning tools used to classify objects according to their classes, were used for a multi-class classification of the 513 chemical structures of NP according to their biological activities (A-D). The cross-validated models using 2D descriptors showed an accuracy of 70 – 90% and good statistical significance (Kappa statistic and ROC area). Additional 2,948 chemical structures of NP from Analyticon Discovery GmbH (Germany) with unknown biological activities were used as external data set. The RF and k-NNN models were suitable to predict new NP hits against multiple targets that can be further investigated by docking and biological assays.

The Insecticide Synergist Piperonyl Butoxide Inhibits Hedgehog Signaling: Assessing Chemical Risks

The spread of chemicals, including insecticides, into the environment often raises public health concerns, as exemplified by a recent epidemiologic study associating in utero piperonyl butoxide (PBO) exposure with delayed mental development. The insecticide synergist PBO is listed among the top 10 chemicals detected in indoor dust; a systematic assessment of risks from PBO exposure, as for many toxicants unfortunately, may be underdeveloped when important biological targets that can cause toxicity are unknown. Hedgehog/Smoothened signaling is critical in neurological development. This study was designed to use novel high-throughput in vitro drug screening technology to identify modulators of Hedgehog signaling in environmental chemicals to assist the assessment of their potential risks. A directed library of 1408 environmental toxicants was screened for Hedgehog/Smoothened antagonist activity using a high-content assay that evaluated the interaction between Smoothened and βarrestin2 green fluorescent protein. PBO was identified as a Hedgehog/Smoothened antagonist capable of inhibiting Hedgehog signaling. We found that PBO bound Smoothened and blocked Smoothened overexpression-induced Gli-luciferase reporter activity but had no effect on Gli-1 downstream transcriptional factor-induced Gli activity. PBO inhibited Sonic Hedgehog ligand-induced Gli signaling and mouse cerebellar granular precursor cell proliferation. Moreover, PBO disrupted zebrafish development. Our findings demonstrate the value of high-throughput target-based screening strategies that can successfully evaluate large numbers of environmental toxicants and identify key targets and unknown biological activity that is helpful in properly assessing potential risks.

Regulation of RUNX2 transcription factor–DNA interactions and cell proliferation by vitamin D3 (cholecalciferol) prohormone activity

The fat-soluble prohormone cholecalciferol (Vitamin D3) is a precursor of the circulating 25-OH Vitamin D3, which is converted by 1α-hydroxylase to the biologically active 1,25-OH Vitamin D3. Active Vitamin D3 interacts with the Vitamin D receptor (VDR), a transcription factor that plays an important role in calcium mobilization and bone formation. RUNX2 is a DNA-binding transcription factor that regulates target genes important in bone formation, angiogenesis, and cancer metastasis. Using computer-assisted drug design (CADD) and a microtiter plate-based DNA-binding enzyme-linked immunosorbent assay (D-ELISA) to measure nuclear RUNX2 DNA binding, we have found that Vitamin D3 prohormones can modulate RUNX2 DNA binding, which was dose-dependent and sensitive to trypsin, salt, and phosphatase treatment. Unlabeled oligonucleotide or truncated, dominant negative RUNX2 proteins were competitive inhibitors of RUNX2 DNA binding. The RUNX2 heterodimeric partner, Cbfβ, was detected in the binding complexes with specific antibodies. Evaluation of several RUNX2:DNA targeted small molecules predicted by CADD screening revealed a previously unknown biological activity of the inactive Vitamin D3 precursor, cholecalciferol. Cholecalciferol modulated RUNX2:DNA binding at nanomolar concentrations even in cells with low VDR. Cholecalciferol and 25-OH Vitamin D3 prohormones were selective inhibitors of RUNX2-positive endothelial, bone, and breast cancer cell proliferation, but not of cells lacking RUNX2 expression. These compounds may have application in modulating RUNX2 activity in an angiogenic setting, in metastatic cells, and to promote bone formation in disease-mediated osteoporosis. The combination CADD discovery and D-ELISA screening approaches allows the testing of other novel derivatives of Vitamin D and/or transcriptional inhibitors with the potential to regulate DNA binding and biological function.

Antioxidant activity of methimazole–copper(II) bioactive species and spectroscopic investigations on the mechanism of its interaction with Bovine Serum Albumin

Free radical-mediated oxidative stress has been implicated in numerous autoimmune disorders including Graves’ disease. Hyperthyroidism results in a marked increase in intracellular antioxidant enzymes including superoxide dismutase. The later activity is significantly increased in untreated Graves’ patients while treatment with methimazole results in normalization of the free radical and antioxidant activity indices. In this context, and considering the findings mentioned above, the aim of the present study was to evaluate unknown biological activities of methimazole and its methimazole–copper(II) complex investigating their superoxide scavenger power. Under the applied experimental conditions, methimazole did not show superoxide dismutase (SOD) activity while the copper complexes exhibited a strong superoxide radical scavenging capacity. Bearing in mind that the capacity of drugs to bind and/or interact with albumin is essential for their pharmacokinetic and pharmacodynamic properties, a complete investigation of the binding ability of both compounds by using Fourier transform infrared (FT-IR), Raman and Fluorescence spectroscopies, and UV–Vis spectrophotometry was included. Besides, in order to probe the copper ligand environment the EPR spectra of such compounds were analyzed.

Chemical genetics approach to restoring p27Kip1 reveals novel compounds with antiproliferative activity in prostate cancer cells

BackgroundThe cyclin-dependent kinase (CDK) inhibitor p27Kip1 is downregulated in a majority of human cancers due to ectopic proteolysis by the ubiquitin-proteasome pathway. The expression of p27 is subject to multiple mechanisms of control involving several transcription factors, kinase pathways and at least three different ubiquitin ligases (SCFSKP2, KPC, Pirh2), which regulate p27 transcription, translation, protein stability and subcellular localization. Using a chemical genetics approach, we have asked whether this control network can be modulated by small molecules such that p27 protein expression is restored in cancer cells.ResultsWe developed a cell-based assay for measuring the levels of endogenous nuclear p27 in a high throughput screening format employing LNCaP prostate cancer cells engineered to overexpress SKP2. The assay platform was optimized to Z' factors of 0.48 - 0.6 and piloted by screening a total of 7368 chemical compounds. During the course of this work, we discovered two small molecules of previously unknown biological activity, SMIP001 and SMIP004, which increase the nuclear level of p27 at low micromolar concentrations. SMIPs (small molecule inhibitors of p27 depletion) also upregulate p21Cip1, inhibit cellular CDK2 activity, induce G1 delay, inhibit colony formation in soft agar and exhibit preferential cytotoxicity in LNCaP cells relative to normal human fibroblasts. Unlike SMIP001, SMIP004 was found to downregulate SKP2 and to stabilize p27, although neither SMIP is a proteasome inhibitor. Whereas the screening endpoint - nuclear p27 - was robustly modulated by the compounds, SMIP-mediated cell cycle arrest and apoptosis were not strictly dependent on p27 and p21 - a finding that is explained by parallel inhibitory effects of SMIPs on positive cell cycle regulators, including cyclins E and A, and CDK4.ConclusionsOur data provide proof-of-principle that the screening platform we developed, using endogenous nuclear p27 as an endpoint, presents an effective means of identifying bioactive molecules with cancer selective antiproliferative activity. This approach, when applied to larger and more diverse sets of compounds with refined drug-like properties, bears the potential of revealing both unknown cellular pathways globally impinging on p27 and novel leads for chemotherapeutics targeting a prominent molecular defect of human cancers.

Antimicrobial peptides from Phyllomedusa frogs: from biomolecular diversity to potential nanotechnologic medical applications

Screening for new bioactive peptides in South American anurans has been pioneered in frogs of the genus Phyllomedusa. All frogs of this genus have venomous skin secretions, i.e., a complex mixture of bioactive peptides against potential predators and pathogens that presumably evolved in a scenario of predator-prey interaction and defense against microbial invasion. For every new anuran species studied new peptides are found, with homologies to hormones, neurotransmitters, antimicrobials, and several other peptides with unknown biological activity. From Vittorio Erspamer findings, this genus has been reported as a "treasure store" of bioactive peptides, and several groups focus their research on these species. From 1966 to 2009, more than 200 peptide sequences from different Phyllomedusa species were deposited in UniProt and other databases. During the last decade, the emergence of high-throughput molecular technologies involving de novo peptide sequencing via tandem mass spectrometry, cDNA cloning, pharmacological screening, and surface plasmon resonance applied to peptide discovery, led to fast structural data acquisition and the generation of peptide molecular libraries. Research groups on bioactive peptides in Brazil using these new technologies, accounted for the exponential increase of new molecules described in the last decade, much higher than in any previous decades. Recently, these secretions were also reported as a rich source of multiple antimicrobial peptides effective against multidrug resistant strains of bacteria, fungi, protozoa, and virus, providing instructive lessons for the development of new and more efficient nanotechnological-based therapies for infectious diseases treatment. Therefore, novel drugs arising from the identification and analysis of bioactive peptides from South American anuran biodiversity have a promising future role on nanobiotechnology.

A Novel “Clip-and-link” Activity of Repeat in Toxin (RTX) Proteins from Gram-negative Pathogens: COVALENT PROTEIN CROSS-LINKING BY AN ASP-LYS ISOPEPTIDE BOND UPON CALCIUM-DEPENDENT PROCESSING AT AN ASP-PRO BOND

Clinical isolates of Neisseria meningitidis produce a repeat in toxin (RTX) protein, FrpC, of unknown biological activity. Here we show that physiological concentrations of calcium ions induce a novel type of autocatalytic cleavage of the peptide bond between residues Asp(414) and Pro(415) of FrpC that is insensitive to inhibitors of serine, cysteine, aspartate, and metalloproteases. Moreover, as a result of processing, the newly generated amino-terminal fragment of FrpC can be covalently linked to another protein molecule by a novel type of Asp-Lys isopeptide bond that forms between the carboxyl group of its carboxyl-terminal Asp(414) residue and the epsilon-amino group of an internal lysine of another FrpC molecule. Point substitutions of negatively charged residues possibly involved in calcium binding (D499K, D510A, D521K, and E532A) dramatically reduced the self-processing activity of FrpC. The segment necessary and sufficient for FrpC processing was localized by deletion mutagenesis within residues 400-657, and sequences homologous to this segment were identified in several other RTX proteins. The same type of calcium-dependent processing and cross-linking activity was observed also for the purified ApxIVA protein of Actinobacillus pleuropneumoniae. These results define a protein cleavage and cross-linking module of a new class of RTX proteins of Gram-negative pathogens of man, animals, and plants. In the calcium-rich environments colonized by these bacteria this novel activity is likely to be of biological importance.

Purification of kappa-casien glycomacropeptide from sweet whey with undetectable level of phenylalanine.

Glycomacropeptide (GMP) found in sweet whey is a biologically active compound released from kappa-casein by the action of chymosin during cheese making. This study was undertaken to purify GMP from sweet whey as a research chemical on a laboratory scale. Glycomacropeptide was isolated from proteins and other non-GMP compounds by deproteinization with trichloroacetic acid and gel chromatography on Sephacryl S-200. The purified GMP accounted for 0.12% of dry sweet whey powder and contained 107.0, 50.9, 61.2 and 4.3 microg, respectively, of sialic acid, galactose, galactosamine and phosphorus per mg dry weight. The GMP was of high purity, with its amino acid composition showing undetectable levels of phenylalanine, tyrosine and arginine, the amino acids that do not occur in bovine GMP. On gel electrophoresis, the GMP showed a single broad band with an average mobility faster than that of carbonic anhydrase (molecular weight = 31 kDa). The purified GMP may be used as a standard glycopeptide in chromatography and electrophoresis and may also be used to test various known or unknown properties and biological activities of this compound.

Characterization of a monoclonal antibody to bovine xanthine oxidase.

The isolation of a hybridoma cell line, C-41, secreting monoclonal antibody to bovine xanthine oxidase (EC 1.2.3.2), is described. The specificity of this antibody was determined by solid-phase immunoassay, immunoblotting procedures, affinity chromatography, immunoelectrophoresis and precipitation techniques. The results are compared with those obtained in similar specificity studies on a previously described monoclonal antibody secreted by hybridoma cell line A-94 [Mather, Nace, Johnson & Goldsby (1980) Biochem. J. 188, 925-928]. This latter antibody appears to bind to xanthine oxidase only when the enzyme is immobilized on a solid support such as a plastic plate or nitrocellulose paper. Potential problems in the determination of the specificity of monoclonal antibodies, especially towards membrane proteins of unknown biological activity, are discussed.

Synthesis and biological activity of vitamin D3 3 beta-sulfate. Role of vitamin D3 sulfates in calcium homeostasis.

To determine the biological activity of vitamin D sulfates, we synthesized vitamin D3 3 beta-sulfate and tested its biological activity in vitamin D-deficient hypocalcemic rats. When vitamin D3 sulfate was administered as a single oral dose of 208,000 or 416,000 pmol (100 micrograms or 200 micrograms), it increased active calcium transport in the duodenum and was also able to mobilize calcium from bone and soft tissue. Dose levels below this failed to elicit a response. Vitamin D3 itself was active at doses as low as 260 pmol when administered in this manner. In order to test the biological activity of vitamin D3 sulfate in various doses when administered chronically, we tested the biological activity of vitamin D3 sulfate after 5 days or oral dosing: vitamin D3 sulfate was active at doses of 52,000 pmol/day (25 microgram), whereas vitamin D3 was active at doses of 65 to 260 pmol/day over a period of 5 days. When administered as a single intravenous dose, vitamin D3 sulfate exhibited no biological activity in doses as high as 52,000 pmol. Vitamin D3, however, was active at a dose of as low as 65 pmol. We conclude that vitamin D3 sulfate, a metabolite of vitamin D3 of heretofore unknown biological activity, is considerably less active than vitamin d3 itself.

Murine leukemia viruses containing an ∼30 S RNA subunit of unknown biological activity, in addition to the 38 S subunit of the viral genome

The 50–70 S RNA complex of a clone of Kirsten murine leukemia virus (Ki-MLV), propagated in NIH 3T3 cells, as well as that of a Moloney (Mo)-MLV, propagated in a mouse thymus-bone marrow cell line, contained an ∼30 S RNA of unknown biological activity in addition to 38 S genomic RNA. The ∼30 S RNA shares with 38 S virion RNA the ability to form 50–70 S RNA complexes and to be packaged by virus particles. The morphology of the cell lines propagating these viruses was indistinguishable from that of uninfected controls.

Properties and function of indoleamine 2,3-dioxygenase.

Available evidence indicates that indoleamine 2,3-dioxygenase, which is particularly abundant in the lung, colon, and intestine, catalyzes the oxidative ring cleavage of the pyrrole moiety of not only tryptophan and 5-hydroxytryptophan but also serotonin, melatonin, and other indoleamine derivatives. Our recent experiments with slices of intestine, together with those with organ culture of pineal glands, perfusion of heart and lung preparations and intact animals, which I did not have time to mention today, indicate that the oxygenative ring cleavages of various indoleamines do occur in vivo to a significant extent. Secondly, this enzyme appears to utilize superoxide anion rather than molecular oxygen. If superoxide anion is demonstrated, unequivocally, to be the true substrate of this enzyme, not only in vitro but also in vivo, then indoleamine 2,3-dioxygenase may be the first example of a new class of enzymes which utilize superoxide anion as an oxidizing agent just as peroxidases utilize hydrogen peroxide. Lastly, this novel enzyme produced a new class of biogenic amines, which may be referred to, generically, as "anthraniloylamines." Although their natural occurrence has not yet been established, they may have hitherto unknown biological or pharmacological activities and are currently under investigation in our laboratory.

Teratogenic compounds of veratrum californicum (durand)—IV. First isolation of veratramine and alkaloid Q and A reliable method for isolation of cyclopamine

The known steroidal, teratogenic alkaloid veratramine and another alkaloid, designated alkaloid Q with as yet unknown structure and biological activity, were isolated for the first time from Veratrum californicum, a plant that induces teratogenic effects in sheep. A more reliable method than previously available is also described for isolation of cyclopamine, the alkaloid that induces cyclopian teratogenic effects in sheep.

Abnormal trace elements in man: Tellurium

TELLURIUM, atomic number 52, is an element of Group VIA of the Periodic Table, which includes oxygen, sulfur, selenium and polonium, all of biologic interest. Named from the Latin relZus, or earth, tellurium is said to rank seventy-fifth in abundance of the elements on the earth’s crust, 0.002 ppm, [l, 23 and has not been detected by older analytical methods in soils, according to VINOGRADOV, [3] nor in sea water, according to MASON [I] and GOLDSCHMIDT. [4] It has been found, however, in certain coals, ,[4] which suggests that plants may have absorbed the element from soil in ancient times. Under these circumstances, one would have little interest in tellurium as an element worthy of biological attention, except for its effects on persons working directly with its compounds. We were intrigued, however, with the report of 1075 ppm tellurium found by neutron activation analysis in the ash of livers of human subjects, with lesser but still large amounts in bone, kidney, lung, ovary, spleen and testes. [5] It is a strange paradox that an element hitherto undetected in sea water, soils, plants and animals should be present in such large amounts in human tissues. Therefore, we have made an extensive survey of tellurium in foods, waters, vegetation and animal tissues and have analyzed samples of human organs, blood and urine in order to attempt to answer these questions: (1) What is the source of the tellurium? (2) Is the abundance of tellurium in soils greater than has heretofore been thought? (3) Is tellurium a constant constituent of biomaterial? (4) Does tellurium have some unknown biological activity or is it inert? (5) Does tellurium accumulate in mammalian tissues with age, and if so, could it influence a chronic disease? (6) Is human exposure to tellurium a “natural” phenomenon or a result of industrial contamination?