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Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD+, essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD+ for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic. Background: NAMPT catalyzes the rate-limiting reaction in converting nicotinamide to NAD+ in cancers. Results: NAMPT inhibition attenuates glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step, resulting in perturbing metabolic pathways related to glycolysis. Conclusion: The metabolic basis of NAMPT inhibition is the attenuation of glycolysis by reducing NAD+ available to glyceraldehyde 3-phosphate dehydrogenase. Significance: This study sheds new light on how NAMPT regulates cancer metabolism.

Fluorescent analogs were synthesized of the potent PI 3-kinase inhibitors, wortmannin and demethoxyviridin. The esterification of 11-deacetylwortmannin, 17-hydroxywortmannin, and demethoxyviridin with the fluorescent carboxylic acids NBD-sarcosine and 7-dimethylaminocoumarin-4-acetic acid generated six novel fluorescent esters. Potent inhibition of PI 3-kinase-alpha was observed for the derivatives of 11-desacetylwortmannin and demethoxyviridin.

Gene expression from the human immunodeficiency virus 1 (HIV-1) is greatly enhanced by binding of the virally encoded Tat protein to a 59-base RNA stem-loop structure, the Transactivation Responsive Element (TAR), located at the 5'-termini of all viral transcripts. This interaction was investigated in vitro using 32P-labelled TAR and highly purified Tat in which cysteine residues were blocked by sulpitolysis (S-Tat). It is shown that specific complex formation between S-Tat and TAR can occur in the presence of urea, with urea concentrations between 5 and 6 M causing an approximately two-fold increase in the level of binding. Two conditions favoring RNA secondary structure, low temperature (0 degree C) and the presence of divalent cations (Mg2+), diminished the level of specific binding. These observations suggest that the presence of mild denaturants promoted macromolecular refolding or rearrangement in a manner that increased the number of molecules available for binding, and present a general method for studying protein/RNA interactions where analysis has been obstructed by improper protein or RNA conformation.

A method has been developed to prepare, purify, and fully characterize poly-iodinated insulin-like growth factor II (IGF-II) which can then be catalytically deiodinated to produce IGF-II with its native disulfide bonded structure. This method can potentially be adapted to prepare tritiated IGF-II with the use of tritium gas in the hydrogenolysis step. IGF-II was iodinated at all three tyrosines using lactoperoxidase with a three-fold excess of sodium iodide. The iodinated products were purified using reversed-phase HPLC and characterized by peptide mapping. The tyrosine-containing peptides generated by pepsin digestion were characterized by amino acid sequence analysis. Monoand di-iodinated phenylthiohydantoin tyrosine derivatives were synthesized and used to identify the idodination state of the modified tyrosine residues in the sequence analysis. Purified poly-iodinated IGF-II was deiodinated by hydrogenolysis, over a prereduced palladium (II) oxide catalyst to form IGF-II with its native disulfide bonds intact, as shown by peptide mapping.

The Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases that phosphorylate the 3-hydroxyl group of the inositol ring of phosphatidylinositides. Although closely related, experimental evidence suggests that the four Class I PI3Ks may be functionally distinct. To further study their unique biochemical properties, the three human Class Ia PI3K (alpha, beta, and delta) p110 catalytic domains were cloned and co-expressed with the p85alpha regulatory domain in Sf9 cells. None of the p110 subunits were successfully expressed in the absence of p85alpha. Successful expression and purification of each p85alpha/p110 protein required using an excess of the p110 vector over the p85 vector during co-infection of Sf9 cells. Proteins were purified as the p85alpha/p110 complex by nickel affinity chromatography through an N-terminal His-tag on the p110 subunit using an imidazole gradient. The purification yields were high using the optimized ratio of p85/p110 vector and small culture volumes, with 24mg/L cell culture media for p85alpha/p110alpha, 17.5mg/L for p85alpha/p110delta, and 3.5mg/L for p85alpha/p110beta. The identity of each purified isoform was confirmed by mass spectral analysis and immunoblotting. The activities of the three p85alpha/p110 proteins and the Class Ib p110gamma catalytic domain were investigated using phosphatidylinositol 4,5-bisphosphate (PIP2) as the substrate in a PIP2/phosphatidylserine (PS) liposome. All four enzymes exhibited reaction velocities that were dependent on the surface concentration of PIP2. The surface concentrations that gave maximal activity for each human isoform with 0.5mM PIP2 were 2.5mol% PIP2 for p110gamma, 7.5mol% for p85alpha/p110beta, and 10mol% PIP2 for p85alpha/p110alpha and p85alpha/p110delta. The specific activity of p85alpha/p110alpha was three to five times higher than that of the other human isoforms. These kinetic differences may contribute to the unique roles of these isoforms in cells.

The first potent inhibitors of glutamate racemase (MurI) enzyme that show whole cell antibacterial activity are described. Optically pure 4-substituted D-glutamic acid analogues with (2R,4S) stereochemistry and bearing aryl-, heteroaryl-, cinnamyl-, or biaryl-methyl substituents represent a novel class of glutamate racemase inhibitors. Exploration of the D-Glu core led to the identification of lead compounds (-)-8 and 10. 2-Naphthylmethyl derivative 10 was found to be a potent competitive inhibitor of glutamate racemase activity (K(i) = 16 nM, circular dichroism assay; IC(50) = 0.1 microg/mL high-performance liquid chromatography (HPLC) assay). Thorough structure-activity relationship (SAR) studies led to benzothienyl derivatives such as 69 and 74 with increased potency (IC(50) = 0.036 and 0.01 microg/mL, respectively, HPLC assay). These compounds showed potent whole cell antibacterial activity against S. pneumoniae PN-R6, and good correlation with the enzyme assay. Compounds 69, 74 and biaryl derivative 52 showed efficacy in an in vivo murine thigh infection model against Streptococcus pneumoniae. Data described herein suggest that glutamate racemase may be a viable target for developing new antibacterial agents.

Publisher Summary This chapter describes expression and preparation of pure human cytomegalovirus (HCMV) assemblin with the help of a chelating peptide (CP) purification handle. Chelating peptide-immobilized metal ion affinity chromatography (CP-IMAC) uses an engineered metal-binding site or CP at either the N terminus or C terminus of a recombinant protein for a one-step affinity purification using IMAC. The CP sequences are easily incorporated into the protein with recombinant DNA cloning techniques. Cells are lysed with lysozyme and sonicated. Inclusion bodies are collected by centrifugation, lyophilized, and 10 mg/ml of solids dissolved in 0.5 M Tris, 7 M urea, pH 8.2. All purification steps are carried out in buffers containing 7 M urea, which maintains the enzyme in an unfolded inactive conformation. Blocking the cysteine residues as S-sulfonates facilitates the purification by solubilizing more protein from the inclusion bodies and preventing the formation of intermolecular disulfide bonds and aggregates. The activity of HCMV CP-assemblin is measured by following the hydrolysis of FITC, fluorescein isothiocyanate, a labeled peptide mimic of the maturational site of the assembly protein precursor.

We report our experimental results supporting the hypothesis that a specific metal-chelating peptide (CP) on the NH2 terminus of a protein can be used to purify that protein using immobilized metal ion affinity chromatography (IMAC). The potential utility of this approach resides with recombinant proteins since the nucleotide sequence that codes for the protein can be extended to include codons for the chelating peptide and thereby generate the gene for a chimeric CP-protein that can be cloned, expressed, and affinity-purified with immobilized metal ions. The chelating peptide purification handle could then be removed chemically or enzymatically after purification has been achieved to generate a protein with the natural amino acid sequence. The feasibility of using a chelating peptide as a purification handle has been demonstrated using a leuteinizing hormone-releasing hormone (LHRH) analog, 2-10 LHRH, which contains the previously identified chelating peptide, His-Trp, on the NH2 terminus. 2-10 LHRH had a high affinity for a Ni(II) IMAC column due to the NH2-terminal dipeptide sequence His-Trp, forming a coordination complex with Ni(II), whereas the controls, 3-10 LHRH and 4-10 LHRH, lacking the CP sequence, did not bind. Furthermore, 2-10 LHRH could be purified from a mixture of histidine-containing peptides on a Ni(II) IMAC column in one step. His-Trp proinsulin was used as a model of a recombinant CP-protein. The S-sulfonates of His-Trp-proinsulin and proinsulin were isolated from Escherichia coli engineered to overproduce these proteins as trpLE' fusion proteins. His-Trp-proinsulin(SSO3-)6 had a higher affinity for immobilized Ni(II) than proinsulin (SSO3-)6. Both proteins were eluted by decreasing the pH or by introducing a displacing ligand into the buffer. Ni(II) eluted from the column with much higher concentrations of displacing ligand than the proteins.

The complete peptide map of purified folded recombinant human insulin-like growth factor II (rhIGF-II) was determined to verify its sequence and disulfide bonding scheme. Each peptide generated by digestion with pepsin was purified and characterized by amino acid analysis, amino acid sequence analysis, and fast atom bombardment/mass spectrometry. Some peptides were also sequenced using tandem mass spectrometry. The rhIGF-II peptide map was compared to that of rat insulin-like growth factor II and to that of a disulfide-bonded isomer of rhIGF-II. The data obtained in these studies are consistent with the conclusion that the rhIGF-II obtained from Escherichia coli has the correct amino acid composition, sequence, and the native disulfide-bonded structure. The binding affinities of these forms of recombinant IGF-II for IGF carrier proteins were measured in an IGF binding protein assay. The disulfide isomer of rhIGF-II was 160-fold less potent than native rhIGF-II in the competitive protein binding assay. These studies illustrate the need to characterize recombinant polypeptides containing disulfide bonds to allow the native structure to be verified before characterizing the biological properties of such molecules in hopes of elucidating their physiologic functions.

Recently, the sequence of the human receptor for insulin-like growth factor II (IGF-II) was found to be 80% identical [Morgan et al., (1987) Nature 329 , 301–307] to the sequence of a partial clone of the bovine cation-independent mannose-6-phosphate receptor [Lobel et al., (1987) Proc. Natl. Acad. Sci. USA 84 , 2233–2237]. In the present study, the purified receptor for insulin-like growth factor II (IGF-II) was found to react with two different polyclonal antibodies to the purified mannose-6-phosphate receptor. Moreover, mannose-6-phosphate was found to stimulate the binding of labeled IGF-II to the IGF-II receptor by two-fold. This effect had the same specificity and affinity as the reported binding of mannose-6-phosphate to its receptor; mannose-1-phosphate and mannose had no effect on the binding of labeled IGF-II to its receptor, and the half-maximally effective concentration of mannose-6-phosphate was 0.3 mM. Also, mannose-6-phosphate did not affect labeled IGF-II binding to the insulin receptor. These results support the hypothesis that a single protein of Mr∼250,000 binds both IGF-II and mannose-6-phosphate. Furthermore, they indicate that mannose-6-phosphate can modulate the interaction of IGF-II to its receptor.

Human Insulin-like growth factor II (IGF-II) was produced in Escherichia coli as a 113 amino acid chimeric protein, LE1-Met-IGF-II consisting of 45 N-terminal amino acids from the E. coli trp leader peptide and the trpE polypeptide, and 67 C-terminal amino acids for IGF-II. High-level expression of the fusion protein, LE1-Met-IGF-II, was obtained with pCZ21, a plasmid containing a thermoinducible runaway replicon and the kanamycin resistance marker from Tn5. In E. coli, LE1-Met-IGF-II formed granules that were isolated and cleaved with cyanogen bromide to liberate IGF-II. IGF-II was sulfitolyzed, purified by anion exchange chromatography, refolded through a disulfide interchange reaction, and further purified by reverse-phase HPLC and gel filtration. The biological activity of this recombinant human IGF-II was measured in a competitive protein binding assay for IGF-II and by its ability to stimulate amino isobutyric acid uptake and protein synthesis in NCTC 2414 fibroblast cultures.

Several properties of the FeMo-cofactor (co) of nitrogenase in N-methylformamide solution at ambient temperature have been investigated by means of 19F NMR spectroscopy. With C6H5CF3 reference signals the magnetic moment per Mo atom was found to be approximately equal to 3.9 BM, consistent with S = 3/2 ground state identified by other spectroscopic methods at low temperature. Reaction of FeMo-co with 1.0 eq of RFS- (RF = p-C6H4CF3, p-C6H4F) afforded isotropically shifted signals indicative of binding to a paramagnetic cluster. By comparison with the spectra of Fe-S and Fe-Mo-S species derivatized with RFS-, including the cubane-type MoFe3S4 clusters with S = 3/2 ground states, it was concluded that the essential FeMo-co cluster structure remains intact and a Fe atom is the probable thiolate binding site. An interaction of FeMo-co with C6H5S- had been detected earlier by low temperature EPR spectroscopy. The binding site assignment is based on large observed isotropic shifts (ca. -12ppm) compared to the much smaller values found for Mo-SRF ligands in MoFe3S4 clusters and anticipated in FeMo-co on the basis of recent spectroscopic results. Isotropic 19F shifts have proven extremely sensitive to electronic and structural features of Fe-S and Fe-Mo-S clusters. The inclusion of a 19F NMR label in FeMo-co should prove of utility in further investigation of cofactor properties and reactions.