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AICARFT is a folate dependent catalytic site within the ATIC gene, part of the purine biosynthetic pathway, a pathway frequently upregulated in cancers. LSN3213128 is a potent (16 nM) anti-folate inhibitor of AICARFT and selective relative to TS, SHMT1, MTHFD1, MTHFD2 and MTHFD2L. Increases in ZMP, accompanied by activation of AMPK and cell growth inhibition, were observed with treatment of LY3213128. These effects on ZMP and proliferation were dependent on folate levels. In human breast MDA-MB-231met2 and lung NCI-H460 cell lines, growth inhibition was rescued by hypoxanthine, but not in the A9 murine cell line which is deficient in purine salvage. In athymic nude mice, LSN3213128 robustly elevates ZMP in MDA-MB-231met2, NCI-H460 and A9 tumors in a time and dose dependent manner. Significant tumor growth inhibition in human breast MDA-MB231met2 and lung NCI-H460 xenografts and in the syngeneic A9 tumor model were observed with oral administration of LSN3213128. Strikingly, AMPK appeared activated within the tumors and did not change even at high levels of intratumoral ZMP after weeks of dosing. These results support the evaluation of LSN3213128 as an antineoplastic agent.

A hallmark of cancer is unbridled proliferation that can result in increased demand for de novo synthesis of purine and pyrimidine bases required for DNA and RNA biosynthesis. These synthetic pathways are frequently upregulated in cancer and involve various folate-dependent enzymes. Antifolates have a proven record as clinically used oncolytic agents. Our recent research efforts have produced LSN 3213128 (compound 28a), a novel, selective, nonclassical, orally bioavailable antifolate with potent and specific inhibitory activity for aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT), an enzyme in the purine biosynthetic pathway. Inhibition of AICARFT with compound 28a results in dramatic elevation of 5-aminoimidazole 4-carboxamide ribonucleotide (ZMP) and growth inhibition in NCI-H460 and MDA-MB-231met2 cancer cell lines. Treatment with this inhibitor in a murine based xenograft model of triple negative breast cancer (TNBC) resulted in tumor growth inhibition.

DNA-dependent RNA polymerase II (RNAP II) largest subunit RPB1 C-terminal domain (CTD) kinases, including CDK9, are serine/threonine kinases known to regulate transcriptional initiation and elongation by phosphorylating Ser 2, 5, and 7 residues on CTD. Given the reported dysregulation of these kinases in some cancers, we asked whether inhibiting CDK9 may induce stress response and preferentially kill tumor cells. Herein, we describe a potent CDK9 inhibitor, LY2857785, that significantly reduces RNAP II CTD phosphorylation and dramatically decreases MCL1 protein levels to result in apoptosis in a variety of leukemia and solid tumor cell lines. This molecule inhibits the growth of a broad panel of cancer cell lines, and is particularly efficacious in leukemia cells, including orthotopic leukemia preclinical models as well as in ex vivo acute myeloid leukemia and chronic lymphocytic leukemia patient tumor samples. Thus, inhibition of CDK9 may represent an interesting approach as a cancer therapeutic target, especially in hematologic malignancies., (©2014 American Association for Cancer Research.)

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.

DNA ligases are the enzymes essential for DNA replication, repair and recombination in all organisms. The bacterial DNA ligases involved in DNA replication require NAD(+) for activity, but eukaryotic and viral DNA ligases require ATP. Because of their essential nature, unique structures and widespread existence in nature, bacterial DNA ligases represent a class of valuable targets for identifying novel and selective antibacterial agents. In this study, we cloned and expressed the ligA gene from Streptococcus pneumoniae, and characterized this ligA-encoded NAD(+)-dependent DNA ligase. We then screened small molecule chemical libraries using a biochemical assay and identified a new small molecule with a structure of 2,4-diamino-7-dimethylamino-pyrimido[4,5-d]pyrimidine. We show that this small molecule is a specific inhibitor of bacterial NAD(+)-dependent DNA ligases. Biochemical studies show that this molecule inhibits NAD(+)-dependent DNA ligases, but not ATP-dependent enzymes. The molecule inhibits NAD(+)-dependent DNA ligases competitively with respect to NAD(+) and specifically inhibits enzyme adenylation, but not DNA adenylation or ligation. Labeling studies establish that this molecule inhibits the incorporation of thymidine into DNA and that overexpression of DNA ligase in the cell abolishes this inhibition. Finally, microbiological studies show that this molecule exhibits a broad spectrum of antibacterial activity. Together, this study shows that this small molecule inhibitor identified is specific to bacterial NAD(+)-dependent DNA ligases, exhibits a broad spectrum of antibacterial activities, and has the potential to be developed into an antibacterial agent.

Many 3-aryl-4-(1,2,3,4-tetrahydro[1,4]diazepino[6,7,1-hi]indol-7-yl)maleimides exhibit potent GSK3 inhibitory activity (<100 nM IC(50)), although few show significant selectivity (>100x) versus CDK2, CDK4, or PKCbetaII. However, combining 3-(imidazo[1,2-a]pyridin-3-yl), 3-(pyrazolo[1,5-a]pyridin-3-yl) or aza-analogs with a 4-(2-acyl-(1,2,3,4-tetrahydro[1,4]diazepino[6,7,1-hi]indol-7-yl)) group on the maleimide resulted in very potent inhibitors of GSK3 (160 to >10,000-fold selectivity versus CDK2/4 and PKCbetaII. These compounds also inhibited tau phosphorylation in cells and were effective in lowering plasma glucose in a rat model of type 2 diabetes (ZDF rat).

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.

DNA ligase is an enzyme essential for DNA replication, repair, and recombination in all organisms. Bacterial DNA ligases catalyze a NAD(+)-dependent DNA ligation reaction, i.e., the formation of a phosphodiester bond between adjacent 3'-OH and 5'-phosphate termini of dsDNA. Due to their essential nature, unique cofactor requirement, and widespread existence in nature, bacterial DNA ligases appear to be valuable targets for identifying novel antibacterial agents. To explore bacterial DNA ligases as antibacterial targets and further characterize them, we developed a simple, robust, homogeneous time-resolved fluorescence resonance energy transfer assay (TR-FRET) for measuring Streptococcus pneumoniae DNA ligase activity. This assay involves the use of one dsDNA molecule labeled with biotin and another dsDNA molecule labeled with Cy5, an acceptor fluorophore. During ligation reactions, the donor fluorophore europium (Eu(3+)) labeled with streptavidin was added to the assay mixtures, which bound to the biotin label on the ligated products. This in turn resulted in the FRET from Eu(3+) to Cy5 due to their close proximity. The formation of ligation products was measured by monitoring the emission at 665nm. This assay was validated by the experiments showing that the DNA ligase activity required NAD(+) and MgCl(2), and was inhibited by NMN and AMP, products of the ligase reaction. Using this assay, we determined the K(m) values of the enzyme for dsDNA substrates and NAD(+), and the IC(50) values of NMN and AMP, examined the effects of MgCl(2) and PEG(8000) on the enzyme activity, optimized the concentrations of Eu(3+) in the assay, and validated its utilities for high-throughput screening and biochemical characterizations of this class of enzymes.

Era, an essential GTPase, plays a regulatory role in several cellular processes. The Era protein of Streptococcus pneumoniae has recently been shown to bind to 16S rRNA and the cytoplasmic membrane. However, exact locations of Era responsible for RNA- and membrane-binding were unknown. To identify the regions in Era that interact with the RNA and membrane, the C-terminal part of S. pneumoniae Era was systematically deleted while the N-terminal part, responsible for the GTPase activity of the protein, was kept intact. The resulting truncated Era proteins were purified and characterized. The C-terminal deletion of 9 or 19 amino-acid residues did not affect 16S rRNA-binding activity while further deletions of the C-terminus (29-114 amino-acid residues) abolished the activity. These results indicate that the integrity of the putative KH domain of Era, spanning the amino-acid residues between approximately 22-83 from the C-terminus, is required for 16S rRNA-binding. Furthermore, the Era proteins with a deletion up to 45 residues from the C-terminus retained membrane-binding activity, but longer deletions significantly reduced the activity. These results indicate that part of the putative KH domain is also required for membrane-binding. Thus, these results indicate for the first time that the regions critical for the membrane- and 16S rRNA-binding activities of Era overlap. The era gene with a deletion of 9 or 19 codons from its 3' terminus complemented an Escherichia coli mutant strain deficient in Era production whereas the genes with longer deletions failed to do so, thereby indicating that the KH domain is essential for Era function. Taken together, the results of this study indicate that the putative KH domain is required for 16S rRNA-binding activity and that part of the KH domain is also required for membrane-binding activity. The results also suggest that the interaction between Era and 16S rRNA is essential for bacterial growth.

Novel fluorescent analogs of penicillin V were synthesized and evaluated for efficacy in the detection of penicillin binding proteins (PBPs). These molecules include the full structure of penicillin V, with the potent Bodipy fluorophore attached to the para-position of the penicillin V phenyl group. The green fluorescent Bocillin FL and the near-infrared (IR) fluorescent Bocillin 650/665 probes were shown to bind to PBPs, both purified and from membrane preparations, with high affinity and specificity. These reagents allow for facile detection of 2-4 ng of purified PBP with the aid of a fluorescent scanner.

Acyl carrier protein synthase (AcpS) is an essential enzyme in the biosynthesis of fatty acids in all bacteria. AcpS catalyzes the transfer of 4'-phosphopantetheine from coenzyme A (CoA) to apo-ACP, thus converting apo-ACP to holo-ACP that serves as an acyl carrier for the biosynthesis of fatty acids and lipids. To further understand the physiological role of AcpS, we identified, cloned, and expressed the acpS and acpP genes of Streptococcus pneumoniae and purified both products to homogeneity. Both acpS and acpP form operons with the genes whose functions are required for other cellular metabolism. The acpS gene complements an Escherichia coli mutant defective in the production of AcpS and appears to be essential for the growth of S. pneumoniae. Gel filtration and cross-linking analyses establish that purified AcpS exists as a homotrimer. AcpS activity was significantly stimulated by apo-ACP at concentrations over 10 microm and slightly inhibited at concentrations of 5-10 microm. Double reciprocal analysis of initial velocities of AcpS at various concentrations of CoA or apo-ACP indicated a random or compulsory ordered bi bi type of reaction mechanism. Further analysis of the inhibition kinetics of the product (3',5'-ADP) suggested that it is competitive with respect to CoA but mixed (competitive and noncompetitive) with respect to apo-ACP. Finally, apo-ACP bound tightly to AcpS in the absence of CoA, but CoA failed to do so in the absence of apo-ACP. Together, these results suggest that AcpS may be allosterically regulated by apo-ACP and probably proceeds by an ordered reaction mechanism with the first formation of the AcpS-apo-ACP complex and the subsequent transfer of 4'-phosphopantetheine to the apo-ACP of the complex.

To further understand the role of penicillin-binding protein 2a (PBP 2a) of Streptococcus pneumoniae in penicillin resistance, we confirmed the identity of the protein as PBP 2a. The PBP 2a protein migrated electrophoretically to a position corresponding to that of PBP 2x, PBP 2a, and PBP 2b of S. pneumoniae and was absent in a pbp2a insertional mutant of S. pneumoniae. We found that the affinities of PBP 2a for penicillins were lower than for cephalosporins and a carbapenem. When compared with other S. pneumoniae PBPs, PBP 2a exhibited lower affinities for beta-lactam antibiotics, especially penicillins. Therefore, PBP 2a is a low-affinity PBP for beta-lactam antibiotics in S. pneumoniae.

Era, an essential GTPase, appears to play an important role in the regulation of the cell cycle and protein synthesis of bacteria and mycoplasmas. In this study, native Era, His-tagged Era (His-Era) and glutathione S-transferase (GST)-fusion Era (GST-Era) proteins from Escherichia coli were expressed and purified. It was shown that the GST-Era and His-Era proteins purified by 1-step affinity column chromatographic methods were associated with RNA and exhibited a higher GTPase activity. However, the native Era protein purified by a 3-step column chromatographic method had a much lower GTPase activity and was not associated with RNA which had been removed during purification. Purified GST-Era protein was shown to be present as a high- and a low-molecular-mass forms. The high-molecular-mass form of GST-Era was associated with RNA and exhibited a much higher GTPase activity. Removal of the RNA associated with GST-Era resulted in a significant reduction in the GTPase activity. The RNA associated with GST-Era was shown to be primarily 16S rRNA. A purified native Era protein preparation, when mixed with total cellular RNA, was found to bind to some of the RNA. The native Era protein isolated directly from the cells of a wild-type E. coli strain was also present as a high-molecular-mass form complexed with RNA and RNase treatment converted the high-molecular-mass form into a 32 kDa low-molecular-mass form, a monomer of Era. Furthermore, a C-terminally truncated Era protein, when expressed in E. coli, did not bind RNA. Finally, the GTPase activity of the Era protein free of RNA, but not the Era protein associated with the RNA, was stimulated by acetate and 3-phosphoglycerate. These carbohydrates, however, failed to activate the GTPase activity of the C-terminally truncated Era protein. Thus, the results of this study establish that the C-terminus of Era is essential for the RNA-binding activity and that the RNA and carbohydrates modulate the GTPase activity of Era possibly through a similar mechanism.

The effects of inactivation of the genes encoding penicillin-binding protein 1a (PBP1a), PBP1b, and PBP2a in Streptococcus pneumoniae were examined. Insertional mutants did not exhibit detectable changes in growth rate or morphology, although a pbp1a pbp1b double-disruption mutant grew more slowly than its parent did. Attempts to generate a pbp1a pbp2a double-disruption mutant failed. The pbp2a mutants, but not the other mutants, were more sensitive to moenomycin, a transglycosylase inhibitor. These observations suggest that individually the pbp1a, pbp1b, and pbp2a genes are dispensable but that either pbp1a or pbp2a is required for growth in vitro. These results also suggest that PBP2a is a functional transglycosylase in S. pneumoniae.

Era is an essential membrane-associated GTPase that is present in bacteria and mycoplasmas. Era appears to play an important role in the regulation of the bacterial cell cycle. In this study, we expressed the native and glutathione S-transferase (GST) fusion forms of Streptococcus pneumoniae Era in Escherichia coli and purified both proteins to homogeneity. We showed that RNA was copurified with the GST-Era protein of S. pneumoniae during affinity purification and remained associated with the protein after removal of the GST tag by thrombin cleavage. The thrombin-treated and untreated GST-Era proteins could bind and hydrolyze GTP and exhibited similar kinetic properties (dissociation constant [kD], Km, and Vmax). However, the native Era protein purified by using different chromatographic columns had a much lower GTPase activity than did GST-Era, although it had a similar k(D). In addition, RNA was not associated with the protein. Purified GST-Era protein was shown to be present as high (600-kDa)- and low (120-kDa)-molecular-mass forms. The high-molecular-mass form of GST-Era was associated with RNA and exhibited a very high GTPase activity. Approximately 40% of purified GST-Era protein was associated with RNA, and removal of the RNA resulted in a significant reduction in GTPase activity. The RNA associated with GST-Era was shown to be predominantly 16S rRNA. The native Era protein isolated directly from S. pneumoniae was also present as a high-molecular-mass species (600 kDa) complexed with RNA. Together, our results suggest that 16S rRNA is associated with Era and might stimulate its GTPase activity.

Penicillin-binding proteins (PBPs), targets of beta-lactam antibiotics, are membrane-bound enzymes essential for the biosynthesis of the bacterial cell wall. PBPs possess transpeptidase and transglycosylase activities responsible for the final steps of the bacterial cell wall cross-linking and polymerization, respectively. To facilitate our structural studies of PBPs, we constructed a 5'-truncated version (lacking bp from 1 to 231 encoding the N-terminal part of the protein including the transmembrane domain) of the pbp2a gene of Streptococcus pneumoniae and expressed the truncated gene product as a GST fusion protein in Escherichia coli. This GST fusion form of PBP2a, designated GST-PBP2a*, was expressed almost exclusively as inclusion bodies. Using a combination of high- and low-speed centrifugation, large amounts of purified inclusion bodies were obtained. These purified inclusion bodies were refolded into a soluble and enzymatically active enzyme using a single-step refolding method consisting of solubilization of the inclusion bodies with urea and direct dialysis of the solubilized preparations. Using these purification and refolding methods, approximately 37 mg of soluble GST-PBP2a* protein was obtained from 1 liter of culture. The identity of this refolded PBP2a* protein was confirmed by N-terminal sequencing. The refolded PBP2a*, with or without the GST-tag, was found to bind to BOCILLIN FL, a beta-lactam, and to hydrolyze S2d, an analog of the bacterial cell wall stem peptides. The S2d hydrolysis activity of PBP2a* was inhibited by penicillin G. In conclusion, using this expression system, and the purification and refolding methods, large amounts of the soluble GST-PBP2a* protein were obtained and shown to be enzymatically active., (Copyright 1999 Academic Press.)

We describe a new, sensitive, rapid, and nonradioactive method involving the use of the commercially available BOCILLIN FL, a fluorescent penicillin, as a labeling reagent for the detection and study of penicillin-binding proteins (PBPs). This method allowed rapid detection of 30 ng of a purified PBP protein under UV light and of 2 to 4 ng of the protein with the aid of a FluorImager. This method also allowed rapid determination of the PBP profiles of Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pneumoniae. The PBP profiles obtained are virtually identical to those reported previously with 3H-, 14C-, or 125I-labeled penicillin. Using this method enabled us to determine the 50% inhibitory concentrations of the penicillin-sensitive and -resistant PBP2x proteins of S. pneumoniae for penicillin G, thereby allowing a direct evaluation of their relative affinities for penicillin G. Finally, this method also allowed us to compare relative affinities of a PBP2x protein for different beta-lactam antibiotics with the aid of fluorescence polarization technology and to monitor a PBP2x protein during purification.

Era, an essential GTPase, is present in many bacteria and Mycoplasma spp. and appears to play a major role in the cell cycle and other cellular processes. To further understand its function, an era gene from Streptococcus pneumoniae was identified and cloned, and a mutant era gene with a deletion of 68 codons from its 3'-terminus was constructed. The truncated Era protein was then purified and characterized, and the ability of the truncated era gene to complement an Escherichia coli mutant strain defective in Era production was examined. Like the full-length Era protein, the truncated Era protein was able to bind and hydrolyse GTP, but its binding activity was increased twofold and its hydrolytic activity was reduced sevenfold when compared with those of the full-length Era protein. Unlike the full-length Era protein, the truncated Era protein lost its ability to bind to the E. coli cytoplasmic membrane. The full-length era gene was able to complement the E. coli mutant deficient in Era production when carried on pACYC184, while the truncated era gene failed to do so when carried on pACYC184, pBR322 or pUC18. The cellular amounts of the truncated Era and the full-length Era proteins in E. coli and S. pneumoniae, respectively, were then determined by Western blot analysis. In addition, the minimal amount of the S. pneumoniae Era protein needed for complementation of the E. coli mutant was also measured. Taken together, these results suggest that the C-terminus of the Era protein might be responsible for the binding of the protein to the cytoplasmic membrane and be essential for function.

A strong transcriptional pause delays human RNA polymerase II three nt after the last potentially paired base in HIV-1 TAR, the RNA structure that binds the transactivator protein Tat. We report here that the HIV-1 pause depends in part on an alternative RNA structure (the HIV-1 pause hairpin) that competes with formation of TAR. By probing the nascent RNA structure in halted transcription complexes, we found that the transcript folds as the pause hairpin before and at the pause, and rearranges to TAR concurrent with or just after escape from the pause. The pause signal triggers a 2 nt reverse translocation by RNA polymerase that may block the active site and be counteracted by formation of TAR. Thus, the HIV-1 pause site modulates nascent RNA rearrangement from a structure that favors pausing to one that both recruits Tat and promotes escape from the pause.

To understand the biochemical basis of resistance of bacteria to beta-lactam antibiotics, we purified a penicillin-resistant penicillin-binding protein 2x (R-PBP2x) and a penicillin-sensitive PBP2x (S-PBP2x) enzyme of Streptococcus pneumoniae and characterized their transpeptidase activities, using a thioester analog of stem peptides as a substrate. A comparison of the k(cat)/Km values for the two purified enzymes (3,400 M(-1) s(-1) for S-PBP2x and 11.2 M(-1) s(-1) for R-PBP2x) suggests that they are significantly different kinetically. Implications of this finding are discussed. We also found that the two purified enzymes did not possess a detectable level of beta-lactam hydrolytic activity. Finally, we show that the expression levels of both PBP2x enzymes were similar during different growth phases.

A central enigma of transcriptional regulation is how the normally efficient transcription elongation complex stops at pause and termination signals. One possibility, raised by the discovery that RNA polymerase sometimes contracts its DNA footprint, is that discontinuous movements contribute to recognizing these signals. We report that E. coli RNA polymerase responds to sequences immediately downstream and upstream from the his leader pause site by changing neither its downstream DNA contact nor its upstream RNA contact for 8 bp preceding the pause. This compressed complex isomerizes to a paused conformation by an approximately 10 bp jump of its downstream DNA contact and simultaneous extrusion of an RNA hairpin that stabilizes the paused conformation. We suggest pausing and termination could be alternative outcomes of a similar isomerization that depend on the strength of contacts to 3'-proximal RNA remaining after the jump.