Targeting of the MYCN Protein with Small Molecule c-MYC Inhibitors

Members of the MYC family are the most frequently deregulated oncogenes in human cancer and are often correlated with aggressive disease and/or poorly differentiated tumors. Since patients with MYCN-amplified neuroblastoma have a poor prognosis, targeting MYCN using small molecule inhibitors could represent a promising therapeutic approach. We have previously demonstrated that the small molecule 10058-F4, known to bind to the c-MYC bHLHZip dimerization domain and inhibiting the c-MYC/MAX interaction, also interferes with the MYCN/MAX dimerization in vitro and imparts anti-tumorigenic effects in neuroblastoma tumor models with MYCN overexpression. Our previous work also revealed that MYCN-inhibition leads to mitochondrial dysfunction resulting in accumulation of lipid droplets in neuroblastoma cells. To expand our understanding of how small molecules interfere with MYCN, we have now analyzed the direct binding of 10058-F4, as well as three of its analogs; #474, #764 and 10058-F4(7RH), one metabolite C-m/z 232, and a structurally unrelated c-MYC inhibitor 10074-G5, to the bHLHZip domain of MYCN. We also assessed their ability to induce apoptosis, neurite outgrowth and lipid accumulation in neuroblastoma cells. Interestingly, all c-MYC binding molecules tested also bind MYCN as assayed by surface plasmon resonance. Using a proximity ligation assay, we found reduced interaction between MYCN and MAX after treatment with all molecules except for the 10058-F4 metabolite C-m/z 232 and the non-binder 10058-F4(7RH). Importantly, 10074-G5 and 10058-F4 were the most efficient in inducing neuronal differentiation and lipid accumulation in MYCN-amplified neuroblastoma cells. Together our data demonstrate MYCN-binding properties for a selection of small molecules, and provide functional information that could be of importance for future development of targeted therapies against MYCN-amplified neuroblastoma.     

High Throughput Screen Identifies Small Molecule Inhibitors Specific for Mycobacterium tuberculosis Phosphoserine Phosphatase

The emergence of drug-resistant strains of Mycobacterium tuberculosis makes identification and validation of newer drug targets a global priority. Phosphoserine phosphatase (PSP), a key essential metabolic enzyme involved in conversion of O-phospho-l-serine to l-serine, was characterized in this study. The M. tuberculosis genome harbors all enzymes involved in l-serine biosynthesis including two PSP homologs: Rv0505c (SerB1) and Rv3042c (SerB2). In the present study, we have biochemically characterized SerB2 enzyme and developed malachite green-based high throughput assay system to identify SerB2 inhibitors. We have identified 10 compounds that were structurally different from known PSP inhibitors, and few of these scaffolds were highly specific in their ability to inhibit SerB2 enzyme, were noncytotoxic against mammalian cell lines, and inhibited M. tuberculosis growth in vitro. Surface plasmon resonance experiments demonstrated the relative binding for these inhibitors. The two best hits identified in our screen, clorobiocin and rosaniline, were bactericidal in activity and killed intracellular bacteria in a dose-dependent manner. We have also identified amino acid residues critical for these SerB2-small molecule interactions. This is the first study where we validate that M. tuberculosis SerB2 is a druggable and suitable target to pursue for further high throughput assay system screening.     

Plant-Derived Small Molecule Inhibitors of Neuronal NO-Synthase: Potential Effects on Protein Degradation

Cigarette smoking is known to cause a decrease in NO production in man resulting in a variety of pathological effects, including vascular dysfunction. Aqueous extracts of cigarette and cigarette smoke contain chemical inhibitors to NO-synthases, a heme-containing cytochrome P450 enzymes. More recently, it was shown that freshly harvested leaves from the tobacco plant (Nicotiana tabacum, Solanaceae) also contain chemical inhibitors to neuronal NO-synthase (nNOS). Examination of leaves from 32 other plants representing diverse members of the plant kingdom showed that 17 other plants, besides tobacco, contain these chemical inhibitors. Of all these plants, 16 are members of the core asterids flowering plant group and 6 are members of the Solanaceae family. Although the identity of the chemicals is not known, perhaps the closely related plants contain the same or similar compounds that inhibit nNOS. The inhibitory effects are not attributable to nicotine. The discovery of these chemicals and their further characterization may help to explain the loss of nNOS in smokers. In this addendum, we discuss these results in light of the effect of tobacco-derived chemicals in inhibiting P450 cytochromes, as well as our thoughts on how the inactivation of nNOS leads to its selective downregulation through proteolytic degradation.Key Words: nitric oxide, inhibitors, tobacco, natural products, cytochrome P450, nitric oxide synthase, heat shock proteinsNO-synthase (NOS) is a cytochrome P450 like hemeprotein enzyme that catalyzes the production of NO from L-arginine.^1,2 NO is very important cellular signaling molecule, which plays a role in a variety of physiological processes including blood pressure regulation, neurotransmission, and penile erection. Like other P450 cytochromes, the catalytic site of NOS contains a heme prosthetic group that upon reduction and complexation with CO forms a chromophore that absorbs in the 450 nm region. Thus, although not closely related with respect to amino acid sequence to other P450 enzymes, the catalytic core of NOS and the chemistry involved in catalysis by NOS are highly similar to other P450 cytochromes.The cytochrome P450 enzymes comprise a very large family of enzymes that are responsible for metabolism of drugs and other xenobiotics as well as for synthesis of many hormones. There are over 2,000 P450 genes characterized in animals with over 50 genes in humans and over 1,900 genes in plants. The diversity of the P450 family is thought to reflect a complex “battle” between plants and animals.³ That is, plants are thought to have developed new alkaloids with new enzymes to protect themselves against animals whereas the animals developed new enzymes to metabolize these newly created plant toxins. This is thought to explain why the diversity in CYP genes increased dramatically approximately 400 millions years ago when organisms moved from the oceans to land.^3,4 Consistent with this hypothesis, chemicals in plants have been documented to inhibit mammalian P450 cytochromes. More importantly for humans, a variety of plant products have been documented to interact clinically with P450 cytochromes.⁵ For example, furanocoumarins found in grapefruit juice are known to enhance the oral bioavailability of felodipine, a calcium channel blocker, by inhibiting intestinal P450 metabolism of the drug.⁶In the case of tobacco-based cigarettes, hydrophobic chemicals in cigarettes have been found to inhibit P450 cytochromes. In particular, aromatase, the terminal P450 enzyme responsible for estrogen synthesis in humans, has been shown to be inhibited by tobacco smoke.⁷ Acyl derivatives of noricotine and anabasine were found to be active inhibitors of aromatase. In the case of NOS, studies by Heitzer et al.⁸ showed the amelioration of the vascular dysfunction in smokers by tetrahydrobiopterin and the authors concluded that there was a loss of endothelial NO synthesis caused by smoking. Moreover, Xie et al.⁹ showed that exposing rats to cigarette smoke led to the loss of both neuronal NO-synthase (nNOS) activity and protein. This observation reminded us of our earlier studies showing that guanabenz is a time-dependent irreversible inhibitor of nNOS that causes a loss of the penile nNOS activity and protein when given to rats.¹⁰ Thus, based on these findings, we wondered if chemicals in cigarettes could interact with NOS and explain the loss of NOS. We discovered that aqueous extracts of cigarettes and cigarette smoke could inhibit nNOS¹¹ and endothelial NOS.¹² More recently, extracts from freshly harvested tobacco leaves and leaves of various other plants from diverse phylogeny were also examined.¹³Tobacco leaves contained inhibitors to nNOS, indicating that these compounds did not necessarily arise from the curing and processing of the tobacco leaves to make cigarettes or from burning of the cigarettes. Moreover, other extracts made from other plants including 6 from the Solanacea family were found to have inhibitory activity. Thus, we believe that some common chemical or related chemicals exist that inhibits nNOS. The inhibitors are small molecules with hydrophilic and cationic qualities. In comparison, the inhibitors to aromatase are more hydrophobic than those of nNOS, likely reflecting the hydrophobic nature of the active site of aromatase, which accepts steroidal androgens. On the other hand, nNOS accepts L-arginine, a cationic water-soluble compound. The water-soluble nature of the inhibitors has so far hindered the isolation and characterization of the nNOS inhibitors, and thus we do not know the identity of these chemicals.The irreversible nature of the interaction of tobacco inhibitors with nNOS found in our studies may be important in understanding the long-term consequences of smoking. The irreversible inactivation of nNOS by various guanidine-based compounds is known to cause the enhanced proteasomal degradation of nNOS.¹⁴ The dysfunctional nNOS is known to be selectively ubiquitinated¹⁵ by a process involving Hsp70 and CHIP¹⁶ (Fig. 1). The degradation is also accelerated by inhibition of Hsp90, indicating that NOS is also regulated by the Hsp90-based chaperones.¹⁴ The loss of nNOS protein due to cigarette smoke suggests a similar process may be occurring with smokers.Open in a separate windowFigure 1Ubiquitination and Degradation of Inactivated nNOS. The nNOS is inactivated by chemicals found in some plants by alterations directed at the heme-containing active site of nNOS. This dysfunctional form of nNOS is directed for ubiquitination by an E3 ligase, such as CHIP (C-terminal Hsp-interacting protein), hsp70, and E2 ligase enzyme. The ubiquitinated nNOS is then recognized for proteasomal degradation. Closed circles represent ubiquitin molecules.The discovery of nNOS inhibitors in plants could be adequately interpreted in the context of plant-animal warfare. However, in light of the recent discovery of plant NOS,¹⁷ and in particular NOS in tobacco cells,¹⁸ it is possible that these inhibitors are endogenous modulators of the plant NOS. In this respect, an endogenous inhibitor has been identified for mammalian NOS.¹⁹ Furthermore, considering that the Hsp90-and Hsp70-based chaperones, ubiquitin, CHIP, and proteasome are found in plants and serve similar functions,^20–23 the NOS in plants may be similarly regulated. Thus, the mechanism outlined in Figure 1 for nNOS may also be pertinent to regulation of plant NOS. We believe this process reflects a fundamental biological process of protein quality control that is greatly affected by the binding of small molecules to the target protein.     

Small Molecule Proprotein Convertase Inhibitors for Inhibition of Embryo Implantation

Uterine proprotein convertase (PC) 6 plays a critical role in embryo implantation and is pivotal for pregnancy establishment. Inhibition of PC6 may provide a novel approach for the development of non-hormonal and female-controlled contraceptives. We investigated a class of five synthetic non-peptidic small molecule compounds that were previously reported as potent inhibitors of furin, another PC member. We examined (i) the potency of these compounds in inhibiting PC6 activity in vitro; (ii) their binding modes in the PC6 active site in silico; (iii) their efficacy in inhibiting PC6-dependent cellular processes essential for embryo implantation using human cell-based models. All five compounds showed potent inhibition of PC6 activity in vitro, and in silico docking demonstrated that these inhibitors could adopt a similar binding mode in the PC6 active site. However, when these compounds were tested for their inhibition of decidualization of primary human endometrial stromal cells, a PC6-dependent cellular process critical for embryo implantation, only one (compound 1o) showed potent inhibition. The lack of activity in the cell-based assay may reflect the inability of the compounds to penetrate the cell membrane. Because compound''s lipophilicity is linked to cell penetration, a measurement of lipophilicity (logP) was calculated for each compound. Compound 1o is unique as it appears the most lipophilic among the five compounds. Compound 1o also inhibited another crucial PC6-dependent process, the attachment of human trophoblast spheroids to endometrial epithelial cells (a model for human embryo attachment). We thus identified compound 1o as a potent small molecule PC6 inhibitor with pharmaceutical potential to inhibit embryo implantation. Our findings also highlight that human cell-based functional models are vital to complement the biochemical and in silico analyses in the selection of promising drug candidates. Further investigations for compound 1o are warranted in animal models to test its utility as an implantation-inhibiting contraceptive drug.     

The C-terminal 50 Amino Acid Residues of Dengue NS3 Protein Are Important for NS3-NS5 Interaction and Viral Replication

Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome synthesis, methylation of the 5′-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566–585 disrupts NS3-NS5 interaction but not the null-peptide bearing the N570A mutation. Small angle x-ray scattering study on NS3(172–618) helicase and covalently linked NS3(172–618)-NS5(320–341) reveals a rigid and compact formation of the latter, indicating that peptide NS5(320–341) engages in specific and discrete interaction with NS3. Significantly, NS3:Asn-570 to alanine mutation introduced into an infectious DENV2 cDNA clone did not yield detectable virus by plaque assay even though intracellular double-stranded RNA was detected by immunofluorescence. Detection of increased negative-strand RNA synthesis by real time RT-PCR for the NS3:N570A mutant suggests that NS3-NS5 interaction plays an important role in the balanced synthesis of positive- and negative-strand RNA for robust viral replication. Dengue virus infection has become a global concern, and the lack of safe vaccines or antiviral treatments urgently needs to be addressed. NS3 and NS5 are highly conserved among the four serotypes, and the protein sequence around the pinpointed amino acids from the NS3 and NS5 regions are also conserved. The identification of the functionally essential interaction between the two proteins by biochemical and reverse genetics methods paves the way for rational drug design efforts to inhibit viral RNA synthesis.     

Molecular Probing of the HPV-16 E6 Protein Alpha Helix Binding Groove with Small Molecule Inhibitors

The human papillomavirus (HPV) HPV E6 protein has emerged as a central oncoprotein in HPV-associated cancers in which sustained expression is required for tumor progression. A majority of the E6 protein interactions within the human proteome use an alpha-helix groove interface for binding. The UBE3A/E6AP HECT domain ubiquitin ligase binds E6 at this helix-groove interface. This enables formation of a trimeric complex with p53, resulting in destruction of this tumor suppressor. While recent x-ray crystal structures are useful, examples of small molecule probes that can modulate protein interactions at this interface are limited. To develop insights useful for potential structure-based design of ligands for HPV E6, a series of 2,6-disubstituted benzopyranones were prepared and tested as competitive antagonists of E6-E6AP helix-groove interactions. These small molecule probes were used in both binding and functional assays to evaluate recognition features of the E6 protein. Evidence for an ionic functional group interaction within the helix groove was implicated by the structure-activity among the highest affinity ligands. The molecular topographies of these protein-ligand interactions were evaluated by comparing the binding and activities of single amino acid E6 mutants with the results of molecular dynamic simulations. A group of arginine residues that form a rim-cap over the E6 helix groove offer compensatory roles in binding and recognition of the small molecule probes. The flexibility and impact on the overall helix-groove shape dictated by these residues offer new insights for structure-based targeting of HPV E6.     

On the Importance of Amino Acid Sequence and Spatial Proximity of Interacting Residues for Protein Folding

Abstract

Membrane proteins can be classified among the following five types: (1) type I membrane protein. (2) type II membrane protein. (3) multipass transmembrane proteins. (4) lipid chain- anchored membrane proteins, and (5) GPI-anchored membrane proteins. T. Kohonen's self-organization model which is a typical neural network is applied for predicting the type of a given membrane protein based on its amino acid composition. As a result, the high rates of self-consistency (94.80%) and cross-validation (77.76%), and stronger fault-tolerant ability were obtained.     

Pulsed EPR Determination of Water Accessibility to Spin-Labeled Amino Acid Residues in LHCIIb

Membrane proteins reside in a structured environment in which some of their residues are accessible to water, some are in contact with alkyl chains of lipid molecules, and some are buried in the protein. Water accessibility of residues may change during folding or function-related structural dynamics. Several techniques based on the combination of pulsed electron paramagnetic resonance (EPR) with site-directed spin labeling can be used to quantify such water accessibility. Accessibility parameters for different residues in major plant light-harvesting complex IIb are determined by electron spin echo envelope modulation spectroscopy in the presence of deuterated water, deuterium contrast in transversal relaxation rates, analysis of longitudinal relaxation rates, and line shape analysis of electron-spin-echo-detected EPR spectra as well as by the conventional techniques of measuring the maximum hyperfine splitting and progressive saturation in continuous-wave EPR. Systematic comparison of these parameters allows for a more detailed characterization of the environment of the spin-labeled residues. These techniques are applicable independently of protein size and require ∼10-20 nmol of singly spin-labeled protein per sample. For a residue close to the N-terminus, in a domain unresolved in the existing x-ray structures of light-harvesting complex IIb, all methods indicate high water accessibility.     

Identification of Amino Acid Residues Responsible for the Enantioselectivity and Amide Formation Capacity of the Arylacetonitrilase from Pseudomonas fluorescens EBC191

The nitrilase from Pseudomonas fluorescens EBC191 converted (R,S)-mandelonitrile with a low enantioselectivity to (R)-mandelic acid and (S)-mandeloamide in a ratio of about 4:1. In contrast, the same substrate was hydrolyzed by the homologous nitrilase from Alcaligenes faecalis ATCC 8750 almost exclusively to (R)-mandelic acid. A chimeric enzyme between both nitrilases was constructed, which represented in total 16 amino acid exchanges in the central part of the nitrilase from P. fluorescens EBC191. The chimeric enzyme clearly resembled the nitrilase from A. faecalis ATCC 8750 in its turnover characteristics for (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile (2-PPN) and demonstrated an even higher enantioselectivity for the formation of (R)-mandelic acid than the nitrilase from A. faecalis. An alanine residue (Ala165) in direct proximity to the catalytically active cysteine residue was replaced in the nitrilase from P. fluorescens by a tryptophan residue (as found in the nitrilase from A. faecalis ATCC 8750 and most other bacterial nitrilases) and several other amino acid residues. Those enzyme variants that possessed a larger substituent in position 165 (tryptophan, phenylalanine, tyrosine, or histidine) converted racemic mandelonitrile and 2-PPN to increased amounts of the R enantiomers of the corresponding acids. The enzyme variant Ala165His showed a significantly increased relative activity for mandelonitrile (compared to 2-PPN), and the opposite was found for the enzyme variants carrying aromatic residues in the relevant position. The mutant forms carrying an aromatic substituent in position 165 generally formed significantly reduced amounts of mandeloamide from mandelonitrile. The important effect of the corresponding amino acid residue on the reaction specificity and enantiospecificity of arylacetonitrilases was confirmed by the construction of a Trp164Ala variant of the nitrilase from A. faecalis ATCC 8750. This point mutation converted the highly R-specific nitrilase into an enzyme that converted (R,S)-mandelonitrile preferentially to (S)-mandeloamide.Nitrilases hydrolyze organic nitriles (R-C☰N) to the corresponding carboxylic acids and ammonia. These enzymes have been isolated from various sources, such as bacteria, fungi, and plants. Commercially, they are a very interesting group of enzymes, because nitriles are important intermediates in the chemical industry and several biotransformations have been described that utilize the chemo-, regio-, or enantioselectivity of nitrilases (2, 6, 16, 20, 22, 29).There is an informal classification that groups nitrilases according to their substrate specificities into “benzonitrilases,” “aliphatic nitrilases,” and “arylacetonitrilases” (17, 23). The arylacetonitrilases convert substrates, such as phenylacetonitrile and α-substituted arylacetonitriles (e.g., 2-phenylpropionitrile [2-PPN], mandelonitrile [2-hydroxyphenylacetonitrile], or phenylglycinonitrile [2-aminophenylacetonitrile]). This group of nitrilases is especially interesting for applications in biotechnology because these enzymes can enantioselectively hydrolyze α-substituted racemic nitriles to optically active carboxylic acids and thus in principle allow the production of the enantiomers of α-amino-, α-hydroxy-, and α-methylcarboxylic acids (1, 3, 10, 34). This trait has been used for the industrial production of (substituted) (R)-mandelic acid(s) from racemic (substituted) mandelonitrile(s) by dynamic kinetic resolution processes using different microorganisms (often strains of Alcaligenes faecalis) (19, 34; M. Ress-Löschke, T. Friedrich, B. Hauer, and R. Mattes, 1998, DE19848129A1, German Patent Office). An enantioselective nitrilase from A. faecalis ATCC 8750 has been purified and characterized, and the encoding gene has been cloned (4, 11, 26, 33).In previous work by our group, a different arylacetonitrilase was obtained from Pseudomonas fluorescens EBC191 (18). This enzyme converted various phenylacetonitriles (e.g., 2-PPN, O-acetoxymandelonitrile, or mandelonitrile), and also aliphatic 2-acetoxynitriles, with moderate enantioselectivities into the corresponding α-substituted carboxylic acids. Furthermore, with some substrates, significant amounts of the corresponding amides were also formed (5, 8, 12, 21, 27).The gene encoding the nitrilase from P. fluorescens EBC191 was recently cloned, and it was found that the nitrilases from P. fluorescens EBC191 and A. faecalis ATCC 8750 are clearly homologous to each other (12). Nevertheless, the two enzymes differ significantly in their catalytic abilities. Thus, the enzyme from A. faecalis ATCC 8750 converts racemic mandelonitrile to (R)-mandelic acid with a high enantioselectivity and forms almost no mandeloamide as a side product. In contrast, the enzyme from P. fluorescens demonstrates only a low degree of enantioselectivity for the formation of (R)-mandelic acid and forms a large amount of mandeloamide (about 16% of the totally converted mandelonitrile). We are therefore currently trying to investigate the molecular basis for these differences in order to improve the substrate specificity and enantiospecificity of nitrilases. In a previous study, we analyzed the effects of various carboxy-terminal mutations on the nitrilase of P. fluorescens EBC191. These experiments showed that deletions of 47 to 67 amino acids from the carboxy terminus of the nitrilase resulted in variant forms that demonstrated, with mandelonitrile and 2-PPN as substrates, increased amide formation and increased formation of the R acids associated with lower specific activities. Although these carboxy-terminal mutants showed increased enantioselectivity for the formation of (R)-mandelic acid, the observed enantioselectivities were still much lower than those observed with the nitrilase from A. faecalis ATCC 8750 and were also associated with increased amide formation (13). Therefore, in the present study, additional mutants were generated in order to analyze the effects of amino acid exchanges close to the catalytic center of the nitrilase.     

Key Amino Acid Residues Responsible for the Color of Green and Yellow Fluorescent Proteins from the Coral Polyp Zoanthus sp.

Site-directed mutagenesis was used to study the structural basis of color diversity of fluorescent proteins by the example of two closely related proteins from one organism (coral polyp Zoanthus sp.), one of which produces green and the other, yellow fluorescence. As a result, the following conversions of emission colors were performed: from yellow to green, from yellow to a dual color (yellow and green), and from green to yellow. The saltatory character of the spectral transitions and the manifestation of the dual-color fluorescence suggest that chemically different fluorophores are responsible for the green and yellow fluorescence. The simultaneous presence of three residues, Gly63, Lys65, and Asp68, is necessary for the efficient formation of the yellow rather than green fluorophore.     

Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase

Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the ‘DFG-out’ inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the ‘gatekeeper’ V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET.     

Search for Functionally Significant Motifs and Amino Acid Residues of Actin

The scientific interest to the structural and functional properties of actin is determined by its abundance in cells. Being an important component of the cytoskeleton, actin is involved in many protein-protein interactions. Using crystal structures and molecular models, we have mapped the amino acid residues that are involved in these interactions and form the ATP-binding site of the actin monomer. Moreover, using mass spectrometry and high-performance liquid chromatography methods, we have discovered the regions of the amino acid sequence of actin that form the core of the actin fibril. According to the bioinformatic analysis, these regions are amyloidogenic and are located in the C-terminal region and in the hinge between the first and third subdomains. The data obtained are applicable to chordate actin, because multiple alignment revealed highly conserved amino acid sequences. In turn, the comparison of the chordate actin with the bacterial homologs showed the presence of numerous amino acid substitutions and insertions.     

Identification of Critical Amino Acid Residues for Human iNOS Functional Activity

Nitric oxide (NO) is a short-lived signaling molecule that mediates a variety of biological functions, including vascular homeostasis, neurotransmission, antimicrobial defense and antitumor activities. Three known NOS isoforms (eNOS, nNOS and iNOS) have been cloned and sequenced. Here, we show that upon expression in Escherichia coli using a novel expression vector, an iNOS sequence containing three mutations (A805D, F831S and L832P) within the iNOS reductase domain produced very little functionally active iNOS protein compared to the wild type (wt) iNOS. Each of these point mutations also was individually constructed into the wt iNOS sequence. The activity of the iNOS protein containing the A805D mutation was comparable to wt, while a drastic reduction in iNOS activity was observed for the F831S and L832P mutants. A comparison of the molecular models of the reductase domain of the wt and mutant iNOS revealed a reduced core packing density for the F831S and L832P mutations compared to wt. In addition, the modeling also suggests altered hydrogen bonding, van der Waals and hydrophobic interactions of these mutants.     

Plasma Amino Acid Profiling Identifies Specific Amino Acid Associations with Cardiovascular Function in Patients with Systolic Heart Failure

BackgroundThe heart has close interactions with other organs’ functions and concomitant systemic factors such as oxidative stress, nitric oxide (NO), inflammation, and nutrition in systolic heart failure (HF). Recently, plasma amino acid (AA) profiling as a systemic metabolic indicator has attracted considerable attention in predicting the future risk of human cardiometabolic diseases, but it has been scarcely studied in HF.MethodsThirty-eight stable but greater than New York Heart Association class II symptomatic patients with left ventricular (LV) ejection fraction <45% and 33 asymptomatic individuals with normal B-type natriuretic peptide (BNP) value were registered as the HF and control groups, respectively. We analyzed fasting plasma concentrations of 41 AAs using high-performance liquid chromatography, serum NO metabolite concentration, hydroperoxide and high-sensitivity C-reactive protein measurements, echocardiography, and flow-mediated dilatation.ResultsWe found that 17 AAs and two ratios significantly changed in the HF group compared with those in the control group (p < 0.05). In the HF group, subsequent univariate and stepwise multivariate analyses with clinical variables revealed that Fischer ratio and five specific AAs, ie, monoethanolamine, methionine, tyrosine, 1-methylhistidine, and histidine have significant correlation with BNP, LV ejection fraction, LV end-diastolic volume index, inferior vena cava diameter, the ratio of early diastolic velocity of the mitral inflow to mitral annulus, and BNP, respectively (p < 0.05). Interestingly, further exploratory factor analysis categorized these AAs into hepatic-related (monoethanolamine, tyrosine, and Fischer ratio) and skeletal muscle-related (histidine, methionine, and 1-methylhistidine) components. Some categorized AAs showed unique correlations with concomitant factors: monoethanolamine, tyrosine, and Fischer ratio with serum NO concentration; histidine with serum albumin; and 1-methylhistidine with flow-mediated dilatation (p < 0.05).ConclusionsPlasma AA profiling identified correlations of specific AAs with cardiac function and concomitant factors, highlighting the cardio-hepatic-skeletal muscle axis in patients with systolic HF.     

Enhanced SH3/Linker Interaction Overcomes Abl Kinase Activation by Gatekeeper and Myristic Acid Binding Pocket Mutations and Increases Sensitivity to Small Molecule Inhibitors

Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control.