Mechanistical Insights into the Ultrasensitive Detection of Radioactive and Chemotoxic UO22+ Ions by a Porous Anionic Co-Metal-Organic Framework.

Development of a simple, cost-efficient, and portable UO22+ sensory probe with high selectivity and sensitivity is highly desirable in the context of monitoring radioactive contaminants. Herein, we report a luminescent Co-based metal-organic framework (MOF), {[Me2NH2]0.5[Co(DATRz)0.5(NH2BDC)]·xG}n (1), equipped with abundant amino functionalities for the selective detection of uranyl cations. The ionic structure consists of two types of channels decorated with plentiful Lewis basic amino moieties, which trigger a stronger acid-base interaction with the diffused cationic units and thus can selectively quench the fluorescence intensity in the presence of other interfering ions. Furthermore, the limit of detection for selective UO22+ sensing was achieved to be as low as 0.13 µM (30.94 ppb) with rapid responsiveness and multiple recyclabilities, demonstrating its excellent efficacy. Density functional theory (DFT) calculations further unraveled the preferred binding sites of the UO22+ ions in the tubular channel of the MOF structure. Orbital hybridization between NH2BDC/DATRz and UO22+ together with its significantly large electron-accepting ability is identified as responsible for the luminescence quenching. More importantly, the prepared 1@PVDF {poly(vinylidene difluoride)} mixed-matrix membrane (MMM) displayed good fluorescence activity comparable to 1, which is of great significance for their practical employment as MOF-based luminosensors in real-world sensing application.

Variation in Catalytic Efficacies of a 2D pH-Stable MOF by Altering Activation Methods.

Although it is well-known that the Lewis acidity of Metal-Organic Frameworks (MOFs) can effectively enhance their catalytic activity in organic transformations, access to these Lewis-acidic sites remains a key hurdle to widespread applications of Lewis-acidic catalysis by MOFs. Easy accessibility of strong Lewis acidic sites onto 2D MOFs by using proper activation methods can be a cornerstone in attaining desired catalytic performance. Herein, we report a new 2D chemically stable MOF, IITKGP-60, which displayed excellent framework robustness over a wide pH range (2-12). Benefiting from the abundant open metal sites (OMSs) and framework robustness, the catalytic activity of the developed material was explored in one-pot three-component Strecker reaction and Knoevenagel condensation reaction. Moreover, the developed catalyst is superior in catalyzing the reactions involving sterically hindered substrate (1-naphthaldehyde) with high turnover number. A comparative catalytic study was conducted using different activation methods (chloroform and methanol exchanged activated samples), highlighting the significant effect of activation methods on its catalytic performances. The sustainable synthetic pathway under solvent-free conditions for a broad scope of substrates using low catalyst loading and excellent recyclability made the developed pH-stable framework a promising heterogeneous catalyst.

A Highly Chemically Robust 3D Interpenetrated MOF Heterogeneous Catalyst for the Synthesis of Hantzsch 1,4-Dihydropyridines and Drug Molecules.

Metal-organic frameworks (MOFs) have attracted immense attention as efficient heterogeneous catalysts over other solid catalysts, however, their chemical environment instability often limits their catalytic potential. Herein, utilizing a flexible unexplored tetra-acid ligand and employing the mixed ligand approach, a 3D interpenetrated robust framework is strategically developed, IITKGP-51 (IITKGP stands for Indian Institute of Technology Kharagpur), which retained its crystallinity over a wide range of pH solution (4-12). Having ample open metal sites (OMSs), IITKGP-51 is explored as a heterogeneous catalyst in one-pot Hantzsch condensation reaction, with low catalyst loading for a broad range of substrates. The synthesis of drug molecules remains one of the most significant and emergent areas of organic and medicinal chemistry. Considering such practical utility, biologically important Nemadipine B and Nifedipine drug molecules (calcium channel protein inhibitor) are synthesized for the first time by using this catalyst and fully characterized via SC-XRD and other spectroscopic methods. This report inaugurates the usage of a MOF material as a catalyst for the synthesis of drug molecules.

Highly Robust Metal-Organic Framework for Efficiently Catalyzing Knoevenagel Condensation and the Strecker Reaction under Solvent-Free Conditions.

Metal-organic frameworks (MOFs) have been recognized as one of the most promising porous materials and offer great opportunities for the rational design of new catalytic solids having great structural diversity and functional tunability. Despite numerous inherent merits, their chemical environment instability limits their practical usage and demands further exploration. Herein, by employing the mixed-ligand approach, we have designed and developed a robust 3D Co-MOF, [Co2(µ2-O)(TDC)2(L)(H2O)2]·2DMF (H2TDC = 2,5-thiophenedicarboxylic acid, L = 3,3'-azobispyridine), IITKGP-50 (IITKGP stands for the Indian Institute of Technology Kharagpur), which exhibited excellent framework robustness not only in water but also in a wide range of aqueous pH solutions (pH = 2-12). Taking advantage of superior framework robustness and the presence of high-density open metal sites, IITKGP-50 was further explored in catalyzing the two-component Knoevenagel condensation reaction and three-component Strecker reactions. Moreover, to verify the size selectivity of IITKGP-50, smaller to bulkier substrates in comparison with the MOF's pore cavity (8.1 × 5.6 Å2) were employed, in which relatively lesser conversions for the sterically bulkier aldehyde derivatives confirmed that the catalytic cycle occurs inside the pore cavity. The easy scalability, lower catalyst loading compared to that of benchmark MOFs, magnificent conversion rate over a wide range of substrates, and excellent recyclability without significant performance loss made IITKGP-50 a promising heterogeneous catalyst candidate.

pH-Stable Zn(II) Coordination Polymer as a Multiresponsive Turn-On and Turn-Off Fluorescent Sensor for Aqueous Medium Detection of Al(III) and Cr(VI) Oxo-Anions.

Nowadays, coordination polymers (CPs) are promising candidates as sensory materials for their high sensitivity, improved selectivity, fast responsive nature, as well as good recyclability. However, poor chemical stability often makes their practical usage limited. Herein, employing a mixed ligand approach, we constructed a chemically robust CP, {[Zn2L2(DPA)2]·3H2O}n (IITKGP-70, IITKGP stands for the Indian Institute of Technology Kharagpur), which exhibited excellent framework robustness not only in water but also over a broad range of pH solutions (pH = 3-11). The developed framework displayed high selectivity and sensitivity for the detection of trivalent Al3+ ions and toxic hexavalent Cr(VI)-oxo anions in an aqueous medium. The developed framework exhibited an aqueous medium Al3+ turn-on phenomenon with a limit of detection (LOD) value of 1.29 µM, whereas a turn-off effect was observed for toxic oxo-anions (Cr2O72- and CrO42-) having LOD values of 0.27 and 0.71 µM, respectively. Both turn-on and turn-off mechanisms are speculated via spectroscopic methods coupled with several ex situ studies. Such a multiresponsive nature (both turn-on and turn-off) for aqueous medium detection of targeted cations and anions simultaneously in a single platform coupled with high robustness, ease of scalability, recyclability, and fast-responsive nature makes IITKGP-70 highly fascinating as a sensory material for real-world applications.

A Water-Stable Cationic SIFSIX MOF for Luminescent Probing of Cr2 O7 2- via Single-Crystal to Single-Crystal Transformation.

The sensing and monitoring of toxic oxo-anion contaminants in water are of significant importance to biological and environmental systems. A rare hydro-stable SIFSIX metal-organic framework, SiF6 @MOF-1, {[Cu(L)2 (H2 O)2 ]·(SiF6 )(H2 O)}n , with exchangeable SiF6 2- anion in its pore is strategically designed and synthesized, exhibiting selective detection of toxic Cr2 O7 2- oxo-anion in an aqueous medium having high sensitivity, selectivity, and recyclability through fluorescence quenching phenomena. More importantly, the recognition and ion exchange mechanism is unveiled through the rarely explored single-crystal-to-single crystal (SC-SC) fashion with well-resolved structures. A thorough SC-SC study with interfering anions (Cl- , F- , I- , NO3 - , HCO3 - , SO4 2- , SCN- , IO3 - ) revealed no such transformations to take place, as per line with quenching studies. Density functional theory calculations revealed that despite a lesser binding affinity, Cr2 O7 2- shows strong orbital mixing and large driving forces for electron transfer than SiF6 2- , and thus enlightens the fluorescence quenching mechanism. This work inaugurates the usage of a SIFSIX MOF toward sensing application domain under aqueous medium where hydrolytic stability is a prime concern for their plausible implementation as sensor materials.

Stereodivergent Zweifel Olefination and its Mechanistic Dichotomy.

Stereoselective Zweifel olefination using boronate complexes carrying two different reactive π-systems was achieved to synthesize vinyl heteroarenes and conjugated 1,3-dienes in good yield and up to 100 % stereoselectivity, which remains unexplored until now. Most importantly, we report the unprecedented formation of E vs. Z-vinyl heteroarenes for different heteroarenes under identical conditions. Density functional theory (DFT) investigations unveil the mechanistic dichotomy between olefin and heteroarene activation followed by 1,2-migration, leading to E or Z-vinyl heteroarenes respectively. We also report a previously unknown reversal of stereoselectivity by using 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) as an electrophile. The Zweifel olefination using a boronate complex that carries two different olefins was previously unexplored due to significant challenges associated with the site-selective activation of olefins. We have solved this problem and reported the site-selective activation of olefins for the stereoselective synthesis of 1,3-dienes.

A "Thermodynamically Stable" 2D Nickel Metal-Organic Framework over a Wide pH Range with Scalable Preparation for Efficient C2 s over C1 Hydrocarbon Separations.

The design and construction of "thermodynamically stable" metal-organic frameworks (MOFs) that can survive in liquid water, boiling water, and acidic/basic solutions over a wide pH range is highly desirable for many practical applications, especially adsorption-based gas separations with obvious scalable preparations. Herein, a new thermodynamically stable Ni MOF, {[Ni(L)(1,4-NDC)(H2 O)2 ]}n (IITKGP-20; L=4,4'-azobispyridine; 1,4-NDC=1,4-naphthalene dicarboxylic acid; IITKGP stands for the Indian Institute of Technology Kharagpur), has been designed that displays moderate porosity with a BET surface area of 218 m2 g-1 and micropores along the [10-1] direction. As an alternative to a cost-intensive, cryogenic, high-pressure distillation process for the separation of hydrocarbons, MOFs have recently shown promise for such separations. Thus, towards an application standpoint, this MOF exhibits a higher uptake of C2 hydrocarbons over that of C1 hydrocarbon under ambient conditions, with one of the highest selectivities based on the ideal adsorbed solution theory (IAST) method. A combination of two strategies (the presence of stronger metal-N coordination of the spacer and the hydrophobicity of the aromatic moiety of the organic ligand) possibly makes the framework highly robust, even stable in boiling water and over a wide range of pH 2-10, and represents the first example of a thermodynamically stable MOF displaying a 2D structural network. Moreover, this material is easily scalable by heating the reaction mixture at reflux overnight. Because such separations are performed in the presence of water vapor and acidic gases, there is a great need to explore thermodynamically stable MOFs that retain not only structural integrity, but also the porosity of the frameworks.

Two Closely Related Zn(II)-MOFs for Their Large Difference in CO2 Uptake Capacities and Selective CO2 Sorption.

Two azo functionalized Zn(II)-based MOFs, {[Zn(SDB)(3,3'-L)0.5]·xG}n, IITKGP-13A, and {[Zn2(SDB)2(4,4'-L)]·xG}n, IITKGP-13B (IITKGP stands for Indian Institute of Technology Kharagpur), have been constructed through the self-assembly of isomeric N,N'-donor spacers (3,3'-L = 3,3'-azobispyridine and 4,4'-L = 4,4'-azobispyridine) with organic ligand 4,4'-sulfonyldibenzoic acid (SDBH2) and Zn(NO3)2·6H2O (G represents disordered solvent molecules). Single-crystal X-ray diffraction studies reveal the 2D structure with sql topology for both MOFs. However, the subtle change in positions of coordinating N atoms of spacers makes IITKGP-13A noninterpenetrated, while IITKGP-13B bears a 2-fold interpenetrated structure. IITKGP-13A exhibits higher uptake of CO2 over CH4 and N2 with high IAST selectivities for mixed CO2/CH4 (50:50, biogas) and CO2/N2 (15:85, flue gas) gas systems. In contrast, IITKGP-13B takes up very low amount of CO2 gas (0.4 mmol g-1) compared to IITKGP-13A (1.65 mmol g-1) at 295 K. Density functional theory (DFT)-based electronic structure calculations have been performed to explain the origin of the large differences in CO2 uptake capacity between the two MOFs at the atomistic level. The results show that the value of the change in enthalpy (ΔH) at 298 K temperature and 1 bar pressure for the CO2 adsorption is more negative in IITKGP-13A as compared to that in IITKGP-13B, thus indicating that CO2 molecules are more favored to get adsorbed in IITKGP-13A than in IITKGP-13B. The computed values for the Gibbs' free energy change (ΔG) for the CO2 adsorption are positive for both of the MOFs, but a higher value is observed for the IITKGP-13B. The noncovalent types of interactions are the main contribution toward the attractive energies between the host MOF frameworks and guest CO2 molecules, which has been studied with the help of energy decomposition analysis (EDA).

A distinctive histidine residue is essential for in vivo glycation-inactivation of human CD59 transgenically expressed in mice erythrocytes: Implications for human diabetes complications.

Clinical and experimental evidences support a link between the complement system and the pathogenesis of diabetes complications. CD59, an extracellular cell membrane-anchored protein, inhibits formation of the membrane attack complex (MAC), the main effector of complement-mediated tissue damage. This complement regulatory activity of human CD59 (hCD59) is inhibited by hyperglycemia-induced ɛ-amino glycation of Lys41 . Biochemical and structural analyses of glycated proteins with known three-dimensional structure revealed that glycation of ɛ-amino lysyl residues occurs predominantly at "glycation motives" that include lysyl/lysyl pairs or proximity of a histidyl residue, in which the imidazolyl moiety is ≈ 5Å from the ɛ-amino group. hCD59 contains a distinctive Lys41 /His44 putative glycation motif within its active site. In a model of transgenic diabetic mice expressing in erythrocytes either the wild type or a H44Q mutant form of hCD59, we demonstrate in vivo that the His44 is required for Lys41 glycation and consequent functional inactivation of hCD59, as evidenced using a mouse erythrocytes hemolytic assay. Since (1) the His44 residue is not present in CD59 from other animal species and (2) humans are particularly prone to develop complications of diabetes, our results indicate that the Lys41 /His44 glycation motif in human CD59 may confer humans a higher risk of developing vascular disease in response to hyperglycemia.

Development of 1-((1,4-trans)-4-Aryloxycyclohexyl)-3-arylurea Activators of Heme-Regulated Inhibitor as Selective Activators of the Eukaryotic Initiation Factor 2 Alpha (eIF2α) Phosphorylation Arm of the Integrated Endoplasmic Reticulum Stress Response.

Heme-regulated inhibitor (HRI), an eukaryotic translation initiation factor 2 alpha (eIF2α) kinase, plays critical roles in cell proliferation, differentiation, adaptation to stress, and hemoglobin disorders. HRI phosphorylates eIF2α, which couples cellular signals, including endoplasmic reticulum (ER) stress, to translation. We previously identified 1,3-diarylureas and 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) as specific activators of HRI that trigger the eIF2α phosphorylation arm of ER stress response as molecular probes for studying HRI biology and its potential as a druggable target. To develop drug-like cHAUs needed for in vivo studies, we undertook bioassay-guided structure-activity relationship studies and tested them in the surrogate eIF2α phosphorylation and cell proliferation assays. We further evaluated some of these cHAUs in endogenous eIF2α phosphorylation and in the expression of the transcription factor C/EBP homologous protein (CHOP) and its mRNA, demonstrating significantly improved solubility and/or potencies. These cHAUs are excellent candidates for lead optimization for development of investigational new drugs that potently and specifically activate HRI.

Plasma Glycated CD59, a Novel Biomarker for Detection of Pregnancy-Induced Glucose Intolerance.

OBJECTIVE: Plasma glycated CD59 (pGCD59) is an emerging biomarker in diabetes. We assessed whether pGCD59 could predict the following: the results of the glucose challenge test (GCT) for screening of gestational diabetes mellitus (GDM) (primary analysis); and the diagnosis of GDM and prevalence of large for gestational age (LGA) newborns (secondary analyses). RESEARCH DESIGN AND METHODS: Case-control study of 1,000 plasma samples from women receiving standard prenatal care, 500 women having a normal GCT (control subjects) and 500 women with a failed GCT and a subsequent oral glucose tolerance test (case patients). RESULTS: Compared with control subjects, the median (interquartile range) pGCD59 value was 8.5-fold higher in case patients and 10-fold higher in GDM patients, as follows: control subjects 0.33 (0.19); case patients 2.79 (1.4); GDM patients 3.23 (1.43) (P < 0.001); area under the receiver operating characteristic curve 0.92. LGA prevalence was 4.3% in the lowest quartile and 13.5% in the highest quartile of pGCD59. CONCLUSIONS: One pGCD59 measurement during weeks 24-28 identifies pregnancy-induced glucose intolerance with high sensitivity and specificity and can potentially identify the risk for LGA.

Deficiency of the complement regulatory protein CD59 accelerates the development of diabetes-induced atherosclerosis in mice.

AIMS: Clinical and experimental evidence supports a strong link between the complement system, complement regulatory proteins and the pathogenesis of diabetes vascular complications. We previously reported that the complement regulatory protein CD59 is inactivated by glycation in humans with diabetes. Our objective for this study is to assess experimentally how the deficiency of CD59 impacts the development of diabetic atherosclerosis in vivo. METHODS: We crossed mCD59 sufficient and deficient mice into the ApoE-/- background to generate mCd59ab+/+/ApoE-/- and mCd59ab-/-/ApoE-/- mice, and induced diabetes by multiple low dose injections of streptozotocin. Atherosclerosis was detected by hematoxylin and eosin (H&E) and oil red-O staining. Membrane attack complex (MAC) deposition and macrophage infiltration were detected by immunostaining. RESULTS: Diabetic mCD59 deficient (mCD59ab-/-/ApoE-/-) mice developed nearly 100% larger atherosclerotic lesion areas in the aorta (7.5%±0.6 vs 3.6%±0.7; p<0.005) and in the aortic roots (H&E: 26.2%±1.9 vs. 14.3%±1.1; p<0.005), in both cases associated with increased lipid (Oil red-O: 14.9%±1.1 vs. 7.8%±1.1; p<0.05) and MAC deposition (6.8%±0.8 vs. 3.0%±0.7; p<0.005) and macrophage infiltration (31.5%±3.7 vs. 16.4%±3.0; p<0.05) in the aortic roots as compared to their diabetic mCD59 sufficient (mCD59ab+/+/ApoE-/-) counterpart. CONCLUSIONS: The deficiency of CD59 accelerates the development of diabetic atherosclerosis.

Role of complement and complement regulatory proteins in the complications of diabetes.

It is well established that the organ damage that complicates human diabetes is caused by prolonged hyperglycemia, but the cellular and molecular mechanisms by which high levels of glucose cause tissue damage in humans are still not fully understood. The prevalent hypothesis explaining the mechanisms that may underlie the pathogenesis of diabetes complications includes overproduction of reactive oxygen species, increased flux through the polyol pathway, overactivity of the hexosamine pathway causing intracellular formation of advanced glycation end products, and activation of protein kinase C isoforms. In addition, experimental and clinical evidence reported in past decades supports a strong link between the complement system, complement regulatory proteins, and the pathogenesis of diabetes complications. In this article, we summarize the body of evidence that supports a role for the complement system and complement regulatory proteins in the pathogenesis of diabetic vascular complications, with specific emphasis on the role of the membrane attack complex (MAC) and of CD59, an extracellular cell membrane-anchored inhibitor of MAC formation that is inactivated by nonenzymatic glycation. We discuss a pathogenic model of human diabetic complications in which a combination of CD59 inactivation by glycation and hyperglycemia-induced complement activation increases MAC deposition, activates pathways of intracellular signaling, and induces the release of proinflammatory, prothrombotic cytokines and growth factors. Combined, complement-dependent and complement-independent mechanisms induced by high glucose promote inflammation, proliferation, and thrombosis as characteristically seen in the target organs of diabetes complications.

Synthesis of rigidified eIF4E/eIF4G inhibitor-1 (4EGI-1) mimetic and their in vitro characterization as inhibitors of protein-protein interaction.

The 4EGI-1 is the prototypic inhibitor of eIF4E/eIF4G interaction, a potent inhibitor of translation initiation in vitro and in vivo and an efficacious anticancer agent in animal models of human cancers. We report on the design, synthesis, and in vitro characterization of a series of rigidified mimetic of this prototypic inhibitor in which the phenyl in the 2-(4-(3,4-dichlorophenyl)thiazol-2-yl) moiety was bridged into a tricyclic system. The bridge consisted one of the following: ethylene, methylene oxide, methylenesulfide, methylenesulfoxide, and methylenesulfone. Numerous analogues in this series were found to be markedly more potent than the parent prototypic inhibitor in the inhibition of eIF4E/eIF4G interaction, thus preventing the eIF4F complex formation, a rate limiting step in the translation initiation cascade in eukaryotes, and in inhibition of human cancer cell proliferation.

Glycation of the complement regulatory protein CD59 is a novel biomarker for glucose handling in humans.

CONTEXT: Human CD59, an inhibitor of the membrane attack complex of complement, is inactivated by glycation. Glycation inactivation of CD59 enhances complement-mediated injury in target organs of diabetes complications. OBJECTIVE: We hypothesized that circulating soluble glycated CD59 (GCD59) represents a novel biomarker of blood glucose handling and aimed to conduct human study protocols to test this hypothesis. DESIGN, SETTING, PARTICIPANTS, AND OUTCOME MEASURES: Using a newly developed ELISA, we measured circulating soluble GCD59 in samples from 3 separate human studies evaluating acute and chronic glucose handling and glucose responses to insulin therapy. Study 1 (normal vs diabetic subjects) evaluated the cross-sectional association between GCD59 and glycated hemoglobin (HbA1c) in 400 subjects with and without type 2 diabetes. Study 2 (oral glucose tolerance test [OGTT] in nondiabetics) evaluated whether fasting GCD59 independently predicted the 2-hour glucose response to an OGTT in 109 subjects without a diagnosis of diabetes. Study 3 (intensified insulin treatment) evaluated the effect of intensification of glycemic control with insulin on GCD59 in 21 poorly controlled individuals with diabetes. RESULTS: In study 1 (normal vs diabetic subjects), GCD59 was independently and positively associated with HbA1c in individuals with and without diabetes (ß = 1.1, P < .0001 and ß = 1.1 P < .001, respectively). In study 2 (OGTT in nondiabetics), a single GCD59 measurement independently predicted the results of the 2-hour OGTT (ß = 19.8, P < .05) after multivariate modeling. In study 3 (intensified insulin treatment), intensification of glucose control with insulin resulted in a concomitant and parallel reduction of average weekly glucose and GCD59 within 2 weeks. CONCLUSIONS: We observed robust relationships between a single measurement of blood levels of GCD59 and both acute (2-hour OGTT) and chronic (HbA1c) measures of glucose handling. Lowering of GCD59 levels closely reflected lowering of average weekly glucose within 2 weeks. The role of GCD59 in the diagnosis, management, and vascular risk stratification in diabetes warrants further investigation.

Structure-activity relationship study of 4EGI-1, small molecule eIF4E/eIF4G protein-protein interaction inhibitors.

Protein-protein interactions are critical for regulating the activity of translation initiation factors and multitude of other cellular process, and form the largest block of untapped albeit most challenging targets for drug development. 4EGI-1, (E/Z)-2-(2-(4-(3,4-dichlorophenyl)thiazol-2-yl)hydrazono)-3-(2-nitrophenyl)propanoic acid, is a hit compound discovered in a screening campaign of small molecule libraries as an inhibitor of translation initiation factors eIF4E and eIF4G protein-protein interaction; it inhibits translation initiation in vitro and in vivo. A series of 4EGI-1-derived thiazol-2-yl hydrazones have been designed and synthesized in order to delineate the structural latitude and improve its binding affinity to eIF4E, and increase its potency in inhibiting the eIF4E/eIF4G interaction. Probing a wide range of substituents on both phenyl rings comprising the 3-phenylpropionic acid and 4-phenylthiazolidine moieties in the context of both E- and Z-isomers of 4EGI-1 led to analogs with enhanced binding affinity and translation initiation inhibitory activities.

3-substituted indazoles as configurationally locked 4EGI-1 mimetics and inhibitors of the eIF4E/eIF4G interaction.

4EGI-1, the prototypic inhibitor of eIF4E/eIF4G interaction, was identified in a high-throughput screening of small-molecule libraries with the aid of a fluorescence polarization assay that measures inhibition of binding of an eIF4G-derived peptide to recombinant eIF4E. As such, the molecular probe 4EGI-1 has potential for the study of molecular mechanisms involved in human disorders characterized by loss of physiological restraints on translation initiation. A hit-to-lead optimization campaign was carried out to overcome the configurational instability in 4EGI-1, which stems from the E-to-Z isomerization of the hydrazone function. We identified compound 1 a, in which the labile hydrazone was incorporated into a rigid indazole scaffold, as a promising rigidified 4EGI-1 mimetic lead. In a structure-activity relationship study directed towards probing the structural latitude of this new chemotype as an inhibitor of eIF4E/eIF4G interaction and translation initiation we identified 1 d, an indazole-based 4EGI-1 mimetic, as a new and improved lead inhibitor of eIF4E/eIF4G interaction and a promising molecular probe candidate for elucidation of the role of cap-dependent translation initiation in a host of pathophysiological states.

A specific and sensitive assay for blood levels of glycated CD59: a novel biomarker for diabetes.

Increasing evidence links the complement system with complications of human diabetes. The complement regulatory protein CD59, an inhibitor of formation of membrane attack complex (MAC), is inhibited by hyperglycemia-induced glycation fostering increased deposition of MAC, a major effector of complement-mediated tissue damage. CD59, an ubiquitous GPI-anchored membrane protein, is shed from cell membranes by phospholipases generating a soluble form present in blood and urine. We established an enzyme-linked immunosorbent assay (ELISA) to measure serum/plasma glycated human CD59 (hCD59) (GCD59) and evaluated its potential as a diabetes biomarker. We used a synthetic peptide strategy to generate (a) a mouse monoclonal antibody to capture hCD59, (b) a rabbit monoclonal antibody to detect GCD59, and (c) a GCD59 surrogate for assay standardization. ELISA conditions were optimized for precision, reproducibility, and clinical sensitivity. The clinical utility of the assay was initially evaluated in 24 subjects with or without diabetes and further validated in a study that included 100 subjects with and 90 subjects without a diagnosis of diabetes. GCD59 (a) was significantly higher in individuals with than in individual without diabetes, (b) was independently associated with HbA1c, and (c) identified individuals with diabetes with high specificity and sensitivity. We report the development and standardization of a novel, sensitive, and specific ELISA for measuring GCD59 in blood. The assay distinguished individuals with diabetes from those without, and showed strong correlation between GCD59 and HbA1c. Because GCD59 likely contributes to the pathogenesis of diabetes complications, measurement of blood levels of GCD59 may be useful in the diagnosis and management of diabetes.

Tumor suppression by small molecule inhibitors of translation initiation.

Translation initiation factors are over-expressed and/or activated in many human cancers and may contribute to their genesis and/or progression. Removal of physiologic restraints on translation initiation causes malignant transformation. Conversely, restoration of physiological restrains on translation initiation reverts malignant phenotypes. Here, we extensively characterize the anti-cancer activity of two small molecule inhibitors of translation initiation: #1181, which targets the eIF2∙GTP∙Met-tRNAi ternary complex, and 4EGI-1, which targets the eIF4F complex. In vitro, both molecules inhibit translation initiation, abrogate preferentially translation of mRNAs coding for oncogenic proteins, and inhibit proliferation of human cancer cells. In vivo, both #1181 and 4EGI-1 strongly inhibit growth of human breast and melanoma cancer xenografts without any apparent macroscopic- or microscopic-toxicity. Mechanistically, #1181 phosphorylates eIF2α while 4EGI-1 disrupts eIF4G/eIF4E interaction in the tumors excised from mice treated with these agents. These data indicate that inhibition of translation initiation is a new paradigm in cancer therapy.