Discovery of a novel IKK-β inhibitor by ligand-based virtual screening techniques

Various inflammatory stimuli that activate the nuclear factor kappa B (NF-κB) signaling pathway converge on a serine/threonine kinase that displays a key role in the activation of NF-κB: the I kappa B kinase β (IKK-β). Therefore, IKK-β is considered an interesting target for combating inflammation and cancer. In our study, we developed a ligand-based pharmacophore model for IKK-β inhibitors. This model was employed to virtually screen commercial databases, giving a focused hit list of candidates. Subsequently, we scored by molecular shape to rank and further prioritized virtual hits by three-dimensional shape-based alignment. One out of ten acquired and biologically tested compounds showed inhibitory activity in the low micromolar range on IKK-β enzymatic activity in vitro and on NF-κB transactivation in intact cells. Compound 8 (2-(1-adamantyl)ethyl 4-[(2,5-dihydroxyphenyl)methylamino]benzoate) represents a novel chemical class of IKK-β inhibitors and shows that the presented model is a valid approach for identification and development of new IKK-β ligands.     

A new class of aggregation inhibitor of amyloid-β peptide based on an O-acyl isopeptide

Inhibition of amyloid β peptide (Aβ) aggregation is a potential therapeutic approach to treat Alzheimer’s disease. We report that an O-acyl isopeptide of Aβ1–42 (1) containing an ester bond at the Gly²⁵-Ser²⁶ moiety inhibits Aβ1–42 fibril formation at equimolar ratio. Inhibitory activity was retained by an N-Me-β-Ala²⁶ derivative (2), in which the ester of 1 was replaced with N-methyl amide to improve chemical stability at physiological pH. Inhibition was verified by fluorescence anisotropy, Western blot, and atomic force microscopy. This report suggests a new class of Aβ aggregation inhibitor based on modification of Aβ1–42 at Gly²⁵-Ser²⁶.     

Screening and identification of a novel class of TGF-β type 1 receptor kinase inhibitor

Transforming growth factor β (TGF-β) type I receptor (activin receptor-like kinase 5, ALK5) has been identified as a promising target for fibrotic diseases. To find a novel inhibitor of ALK5, the authors performed a high-throughput screen of a library of 420,000 compounds using dephosphorylated ALK5. From primary hits of 1521 compounds, 555 compounds were confirmed. In total, 124 compounds were then selected for follow-up based on their unique structures and other properties. Repeated concentration-response testing and final interference assays of the above compounds resulted in the discovery of a structurally novel ALK5 inhibitor (compound 8) (N-(thiophen 2-ylmethyl)-3-(3,4,5 trimethoxyphenyl)imidazo[1,2β]pyridazin 6-amine) with a low IC(50) value of 0.7 μM. Compound 8 also inhibited the TGF-β-induced nuclear translocation of SMAD with an EC(50) value of 0.8 μM. Kinetic analysis revealed that compound 8 inhibited ALK5 via mixed-type inhibition, suggesting that it may bind to ALK5 differently than other published adenosine triphosphate site inhibitors.     

Mechanism of fiber assembly: treatment of Aβ peptide aggregation with a coarse-grained united-residue force field

The growth mechanism of β-amyloid (Aβ) peptide fibrils was studied by a physics-based coarse-grained united-residue model and molecular dynamics (MD) simulations. To identify the mechanism of monomer addition to an Aβ_1-40 fibril, we placed an unstructured monomer at a distance of 20 Å from a fibril template and allowed it to interact freely with the latter. The monomer was not biased towards fibril conformation by either the force field or the MD algorithm. With the use of a coarse-grained model with replica-exchange molecular dynamics, a longer timescale was accessible, making it possible to observe how the monomers probe different binding modes during their search for the fibril conformation. Although different assembly pathways were seen, they all follow a dock-lock mechanism with two distinct locking stages, consistent with experimental data on fibril elongation. Whereas these experiments have not been able to characterize the conformations populating the different stages, we have been able to describe these different stages explicitly by following free monomers as they dock onto a fibril template and to adopt the fibril conformation (i.e., we describe fibril elongation step by step at the molecular level). During the first stage of the assembly (“docking”), the monomer tries different conformations. After docking, the monomer is locked into the fibril through two different locking stages. In the first stage, the monomer forms hydrogen bonds with the fibril template along one of the strands in a two-stranded β-hairpin; in the second stage, hydrogen bonds are formed along the second strand, locking the monomer into the fibril structure. The data reveal a free-energy barrier separating the two locking stages. The importance of hydrophobic interactions and hydrogen bonds in the stability of the Aβ fibril structure was examined by carrying out additional canonical MD simulations of oligomers with different numbers of chains (4-16 chains), with the fibril structure as the initial conformation. The data confirm that the structures are stabilized largely by hydrophobic interactions and show that intermolecular hydrogen bonds are highly stable and contribute to the stability of the oligomers as well.     

Pharmacophore-based discovery of a novel cytosolic phospholipase A(2)α inhibitor

The release of arachidonic acid, a precursor in the production of prostaglandins and leukotrienes, is achieved by activity of the cytosolic phospholipase A(2)α (cPLA(2)α). Signaling mediated by this class of bioactive lipids, which are collectively referred to as eicosanoids, has numerous effects in physiological and pathological processes. Herein, we report the development of a ligand-based pharmacophore model and pharmacophore-based virtual screening of the National Cancer Institute (NCI) database, leading to the identification of 4-(hexadecyloxy)-3-(2-(hydroxyimino)-3-oxobutanamido)benzoic acid (NSC 119957) as cPLA(2)α inhibitor in cell-free and cell-based in vitro assays.     

A novel series of IKKβ inhibitors part I: Initial SAR studies of a HTS hit

A novel series of (E)-1-((2-(1-methyl-1H-imidazol-5-yl) quinolin-4-yl) methylene) thiosemicarbazides was discovered as potent inhibitors of IKKβ. In this Letter we document our early efforts at optimization of the quinoline core, the imidazole and the semithiocarbazone moiety. Most potency gains came from substitution around the 6- and 7-positions of the quinoline ring. Replacement of the semithiocarbazone with a semicarbazone decreased potency but led to some measurable exposure.     

Characterization of Aβ aggregation mechanism probed by congo red

β-Amyloid peptide (1) (Aβ) aggregates are toxic to neuron and the main cause of Alzheimer's disease (AD). The role of congo red (CR) on Aβ aggregation is controversial in aqueous solution. Both prevention and promotion of Aβ aggregation have been proposed, suggesting that CR may interact with Aβ of different structural conformations resulting in different effects on Aβ aggregation behavior. CR with these characteristics can be applied to probe the molecular mechanism of Aβ aggregation. Therefore, in the present study, we used CR as a probe to study the Aβ aggregation behavior in sodium dodecyl sulfate (SDS) condition. Our results show that Aβ(40) adopts two short helices at Q15-S26 and K28-L34 in the SDS environment. CR can interact with the helical form of Aβ(40), and the main interaction site is located at the first helical and hydrophobic core region, residues 17-25, which is assigned as a discordant helix region. Furthermore, CR may prevent Aβ(40) undergoing α-helix to β-strand conversion, and therefore aggregation through stabilizing the helical conformation of discordant helix in SDS environment, suggesting that the discordant helix plays a key role on the conformational stabilization of Aβ. Our present study implies that any factors or molecules that can stabilize the discordant helical conformation may also prevent the Aβ aggregation in membrane associated state. This leads to a new therapeutic strategy for the development of lead compounds to AD.     

A multi-faceted analysis of RutD reveals a novel family of α/β hydrolases

The rut pathway of pyrimidine catabolism is a novel pathway that allows pyrimidine bases to serve as the sole nitrogen source in suboptimal temperatures. The rut operon in E. coli evaded detection until 2006, yet consists of seven proteins named RutA, RutB, etc. through RutG. The operon is comprised of a pyrimidine transporter and six enzymes that cleave and further process the uracil ring. Herein, we report the structure of RutD, a member of the α/β hydrolase superfamily, which is proposed to enhance the rate of hydrolysis of aminoacrylate, a toxic side product of uracil degradation, to malonic semialdehyde. Although this reaction will occur spontaneously in water, the toxicity of aminoacrylate necessitates catalysis by RutD for efficient growth with uracil as a nitrogen source. RutD has a novel and conserved arrangement of residues corresponding to the α/β hydrolase active site, where the nucleophile's spatial position occupied by Ser, Cys, or Asp of the canonical catalytic triad is replaced by histidine. We have used a combination of crystallographic structure determination, modeling and bioinformatics, to propose a novel mechanism for this enzyme. This approach also revealed that RutD represents a previously undescribed family within the α/β hydrolases. We compare and contrast RutD with PcaD, which is the closest structural homolog to RutD. PcaD is a 3‐oxoadipate‐enol‐lactonase with a classic arrangement of residues in the active site. We have modeled a substrate in the PcaD active site and proposed a reaction mechanism. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Characterization of Aβ aggregation mechanism probed by congo red

β-Amyloid peptide ^{1 1}These authors contributed equally to this work. Communicated by Ramaswamy H. Sarma (Aβ) aggregates are toxic to neuron and the main cause of Alzheimer’s disease (AD). The role of congo red (CR) on Aβ aggregation is controversial in aqueous solution. Both prevention and promotion of Aβ aggregation have been proposed, suggesting that CR may interact with Aβ of different structural conformations resulting in different effects on Aβ aggregation behavior. CR with these characteristics can be applied to probe the molecular mechanism of Aβ aggregation. Therefore, in the present study, we used CR as a probe to study the Aβ aggregation behavior in sodium dodecyl sulfate (SDS) condition. Our results show that Aβ₄₀ adopts two short helices at Q15-S26 and K28-L34 in the SDS environment. CR can interact with the helical form of Aβ₄₀, and the main interaction site is located at the first helical and hydrophobic core region, residues 17–25, which is assigned as a discordant helix region. Furthermore, CR may prevent Aβ₄₀ undergoing α-helix to β-strand conversion, and therefore aggregation through stabilizing the helical conformation of discordant helix in SDS environment, suggesting that the discordant helix plays a key role on the conformational stabilization of Aβ. Our present study implies that any factors or molecules that can stabilize the discordant helical conformation may also prevent the Aβ aggregation in membrane associated state. This leads to a new therapeutic strategy for the development of lead compounds to AD.     

Characterization of insulin-degrading enzyme-mediated cleavage of Aβ in distinct aggregation states

To enhance our understanding of the potential therapeutic utility of insulin-degrading enzyme (IDE) in Alzheimer's disease (AD), we studied in vitro IDE-mediated degradation of different amyloid-beta (Aβ) peptide aggregation states. Our findings show that IDE activity is driven by the dynamic equilibrium between Aβ monomers and higher ordered aggregates. We identify Met₃₅–Val₃₆ as a novel IDE cleavage site in the Aβ sequence and show that Aβ fragments resulting from IDE cleavage form non-toxic amorphous aggregates. These findings need to be taken into account in therapeutic strategies designed to increase Aβ clearance in AD patients by modulating IDE activity.     

Virtual ligand screening of α-glucosidase: Identification of a novel potent noncarbohydrate mimetic inhibitor

5-Thiazoleacetamide derivatives of AR122 and AR125 were screened as α-glucosidase inhibitors by in silico high-throughput screening from commercial drug-like small compound libraries. Inhibition of α-glucosidase with AR122 and AR125 is time dependent: with no preincubation, AR122 and AR125 are relatively moderate inhibitors, but interestingly, after a 120 min incubation, they were 50-fold more potent (AR122: IC(50)=2.47 μM and AR125: IC(50)=27.1 μM). Plots of ln [residual α-glucosidase activity %] versus preincubation time show a pseudo-first order kinetics for both inhibitors. Through dialysis of enzyme-inhibitor complexes, no activity recovery was shown. These results suggest that AR122 and AR125 constitute a new class of noncarbohydrate mimetic inhibitor with an irreversible mechanism.     

The co-effect of copper and lipid vesicles on Aβ aggregation

Both metal ions and lipid membranes have a wide distribution in amyloid plaques and play significant roles in AD pathogenesis. Although influences of different metal ions or lipid vesicles on the aggregation of Aβ peptides have been extensively studied, their combined effects are less understood. In this study, we reported a unique effect of copper ion on Aβ aggregation in the presence of lipid vesicles, different from other divalent metal ions. Cu²⁺ in a super stoichiometric amount leads to the rapid formation of β-sheet rich structure, containing abundant low molecular weight (LMW) oligomers. We demonstrated that oligomerization of Aβ40 induced by Cu²⁺ binding was an essential prerequisite for the rapid conformation transition. Overall, the finding provided a new view on the complex triple system of Aβ, copper ion and lipid vesicles, which might help understanding of Aβ pathologies.     

Therapeutic effect of a peptide inhibitor of TGF-β on pulmonary fibrosis

Pulmonary fibrosis encompasses several respiratory diseases characterized by epithelial cell injury, inflammation and fibrosis. Transforming growth factor (TGF)-β1 is one of the main profibrogenic cytokines involved in the pathogenesis of lung fibrosis. It induces fibroblast differentiation into myofibroblasts, which produce high levels of collagen and concomitantly loss of lung elasticity and reduction of the respiratory function. In the present study, we have investigated the effects of P17 (a TGF-β inhibitor peptide) on IMR-90 lung fibroblast differentiation in vitro, as well as on the inhibition of the development of bleomycin-induced pulmonary fibrosis in mice. It was found that in IMR-90 cells, P17 inhibited TGF-β1-induced expression of connective tissue growth factor and α-smooth muscle actin. In vivo, treatment of mice with P17 2days after bleomycin administration decreased lung fibrosis, areas of myofibroblast-like cells and lymphocyte infiltrate. P17 also reduced mRNA expression of collagen type I, fibronectin and the fibronectin splice isoform EDA in the lung, and increased the expression of IFN-γ mRNA. Finally, therapeutic treatment with P17 in mice with already established fibrosis was able to significantly attenuate the progression of lung fibrosis. These results suggest that P17 may be useful in the treatment of pulmonary fibrosis.     

A novel series of IKKβ inhibitors part II: description of a potent and pharmacologically active series of analogs

A novel series of (E)-1-((2-(1-methyl-1H-imidazol-5-yl) quinolin-4-yl) methylene) thiosemicarbazides was discovered as potent inhibitors of IKKβ. In this Letter we document our efforts at further optimization of this series, culminating in 2 with submicromolar potency in a HWB assay and efficacy in a CIA mouse model.     

Characterization of early stage intermediates in the nucleation phase of Aβ aggregation

Alzheimer's disease (AD) is a common form of dementia, which is characterized by the presence of extracellular amyloid plaques comprising the amyloid β peptide (Aβ). Although the mechanism underlying AD pathogenesis remains elusive, accumulating evidence suggests that the process of amyloid fibril formation is a surface-mediated event, which plays an important role in AD onset and progression. In this study, the mechanism of Aβ aggregation on hydrophobic surfaces was investigated with dual polarization interferometry (DPI), which provides real-time information on early stages of the aggregation process. Aggregation was monitored on a hydrophobic C18 surface and a polar silicon oxynitride surface. The DPI results showed a characteristic Aβ aggregation pattern involving a decrease in the density of Aβ at the surface followed by an increase in the thickness on the hydrophobic C18 chip. Most importantly, the DPI measurements provided unique information on the early stages of Aβ aggregation, which is characterized by the presence of initially slow nucleus formation process followed by exponential fibril elongation. The dimensions of the putative nucleus corresponded to a thickness of ～5 nm for both Aβ40 and Aβ42, which may represent about 10-15 molecules. The results thus support the nucleation-dependent polymerization model as indicated by the presence of a nucleation phase followed by an exponential growth phase. These results are the first reported measurements of the real-time changes in Aβ molecular structure during the early stages of amyloid formation at the nanometer level.     

Degradation of fibrin-β amyloid co-aggregate: A novel function attributed to ubiquitin

Human placental extract contains numerous bioactive components that are effective wound healing, antimicrobial and anti-inflammatory agents. During our investigation on the therapeutic potency of human placental extract, we have purified ubiquitin-like molecules that showed strong fibrino(geno)lytic activity. Further investigation confirmed similar potency of ubiquitin purified from adult human erythrocyte. Additionally, ubiquitin efficiently degraded disordered amyloid β 42 peptide (Aβ42) aggregate and fibrin-Aβ42 co-aggregate in vitro and reduced co-aggregate induced cytotoxicity in SH-SY5Y human neuroblastoma cells as compared to plasmin. Ubiquitin also degraded abnormal co-aggregates of fibrin with other plasma proteins such as fibronectin, albumin, lysozyme, tranthyretin and α-synuclein. To elucidate the mechanism of degradation, synthetic peptides (ADG, GKT, DQQ, QRL, LIF, AGK, HLVL) derived from ubiquitin template as well as synthetic ubiquitin (8565.32?Da) were employed. Synthetic ubiquitin completely degraded preformed Aβ 42 aggregate and fibrin-Aβ42 co-aggregate, whereas, the smaller synthetic peptides showed varying degrees of degradation. These observations attribute a novel function of ubiquitin that may be used for degrading abnormal fibrin clots in human body. Thorough investigation might unfold a novel molecular mechanism of ubiquitin in protein homeostasis.     

An Aβ concatemer with altered aggregation propensities

We present an analysis of the conformational and aggregative properties of an Aβ concatemer (Con-Alz) of interest for vaccine development against Alzheimer's disease. Con-Alz consists of 3 copies of the 43 residues of the Aβ peptide separated by the P2 and P30 T-cell epitopes from the tetanus toxin. Even in the presence of high concentrations of denaturants or fluorinated alcohols, Con-Alz has a very high propensity to form aggregates which slowly coalesce over time with changes in secondary, tertiary and quaternary structure. Only micellar concentrations of SDS were able to inhibit aggregation. The increase in the ability to bind the fibril-binding dye ThT increases without lag time, which is characteristic of relatively amorphous aggregates. Confirming this, electron microscopy reveals that Con-Alz adopts a morphology resembling truncated protofibrils after prolonged incubation, but it is unable to assemble into classical amyloid fibrils. Despite its high propensity to aggregate, Con-Alz does not show any significant ability to permeabilize vesicles, which for fibrillating proteins is taken to be a key factor in aggregate cytotoxicity and is attributed to oligomers formed at an early stage in the fibrillation process. Physically linking multiple copies of the Aβ-peptide may thus sterically restrict Con-Alz against forming cytotoxic oligomers, forcing it instead to adopt a less well-organized assembly of intermeshed polypeptide chains.     

NABi,a novel β-sheet breaker,inhibits Aβ aggregation and neuronal toxicity: Therapeutic implications for Alzheimer's disease

Amyloid beta (Aβ) aggregates are an important therapeutic target for Alzheimer's disease (AD), a fatal neurodegenerative disease. To date, AD still remains a big challenge due to no effective treatments. Based on the property that Aβ aggregates have the cross-β-structure, a common structural feature in amyloids, we systemically designed the Aβ-aggregation inhibitor that maintains Aβ-interacting ability but removes toxic part from SOD1 (superoxide dismutase 1)-G93A. We identified NABi (Natural Aβ Binder and Aβ-aggregation inhibitor) composed of β2–3 strands, a novel breaker of Aβ aggregation, which does not self-aggregate and has no cytotoxicity at all. The NABi blocks Aβ-fibril formation in vitro and in vivo and prevents neuronal cell death, a hallmark of AD pathogenesis. Such anti-amyloidogenic properties can provide novel strategies for treating AD. Furthermore, our study provides molecular insights into the design of amyloidogenic inhibitors to cure various neurodegenerative and amyloid-associated diseases, as NABi would regulate aggregation of other toxic β-sheet proteins other than Aβ.