Crystal Structure of Barley Limit Dextrinase-Limit Dextrinase Inhibitor (LD-LDI) Complex Reveals Insights into Mechanism and Diversity of Cereal Type Inhibitors

Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 Å. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs.     

Synthesis and activity study of phosphonamidate dipeptides as potential inhibitors of VanX

In an effort to develop inhibitors of VanX, the phosphonamidate analogs of D-Ala-D-Ala dipeptides, N-[(1-aminoethyl) hydroxyphosphinyl]-glycine (1a), -alanine (1b), -valine (1c), -leucine (1d) and -phenylalanine (1e) were synthesized, characterized and evaluated using recombinant VanX. The crystal structure of the intermediate 6d was obtained (Deposition number: CCDC 839134), and structural analysis revealed that it is orthorhombic with a space group P2(1)2(1)2(1), the bond length of P-N is 1.62? and angle of C-N-P is 123.6°. Phosphonamidate 1(a-e) showed to be inhibitors of VanX with IC(50) values of 0.39, 0.70, 1.12, 2.82, and 4.13mM, respectively, which revealed that the inhibition activities of the phosphonamidates were dependent on the size of R-substituent of them, with the best inhibitor 1a having the smallest substituent. Also, 1a showed antibacterial activity against Staphylococcus aureus (ATCC 25923) with a MIC value of 0.25 μg/ml.     

Probing the isoprenylcysteine carboxyl methyltransferase (Icmt) binding pocket: sulfonamide modified farnesyl cysteine (SMFC) analogs as Icmt inhibitors

Human isoprenylcysteine carboxyl methyltransferase (hIcmt) is a promising anticancer target as it is important for the post-translational modification of oncogenic Ras proteins. We herein report the synthesis and biochemical activity of 41 farnesyl-cysteine based analogs versus hIcmt. We have demonstrated that the amide linkage of a hIcmt substrate can be replaced by a sulfonamide bond to achieve hIcmt inhibition. The most potent sulfonamide-modified farnesyl cysteine analog was 6ag with an IC₅₀ of 8.8 ± 0.5 μM for hIcmt.     

Design, synthesis and docking studies on benzamide derivatives as histone deacetylase inhibitors

A series of benzamide derivatives including two scaffolds were designed and synthesized as potential histone deacetylase inhibitors. Most of synthesized compounds showed moderate enzymatic potency at the same order of magnitude, and compound 12b possessed better potency to the positive control (3.8 μM vs 13.0 μM). It also showed a 50-fold increase in vitro anticancer activity against DU-145 cell-lines. Molecular docking studies were carried out and used to explain the structure-activity relationships observed in vitro. Then we found that the cavity surrounded by ASP104, HIS33, PRO34 and PHE155 may be crucial for the inhibitors’ activity. The docking results provide some useful information for future design of more potent inhibitors.     

Inhibition of the M. tuberculosis 3β-hydroxysteroid dehydrogenase by azasteroids

The cholesterol metabolism pathway in Mycobacterium tuberculosis (M. tb) is a potential source of energy as well as secondary metabolite production that is important for survival of M. tb in the host macrophage. Oxidation and isomerization of 3β-hydroxysterols to 4-en-3-ones is requisite for sterol metabolism and the reaction is catalyzed by 3β-hydroxysteroid dehydrogenase (Rv1106c). Three series of 6-azasteroids and 4-azasteroids were employed to define the substrate preferences of M. tb 3β-hydroxysteroid dehydrogenase. 6-Azasteroids with large, hydrophobic side chains at the C17 position are the most effective inhibitors. Substitutions at C1, C2, C4 and N6 were poorly tolerated. Our structure-activity studies indicate that the 6-aza version of cholesterol is the best and tightest binding competitive inhibitor (K_i = 100 nM) of the steroid substrate and are consistent with cholesterol being the preferred substrate of M. tb 3β-hydroxysteroid dehydrogenase.     

Dual function inhibitors of relevance to chronic obstructive pulmonary disease

The general strategy and rationale underlying the design of COPD therapeutics that possess protease inhibitory activity and are also capable of releasing a species that attenuates inflammation by inhibiting caspase-1, are described. The synthesis and in vitro biochemical evaluation of a dual function molecule that sequentially inhibits HNE and caspase-1 in a time-dependent manner is reported.     

Renin inhibitors for the treatment of hypertension: design and optimization of a novel series of tertiary alcohol-bearing piperidines

The design and optimization of a novel series of renin inhibitor is described herein. Strategically, by committing the necessary resources to the development of synthetic sequences and scaffolds that were most amenable for late stage structural diversification, even as the focus of the SAR campaign moved from one end of the molecule to another, highly potent renin inhibitors could be rapidly identified and profiled.     

Synthesis and evaluation of ferrocenoyl pentapeptide (Fc-KLVFF) as an inhibitor of Alzheimer's Aβ₁-₄₂ fibril formation in vitro

Aggregation and fibril formation of β-amyloid peptides (Aβ) is the key event in the pathogenesis of Alzheimer's disease. Many efforts have been made on the development of effective inhibitors to prevent Aβ fibril formation or disassemble the preformed Aβ fibrils. Peptide inhibitors with sequences homologous to the hydrophobic segments of Aβ can alter the aggregation pathway of Aβ, together with decrease of the cell toxicity. In this study, the conjugate of ferrocenoyl (Fc) with pentapeptide KLVFF (Fc-KLVFF), was synthesized by HBTU/HOBt protocol in solution. The inhibitory effect of Fc-KLVFF on Aβ(1-42) fibril formation was evaluated by thioflavin T fluorescence assay, and confirmed by atomic force microscopy (AFM) and transmission electron microscopy (TEM) analyses. Fc-KLVFF shows high inhibitory effect towards the fibril formation of Aβ(1-42). Additionally, the attachment of ferrocenoyl moiety onto peptides allows us to investigate the interaction between the inhibitor and Aβ(1-42) in real-time by electrochemical method. As expected, tethering of ferrocenoyl moiety onto pentapeptide shows improved lipophilicity and significant resistance towards proteolytic degradation compared to its parent peptide.     

Novel nanomolar imidazo[4,5-b]pyridines as selective nitric oxide synthase (iNOS) inhibitors: SAR and structural insights

Inducible arginine oxidation and subsequent NO production by correspondent synthase (iNOS) are important cellular answers to proinflammatory signals. Prolonged NO production has been proved in higher organisms to cause stroke or septic shock. Several classes of potent NOS inhibitors have been reported, most of them targeting the arginine binding site of the oxygenase domain. Here we disclose the SAR and the rational design of potent and selective iNOS inhibitors which may be useful as anti-inflammatory drugs.