Mechanism of carbamate inactivation of FAAH: implications for the design of covalent inhibitors and in vivo functional probes for enzymes

Fatty acid amide hydrolase (FAAH) regulates a large class of signaling lipids, including the endocannabinoid anandamide. Carbamate inhibitors of FAAH display analgesic and anxiolytic properties in rodents. However, the mechanism by which carbamates inhibit FAAH remains obscure. Here, we provide biochemical evidence that carbamates covalently modify the active site of FAAH by adopting an orientation opposite of that originally predicted from modeling. Based on these results, a series of carbamates was designed that display enhanced potency. One agent was converted into a "click chemistry" probe to comprehensively evaluate the proteome reactivity of FAAH-directed carbamates in vivo. These inhibitors were selective for FAAH in the nervous system, but they reacted with several enzymes in peripheral tissues. The experimental strategy described herein can be used to create in vivo probes for any enzyme susceptible to covalent inhibition.     

Design of potential angiogenin inhibitors

Summary A model of angiogenin has been prepared by homology modelling, based upon the structure of ribonuclease A. This model has been used to postulate an inhibitor based upon the angiostatic agent TAN1120. The complex of ribonuclease A with a dinucleotide has been modelled and used as a guide to the binding orientation of daunomycin—the closest TAN1120 analogue for which the crystal structure and stereochemistry are available.     

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling

Macrocyclic peptides have the potential to address intracellular protein–protein interactions (PPIs) of high value therapeutic targets that have proven largely intractable to small molecules. Here, we report broadly applicable lessons for applying this modality to intracellular targets and specifically for advancing chemical matter to address KRAS, a protein that represents the most common oncogene in human lung, colorectal and pancreatic cancers yet is one of the most challenging targets in human disease. Specifically, we focused on KRpep-2d, an arginine-rich KRAS-binding peptide with a disulfide-mediated macrocyclic linkage and a protease-sensitive backbone. These latter redox and proteolytic labilities obviated cellular activity. Extensive structure–activity relationship studies involving macrocyclic linker replacement, stereochemical inversion, and backbone α-methylation, gave a peptide with on-target cellular activity. However, we uncovered an important generic insight – the arginine-dependent cell entry mechanism limited its therapeutic potential. In particular, we observed a strong correlation between net positive charge and histamine release in an ex vivo assay, thus making this series unsuitable for advancement due to the potentially fatal consequences of mast cell degranulation. This observation should signal to researchers that cationic-mediated cell entry – an approach that has yet to succeed in the clinic despite a long history of attempts – carries significant therapy-limiting safety liabilities. Nonetheless, the cell-active molecules identified here validate a unique inhibitory epitope on KRAS and thus provide valuable molecular templates for the development of therapeutics that are desperately needed to address KRAS-driven cancers – some of the most treatment-resistant human malignancies.

Targeting undruggable intracellular proteins with peptides: novel on-target macrocyclic peptide inhibitors of KRAS with broad inhibition of proliferation of multiple KRAS-dependent cancer cell lines.     

Design and synthesis of aromatic inhibitors of anthranilate synthase

Aromatic analogues of chorismate were synthesised as potential inhibitors of anthranilate synthase. Molecular modelling using GOLD2.1 showed that these analogues docked into the active site of Serratia marcescens anthranilate synthase in the same conformation as chorismate. Most compounds were found to be micromolar inhibitors of S. marcescens anthranilate synthase. The most potent analogue, 3-(1-carboxy-ethoxy)-4-hydroxybenzoate (K(I) 3 microM), included a lactyl ether side chain. This appears to be a good replacement for the enol-pyruvyl side chain of chorismate.     

Design and synthesis of aromatic inhibitors of anthranilate synthase

Anthranilate synthase catalyses the conversion of chorismate to anthranilate, a key step in tryptophan biosynthesis. A series of 3-(1-carboxy-ethoxy) benzoic acids were synthesised as chorismate analogues, with varying functionality at C-4, the position of the departing hydroxyl group in chorismate. Most of the compounds were moderate inhibitors of anthranilate synthase, with inhibition constants between 20-30 microM. The exception was 3-(1-carboxy-ethoxy) benzoic acid, (C-4 = H), for which K(I)= 2.4 microM. These results suggest that a hydrogen bonding interaction with the active site general acid (Glu309) is less important than previously assumed for inhibition of the enzyme by these aromatic chorismate analogues.     

Design and pharmacophoric identification of flavonoid scaffold-based aromatase inhibitors

Aromatase is a crucial enzyme for the catalysis of aromatization reaction at the last and rate-limiting step involved in the conversion of androgenic substrates to an estrogenic substrate. A hormone-dependent breast cancer in postmenopausal woman can be cured by inhibition of estrogen biosynthesis by the help of aromatase inhibitors (AIs). The mode of interactions of flavonones with the active site of aromatase has been studied in search of potent and selective AIs as a substitute of the natural steroidal ligand. Structure-based computational approach namely, molecular docking simulations were performed to investigate the structural features of the docked complex of aromatase and flavonoid ligands. A nonsteroidal flavonoid pharmacophore showing electrostatic and steric features for selective binding within the main pocket of the catalytic active site of aromatase has been identified as an outcome of the study. The binding affinity of quercetin and isoflavone were predicted within aromatase. Isoflavone was used as a negative control to compare its binding affinities with the selected dataset. The predicted binding affinity of negative control isoflavone was in accordance with its in vitro AI efficacy. Isoflavone showed poor binding affinity and ranked last in terms of MolDock score (−86.309 kcal/molÅ) compared to dataset molecules. The generated pharmacophoric information will be helpful for the synthetic chemist to design and synthesize selective AIs with comparable binding affinity to the natural steroidal ligand.     

Design, synthesis and evaluation of new RDP inhibitors

Aminophosphinic acid derivatives were synthesized as potential inhibitors of renal dipeptidase, an enzyme overexpressed in benign and malignant colon tumors. Several compounds showed potent enzyme-inhibitory activity.     

Design and synthesis of carboxylate inhibitors for matrix metalloproteinases

A series of carboxylate compounds were prepared from N(alpha)-substituted 2,3-diaminopropionic acid and were tested for efficacy as matrix metalloproteinase (MMP) inhibitors. During modeling of the initial compound 10a, we utilized three-dimensional structure modeling software (InsightII/Discover Ver. 2.98). Some of the prepared carboxylate derivatives, such as carbamate compounds (12c,d, 22) and sulfonamide compounds (14b,c), proved to be effective MMP-1 inhibitors (with IC50 values of a 10(-6) M order), depending on the substituent at the N(alpha)-position of 2,3-diaminopropionic acid. Some of them were also evaluated for inhibition of stromelysin-1 (MMP-3), and the sulfonamide compound 14c exceeded the lead compound 5b in its MMP-3 inhibitory potency. For the carbamate compounds, we investigated the minimum molecular size at which the MMP-1 inhibitory potency was maintained, and found that this was P3-P1' compound 10b.     

Design and novel synthesis of aryl-heteroaryl-imidazole MAP kinase inhibitors

Inhibitors of the MAP kinase p38, potentially useful for the treatment of rheumatoid arthritis and inflammatory diseases, were found to exhibit antifungal activity. We have developed a new diversity-oriented strategy leading to concise and efficient syntheses of known and new members of this compound class. The strategy is based on carboncarbon cross-coupling reactions using N-protected 4,5-diiodo-imidazoles as the starting templates.     

Design and synthesis of inhibitors of adenosine and AMP deaminases

Nucleosides and nucleotides which are able to undergo covalent hydration in the aglycone ring system are potential inhibitors of the enzymes adenosine deaminase (ADA) and AMP deaminase, respectively. Calculations of the enthalpy of covalent hydration and of enzyme binding energy have been used to design new inhibitors of ADA. The ribosyl triazolotriazine 16, which was synthesized as a result of these calculations, exists predominantly as the covalent hydrate 18 in water and is a potent inhibitor of mammalian ADA (IC(50) 50 nM).     

Design and synthesis of chitin synthase inhibitors as potent fungicides

Chitin is a structural component of fungal cell walls but is absent in vertebrates,mammals,and humans.Chitin synthase is thus an attractive molecular target for developing fungicides.Based on the structure of its donor substrate,UDP-N-acetyl-glucosamine,as well as the modelled structure of the bacterial chitin synthase NodC,we designed a novel scaffold which was then further optimized into a series of chitin synthase inhibitors.The most potent inhibitor,compound 13,exhibited high chitin synthase inhibitory activity with an IC_(50) value of 64.5 μmol/L All of the inhibitors exhibited antifungal activities against the growth of agriculturally-destructive fungi,Fusarium graminearum,Botrytis cinerea.and Colletotrichum lagenarium.This work presents a new scaffold which can be used for the development of novel fungicides.     

药效团检索设计新的HIV-1蛋白酶抑制剂

通过对自建的未开发化合物三维结构库进行药效团检索,得到了4个对HIV-1蛋白酶抑制活化的化合物,通过构象分析发现包含药效团的构象处于优势构象,而且4个结构都含有带两个邻位羟基的苯环和一个间位羰基的药效团以及公共子结构。通过计算发现它们的疏水参数都很小。在考虑满足包含药效团的结构特征和有适中的疏水参数两个因素的前提下,设计出了新的具有潜在抑制HIV-1蛋白酶活性的化合物。它们的结构都比检索得到的四个化合物更为简单,因此易于合成。     

Design,synthesis and evaluation of lactoside-derived galectin-3 inhibitors

Based on docking computation, a panel of lactoside derivatives have been designed as galectin-3 inhibitors. Suitable functional group modifications at C′-3 of methyl lactoside were predicted to supply some additional π–cation, π…H–O, and hydrogen bond interactions between the designed substrates and galectin-3 residues. The selected compounds, giving higher TotalScore in docking calculations, were thus synthesized, and their binding affinities toward galectin-3 were evaluated with SPR assay.     

Design and synthesis of cyclic acylguanidines as BACE1 inhibitors

Based on the lead compound 1 reported in literature, a series of novel BACE1 inhibitors were designed and synthesized, among which compound 11 exhibited a 14-fold improvement in potency over the lead compound 1. This represents a good lead for the discovery of more promising BACE1 inhibitors for the potential treatment of AD.     

Design, synthesis, and screen of cathepsin K inhibitors

We synthesized a series of epoxysuccinic acid derivatives and evaluated their in vitro cathepsin K inhibitory activity The screening results show that the potency of compounds 9e,9d,9p,9j and 9k (IC₅₀≤0.005μmol/L) were equal to or greater than that of the lead compound 9a.Less hydrophobic compounds showed weaker potency,which can be explained by the hydrophobic nature of the cathepsin K binding pockets.