pH‐Responsive Pharmacological Chaperones for Rescuing Mutant Glycosidases

A general approach is reported for the design of small‐molecule competitive inhibitors of lysosomal glycosidases programmed to 1) promote correct folding of mutant enzymes at the endoplasmic reticulum, 2) facilitate trafficking, and 3) undergo dissociation and self‐inactivation at the lysosome. The strategy is based on the incorporation of an orthoester segment into iminosugar conjugates to switch the nature of the aglycone moiety from hydrophobic to hydrophilic in the pH 7 to pH 5 window, which has a dramatic effect on the enzyme binding affinity. As a proof of concept, new highly pH‐responsive glycomimetics targeting human glucocerebrosidase or α‐galactosidase with strong potential as pharmacological chaperones for Gaucher or Fabry disease, respectively, were developed.     

First Discovery of Cholesterol-Lowering Activity of Parthenolide as NPC1L1 Inhibitor

Elevated cholesterol significantly increases the risk of developing atherosclerosis and coronary heart disease. The key to treating hypercholesterolemia is lowering plasma cholesterol levels. There have been no studies on the cholesterol-lowering potential of parthenolide (PTL), a naturally occurring small molecule from Tanacetum parthenium. Here, we first put forth PTL’s cholesterol-lowering ability to inhibit cellular uptake of cholesterol in a dose-dependent manner. Its performance was on par with the positive control drug, ezetimibe. Niemann–Pick C1 Like-1 (NPC1L1) has been identified as a potential therapeutic target for hypercholesterolemia. The interaction of PTL with NPC1L1 could be explained by the results of molecular docking and filipin staining further reinforces this hypothesis. Furthermore, PTL reduced the expression of NPC1L1 in HepG2 cells in a concentration-dependent manner, which suggests that PTL functions as a potential NPC1L1 inhibitor with therapeutic potential for hypercholesterolemia.     

Discovery of a small-molecule inhibitor for kidney ADP-ribosyl cyclase: Implication for intracellular calcium signal mediated by cyclic ADP-ribose

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca2+-mobilizing second messenger, cyclic ADP- ribose (cADPR), from beta-NAD+. A prototype of mammalian ADPR-cyclases is a lymphocyte antigen CD38. Accumulating evidence indicates that ADPR-cyclases other than CD38 are expressed in various cells and organs. In this study, we discovered a small molecule inhibitor of kidney ADPR-cyclase. This compound inhibited kidney ADPR-cyclase activity but not CD38, spleen, heart or brain ADPR-cyclase activity in vitro. Characterization of the compound in a cell-based system revealed that an extracellular calcium-sensing receptor (CaSR)- mediated cADPR production and a later long-lasting increase in intracellular Ca2+ concentration ([Ca2+]i) in mouse mesangial cells were inhibited by the pre-treatment with this compound. In contrast, the compound did not block CD3/TCR-induced cADPR production and the increase of [Ca2+]i in Jurkat T cells, which express CD38 exclusively. The long-lasting Ca2+ signal generated by both receptors was inhibited by pre-treatment with an antagonistic cADPR derivative, 8-Br-cADPR, indicating that the Ca2+ signal is mediated by the ADPR-cyclase metabolite, cADPR. Moreover, among structurally similar compounds tested, the compound inhibited most potently the cADPR production and Ca2+ signal induced by CaSR. These findings provide evidence for existence of a distinct ADPR-cyclase in the kidney and basis for the development of tissue specific inhibitors.     

Iminosugar C-Glycosides Work as Pharmacological Chaperones of NAGLU,a Glycosidase Involved in MPS IIIB Rare Disease**

Mucopolysaccharidosis type IIIB is a devastating neurological disease caused by a lack of the lysosomal enzyme, α-N-acetylglucosaminidase (NAGLU), leading to a toxic accumulation of heparan sulfate. Herein we explored a pharmacological chaperone approach to enhance the residual activity of NAGLU in patient fibroblasts. Capitalizing on the three-dimensional structures of two modest homoiminosugar-based NAGLU inhibitors in complex with bacterial homolog of NAGLU, CpGH89, we have synthesized a library of 17 iminosugar C-glycosides mimicking N-acetyl-D-glucosamine and bearing various pseudo-anomeric substituents of both α- and β-configuration. Elaboration of the aglycon moiety results in low micromolar selective inhibitors of human recombinant NAGLU, but surprisingly it is the non-functionalized and wrongly configured β-homoiminosugar that was proved to act as the most promising pharmacological chaperone, promoting a 2.4 fold activity enhancement of mutant NAGLU at its optimal concentration.     

Computational-Based Discovery of the Anti-Cancer Activities of Pyrrole-Based Compounds Targeting the Colchicine-Binding Site of Tubulin

Despite the tubulin-binding agents (TBAs) that are widely used in the clinic for cancer therapy, tumor resistance to TBAs (both inherited and acquired) significantly impairs their effectiveness, thereby decreasing overall survival (OS) and progression-free survival (PFS) rates, especially for the patients with metastatic, recurrent, and unresectable forms of the disease. Therefore, the development of novel effective drugs interfering with the microtubules’ dynamic state remains a big challenge in current oncology. We report here about the novel ethyl 2-amino-1-(furan-2-carboxamido)-5-(2-aryl/tert-butyl-2-oxoethylidene)-4-oxo-4,5-dihydro-1H-pyrrole-3-carboxylates (EAPCs) exhibiting potent anti-cancer activities against the breast and lung cancer cell lines in vitro. This was due to their ability to inhibit tubulin polymerization and induce cell cycle arrest in M-phase. As an outcome, the EAPC-treated cancer cells exhibited a significant increase in apoptosis, which was evidenced by the expression of cleaved forms of PARP, caspase-3, and increased numbers of Annexin-V-positive cells. By using the in silico molecular modeling methods (e.g., induced-fit docking, binding metadynamics, and unbiased molecular dynamics), we found that EAPC-67 and -70 preferentially bind to the colchicine-binding site of tubulin. Lastly, we have shown that the EAPCs indicated above and colchicine utilizes a similar molecular mechanism to inhibit tubulin polymerization via targeting the T7 loop in the β-chain of tubulin, thereby preventing the conformational changes in the tubulin dimers required for their polymerization. Collectively, we identified the novel and potent TBAs that bind to the colchicine-binding site and disrupt the microtubule network. As a result of these events, the compounds induced a robust cell cycle arrest in M-phase and exhibited potent pro-apoptotic activities against the epithelial cancer cell lines in vitro.     

Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70

Heat Shock Protein 70s (HSP70s) are key molecular chaperones that are overexpressed in many cancers and often associated with metastasis and poor prognosis. It has proven difficult to develop ATP-competitive, drug-like small molecule inhibitors of HSP70s due to the flexible and hydrophilic nature of the HSP70 ATP-binding site and its high affinity for endogenous nucleotides. The aim of this study was to explore the potential for the inhibition of HSP70 through alternative binding sites using fragment-based approaches. A surface plasmon resonance (SPR) fragment screen designed to detect secondary binding sites in HSP70 led to the identification by X-ray crystallography of a cryptic binding site in the nucleotide-binding domain (NBD) of HSP70 adjacent to the ATP-binding site. Fragment binding was confirmed and characterized as ATP-competitive using SPR and ligand-observed NMR methods. Molecular dynamics simulations were applied to understand the interactions with the protein upon ligand binding, and local secondary structure changes consistent with interconversion between the observed crystal structures with and without the cryptic pocket were detected. A virtual high-throughput screen (vHTS) against the cryptic pocket was conducted, and five compounds with diverse chemical scaffolds were confirmed to bind to HSP70 with micromolar affinity by SPR. These results identified and characterized a new targetable site on HSP70. While targeting HSP70 remains challenging, the new site may provide opportunities to develop allosteric ATP-competitive inhibitors with differentiated physicochemical properties from current series.     

Mechanisms of Chaperones as Active Assistant/Protector for Proteins: Insights from NMR Studies

Molecular chaperones are diverse families of proteins that play key roles in protein homeostasis. They assist the folding of client proteins or prevent them from irreversible aggregation under stress conditions. Diverse chaperone families contribute to different aspects of protein homeostasis by interacting with a wide range of client proteins. Despite the vital roles of chaperones in cell survival, the molecular mechanisms underlying chaperone functions remain elusive, due to the non‐specificity of chaperone‐client interactions and the intrinsic flexibility of the clients. Our understanding of the chaperone functional mechanisms, especially regarding chaperone‐client interactions, has greatly expanded in recent years, thanks to the significant contribution from various NMR studies. Solution NMR methods have unique advantages in characterizing disordered protein structures, detecting weak and non‐specific interactions, and probing conformational dynamics of proteins and protein complexes, etc., and therefore are especially powerful in the studies of chaperone structure‐function relationships. In this review, we summarize some of the current knowledge of molecular chaperones, with emphasis on common features of chaperone‐client interactions and examples on a number of specific systems in which solution NMR methods were used to provide essential insights into their functional mechanisms.      

Chaperones Are Necessary for the Expression of Catalytically Active Potato Apyrases in Prokaryotic Cells

NTPDases (nucleoside triphosphate diphosphohydrolases) (also called in plants apyrases) hydrolyze nucleoside 5′-tri- and/or diphosphate bonds producing nucleosides di or monophosphate and inorganic phosphate. For years, studies have been carried out to use both plant and animal enzymes for medicine. Therefore, there is a need to develop an efficient method for the quick production of large amounts of homogeneous proteins with high catalytic activity. Expression of proteins in prokaryotic cells is the most common way for the protein production. The aim of our study was to develop a method of expression of potato apyrase (StAPY4, 5, and 6) genes in bacterial cells under conditions that allowed the production of catalytically active form of these enzymes. Apyrase 4 and 6 were overexpressed in BL21-CodonPlus (DE3) bacteria strain but they were accumulated in inclusion bodies, regardless of the culture conditions and induction method. Co-expression of potato apyrases with molecular chaperones allowed the expression of catalytically active apyrase 5. However, its high nucleotidase activity could be toxic for bacteria and is therefore synthesized in small amounts in cells. Our studies show that each protein requires other conditions for maturation and even small differences in amino acid sequence can essentially affect protein folding regardless of presence of chaperones.     

Site specific biotinylation of the human aldo/keto reductase AKR1A1 for immobilization

New strategies for the specific monolabelling of enzymes play a key role in the development of artificial proteins. Especially for the emerging research field of nanobiotechnology and bioelectronics artificial monofunctionalized redoxproteins are of great interest. The human AKR1A1, an enzyme of the aldo/keto reductase superfamily, has been chosen as subject for the synthesis of an artificially mono biotinylated redoxprotein in order to selectively immobilize this enzyme for bioelectronic applications. To produce monofunctionalized enzyme we applied the strategy of Expressed Protein Ligation (EPL) in combination with solid phase peptide synthesis (SPPS). Accordingly, we used the IMPACT^®-system and cloned the aldo/keto-reductase as fusion protein with an additional intein/chitin binding domain. Through intein mediated splicing we could produce the C-terminal thioester of the aldo/keto-reductase, which maintained its biological activity. Then, the thioester was coupled to Cys-Lys(Ahx-Ahx-biotin)-amide by Native Chemical Ligation, which led to mono-biotinylated protein. The enzyme activity was proven to be intact as shown by various kinetic investigations. Immobilization was performed on avidin coated silica microspheres. Accordingly, for the first time selectively modified AKR1A1 has been immobilized.     

Single-Centre Model for the Active Site of α-Chymotrypsin

The polymer-bound heptapeptide H-Glu-His-Pro-Gly-Ser-Gly-PEGM was designed as a ‘single-centre model’ for the active site of α-chymotrypsin. The peptide was synthesized according to the general principles of the liquid-phase method for peptide synthesis, and its conformational properties were investigated by CD and IR spectroscopy in solution and in the solid state. In harmony with empirical prediction codes, experimental and theoretical conformational considerations, the peptide adopts a β-turn conformation stabilized by H-bonds involving the side chains of Glu, His, and Ser. The development of a H-bonded system similar to the active site of α-chymotrypsin leads to implications with respect to a possible catalytic activity of the model peptide.     

1-Substituted 2-Azaspiro[3.3]heptanes: Overlooked Motifs for Drug Discovery

The 2-substituted piperidine core is found in drugs (18 FDA-approved drugs), however, their spirocyclic analogues remain unknown. Described here is the synthesis of spirocyclic analogues for 2-substituted piperidines and a demonstration of their validation in drug discovery.     

Sirtuin 1-Activating Compounds: Discovery of a Class of Thiazole-Based Derivatives

Sirtuin 1 (SIRT1) is a NAD⁺-dependent deacetylase implicated in various biological and pathological processes, including cancer, diabetes, and cardiovascular diseases. In recent years, SIRT1-activating compounds have been demonstrated to exert cardioprotective effects. Therefore, this enzyme has become a feasible target to treat cardiovascular diseases, and many SIRT1 activators, of a natural or synthetic origin, have been identified. In the present work, we developed thiazole-based SIRT1 activators, which showed remarkably higher SIRT1 activation potencies compared with those of the reference compound resveratrol when tested in enzymatic assays. Thiazole 8, a representative compound of this series, was also subjected to further pharmacological investigations, where it was proven to reduce myocardial damage induced by an in vivo occlusion/reperfusion event, thus confirming its cardioprotective properties. In addition, the cardioprotective effect of compound 8 was significantly higher than that of resveratrol. Molecular modeling studies suggest the binding mode of these derivatives within SIRT1 in the presence of the p53-AMC peptide. These promising results could pave the way to further expand and optimize this chemical class of new and potent SIRT1 activators as potential cardioprotective agents.