Ana Trapero

Polyhydroxylated Bicyclic Isoureas and Guanidines Are Potent Glucocerebrosidase Inhibitors and Nanomolar Enzyme Activity Enhancers in Gaucher Cells

Four diastereomeric series of N-alkylated [6+5] bicyclic isoureas having hydroxyl substituents mimicking glucose hydroxyl groups have been synthesized as potential β-glucocerebrosidase (GCase) inhibitors with the aim of developing pharmacological chaperones for enzyme deficiency in Gaucher disease (GD). The bicyclic compounds differ either by the configuration of the ring fusion carbon atoms or by the nature of the N-alkyl substituents. When assayed for effects on GCase activity, the isoureas displayed selective inhibition of GCase with low micromolar to nanomolar IC(50)s in isolated enzyme experiments. One of the series of isoureas, a family having a specific cis ring fusion, exhibited strong inhibition of recombinant GCase activity with K(i) values in the 2-42 nM range. In addition, the [6+5] bicyclic guanidine derivatives with a substitution pattern analogous to the most active isoureas were also found to be potent inhibitors of GCase with K(i) values between 3 and 10 nM. Interestingly, the active bicyclic isoureas and guanidines also behaved as GCase inhibitors in wild-type human fibroblasts at nanomolar concentrations. The potential of these compounds as pharmaceutical chaperones was determined by analyzing their capacity for increasing GCase activity in GD lymphoblasts derived from N370S and L444P variants, two of the most prevalent Gaucher mutations. Six compounds were selected from the different bicyclic isoureas and guanidines obtained that increased GCase activity by 40-110% in N370S and 10-50% in L444P cells at low micromolar to nanomolar concentrations following a 3 day incubation. These results describe a promising series of potent GCase ligands having the cellular properties required for pharmacological chaperones.

Potent Aminocyclitol Glucocerebrosidase Inhibitors are Subnanomolar Pharmacological Chaperones for Treating Gaucher Disease

Amino-myo-inositol derivatives have been found to be potent inhibitors of glucocerebrosidase (GCase), the β-glucosidase enzyme deficient in Gaucher disease (GD). When tested using lymphoblasts derived from patients with GD homozygous for N370S or L444P mutations, the compounds enhanced GCase activity at very low concentrations. The most potent inhibitor, (1R,2S,3R,4S,5S,6R)-5-(nonylamino)-6-(nonyloxy)cyclohexane-1,2,3,4-tetraol had a K(i) of 1 nM using isolated enzyme and an IC(50) of 4.3 nM when assayed in human fibroblast cell culture. This aminocyclitol produced maximum increases of GCase activities of 90% in N370S lymphoblasts at 1 nM and 40% in L444P at 0.01 nM following a three-day incubation. In addition to inhibitory potency, this compound has the permeability, subcellular distribution, and cell metabolism characteristics that are important for use as a pharmacological chaperone. It is a remarkable finding that picomolar concentrations of aminocyclitols are sufficient to enhance activity in the L444P variant, which produces a severe neuronopathic form of GD without clinical treatment.

A prospect for pyrrolidine iminosugars as antidiabetic α-glucosidase inhibitors.

α-Glucosidase inhibitors are a class of oral antidiabetic agents that have been exploited for the effective management of type 2 diabetes and associated complications for about 20 years. These drugs significantly reduce the postprandial increase in glucose and plasma insulin levels in diabetic patients by inhibiting the activity of α-glucosidases involved in the digestion of carbohydrates. The enzyme inhibition reduces oligosaccharide hydrolysis and depresses intestinal glucose absorption. Currently three drugs (acarbose, miglitol, and voglibose) belonging to this category are in the market and have shown effective clinical use in spite of inducing gastrointestinal disturbance and some other side effects. However, α‐glucosidase inhibitors reduce the cardiovascular harm and the risk of hypoglycaemia associated with other glucose-lowering drug types, demonstrating several comparative advantages over antidiabetics with other mechanism of action. Kato et al. describe a new family of potent pyrrolidine α‐glucosidase inhibitors with promising glucose-lowering activity. The first-in-class compound α-1-C-butyl-LAB is a potent inhibitor of intestinal α-glucosidase enzymes and displays an interesting selectivity and enzymatic profile addressing some of the undesired side effects of this class of antidiabetics. When tested in vivo in the carbohydrate-loading tests, α-1-C-butyl-LAB effectively reduces the rise of plasma glucose after food intake, with a dose about 10 times lower than that required for miglitol. Antidiabetic α-glucosidase inhibitors share some common structural characteristics with a class of bioactive molecules that mimic the sugar structures (Figure 1). Generally, these are cyclic compounds containing a basic amine functionality with several hydroxyl substituents that have a tridimensional arrangement similar to the present in elemental carbohydrates. Sometimes, these structural similarities are translated to the carbohydrate functional roles, and numerous members of these families display interesting biological and enzymatic activities, especially as glycosidase inhibitors. In general, two main classes of carbohydrate mimetics are found: carbasugars, which contain a carbocycle scaffold, and iminosugars, having a saturated nitrogen heterocyclic ring where other heteroatom substituents and hydroxyl groups are attached (Figure 1). Both classes are represented among clinically relevant antidiabetic α-glucosidase inhibitors: acarbose and voglibose are carbasugars with α-amino substituents at the oxygen anomeric position, whereas miglitol is an N-alkyl derivative of deoxynojirimycin (DNJ), the prototypical iminosugar compound. Anomeric mimicry is directly achieved in carbasugars, where heteroatom substituents can be bonded to the carbocycle with identical stereochemistry of the parent sugar anomeric group. In contrast, the presence of an anomeric-like group is less common in iminosugar family because of instability of the N,O-acetal function. Usually these derivatives lack the presence of an anomeric substituent such as in DNJ, or alternatively, ring nitrogen alkylation is introduced as in miglitol. However, nitrogen substituents are not configurationally stable because of fast nitrogen inversion, and therefore, the structural correlation with the rigid anomeric substituent of the sugar is lost. A solution for this has been introduced in the 1-C-iminosugar family, where an “anomeric” substituent is attached by a C−C bond to a position adjacent to the ring nitrogen (Figure 1). This is the approach used in the design of the α-1-C-alkyl-LAB family of compounds that has been synthesized in a straightforward sequence of reactions. The degree of structural similarity between hexoses and cyclohexane carbasugars or piperidine iminosugars is usually high, defining a related binding mode to proteins and enzymes. In contrast, the structural connection between the parent carbohydrate and pyrrolidine iminosugars is less evident, although these compouds give excellent enzymatic inhibitions and biological activities. The docking studies in the paper predict a different binding mode of piperidine iminosugars and α-1-C-butyl-LAB, a molecule with an L‐configuration, opposite that present in the α-D-glucoside substrates (Figure 1). These structural features can explain the similar inhibitory potency on intestinal α-glucosidases of α‐1‐C‐butyl-LAB and the related piperidine iminosugar α-1-C-butyl-DNJ but the better selectivity of the former over the other glycosidases tested. This illustrates that quite often a high degree of similarity of the inhibitors to the carbohydrate substrate is necessary but not sufficient to attain the enzymatic profile necessary for drug applications. Regarding the antidiabetics directed to intestinal α‐glucosidases, the inhibitors must be selective over other glycosyl enzymes and in particular to intracellular α‐glucosidases. These include endoplasmic reticulum α-glucosidase I and II enzymes, involved in the maturation of protein glycosylation and acid α‐lucosidase which operates in lysosomal carbohydrate degradation. Among marketed drugs, the selectivity for intestinal enzymes has been achieved by two different mechanisms. Acarbose and voglibose are poorly absorbed and remain localized in the digestive system until excreted. This confinement prevents its intracellular effects but originates some gastrointestinal complications due to the extensive inhibition of carbohydrate hydrolysis. In contrast, miglitol is fully absorbed, requiring a potent and selective inhibition of intestinal α‐glucosidases to avoid systemic undesired side effects due to inhibition of other glycosidases. On the basis of

Glucocerebrosidase inhibitors for the treatment of Gaucher disease

Gaucher disease is a progressive lysosomal storage disorder caused by a deficiency in the activity of β-glucocerebrosidase and is characterized by the accumulation of the glycosphingolipid glucosylceramide in the lysosomes of macrophages that leads to dysfunction in multiple organ system. An emerging strategy for the treatment of Gaucher disease is pharmacological chaperone therapy, based on the use of β-glucocerebrosidase inhibitors that are capable of enhancing residual hydrolytic activity at subinhibitory concentrations. In this article, the most common lysosomal storage disorder, Gaucher disease, is introduced and the current therapeutic strategies based on the use of enzyme inhibitors to ameliorate this disease are discussed, with a focus on the efforts being made toward finding and optimizing novel molecules as pharmacological chaperones for Gaucher disease that offer the promise to remedy this malady.

The myo-1,2-Diaminocyclitol Scaffold Defines Potent Glucocerebrosidase Activators and Promising Pharmacological Chaperones for Gaucher Disease.

A series of cyclitol derivatives with myo-configuration are β-glucocerebrosidase (GCase) inhibitors and show excellent characteristics for the development of pharmacological chaperones for enzyme deficiency in Gaucher disease (GD). The most potent inhibitor, (1S,2R,3R,4S,5R,6S)-5,6-bis(nonylamino)cyclohexane-1,2,3,4-tetraol, displayed a K i value of 26 nM in isolated enzyme and also inhibited GCase in wild-type (wt) human fibroblasts at nanomolar concentrations. This diaminocyclitol produced maximum increases of GCase activities of 60% in N370S lymphoblasts at 100 nM and 30% in L444P at 1 nM following a 3-day incubation, showing the permeability, subcellular distribution, and cell metabolism characteristics for use as pharmacological chaperone.

Optical control of endogenous receptors and cellular excitability using targeted covalent photoswitches

Light-regulated drugs allow remotely photoswitching biological activity and enable plausible therapies based on small molecules. However, only freely diffusible photochromic ligands have been shown to work directly in endogenous receptors and methods for covalent attachment depend on genetic manipulation. Here we introduce a chemical strategy to covalently conjugate and photoswitch the activity of endogenous proteins and demonstrate its application to the kainate receptor channel GluK1. The approach is based on photoswitchable ligands containing a short-lived, highly reactive anchoring group that is targeted at the protein of interest by ligand affinity. These targeted covalent photoswitches (TCPs) constitute a new class of light-regulated drugs and act as prosthetic molecules that photocontrol the activity of GluK1-expressing neurons, and restore photoresponses in degenerated retina. The modularity of TCPs enables the application to different ligands and opens the way to new therapeutic opportunities.

Synthesis and evaluation of hydroxymethylaminocyclitols as glycosidase inhibitors

Four series of C7N aminocyclitol analogues of glucose were synthesized by stereocontrolled epoxide opening of hydroxyl protected forms of the cyclohexane epoxides cyclophellitol and 1,6-epi-cyclophellitol. The resulting hydroxymethyl substituted aminocyclitols were tested as glycosidase inhibitors. Cyclitols having an amino group in an α configuration at a position equivalent to the anomeric in the sugar were found to be low micromolar inhibitors of the α-glucosidase from bakers yeast with Kis near to 2 μM. On the other hand, N-octyl aminocyclitols having the nitrogen substituents in an α or β configuration were found to be good inhibitors of recombinant β-glucocerebrosidase with Ki values between 8.3 and 17 μM, and also inhibited lysosomal β-glucosidase activity in live cells at low-micromolar concentrations. A computational docking study suggests a differential binding among the different series of β-glucocerebrosidase inhibitors. In agreement with the experimental results, the binding poses obtained indicate that the presence of an alkyl lipid substituent in the inhibitor mimicking one of the lipid chains in the substrate is critical for potency. In contrast, the matching of hydroxymethyl substituents in the aminocyclitols and the parent glucosylceramide does not seem to be strictly necessary for potent inhibition, indicating the risk of simplifying structural analogies in sugar mimetic design.

Allosteric control of an asymmetric transduction in a G protein-coupled receptor heterodimer

GPCRs play critical roles in cell communication. Although GPCRs can form heteromers, their role in signaling remains elusive. Here we used rat metabotropic glutamate (mGlu) receptors as prototypical dimers to study the functional interaction between each subunit. mGluRs can form both constitutive homo- and heterodimers. Whereas both mGlu2 and mGlu4 couple to G proteins, G protein activation is mediated by mGlu4 heptahelical domain (HD) exclusively in mGlu2-4 heterodimers. Such asymmetric transduction results from the action of both the dimeric extracellular domain, and an allosteric activation by the partially-activated non-functional mGlu2 HD. G proteins activation by mGlu2 HD occurs if either the mGlu2 HD is occupied by a positive allosteric modulator or if mGlu4 HD is inhibited by a negative modulator. These data revealed an oriented asymmetry in mGlu heterodimers that can be controlled with allosteric modulators. They provide new insight on the allosteric interaction between subunits in a GPCR dimer.

Adamantane substituted aminocyclitols as pharmacological chaperones for Gaucher disease

Gaucher disease (GD), resulting from deficient lysosomal enzyme β-glucosidase (GCase) activity, is the most common lysosomal storage disorder. We have previously shown that aminocyclitol derivatives displayed selective inhibition of GCase and enhanced GCase activity in N370S and L444P at very low concentrations. In the present study, we combined amino-myo-inositol and amino-scyllo-inositol cores with a hydrophobic alkyl adamantyl amide to afford novel small molecules with enhanced ability to increase GCase activity in GD lymphoblasts. The most potent inhibitor, amino-myo-inositol 2, displayed a Ki value of 250 nM in isolated enzyme. This compound produced a maximum increase of GCase activity of 64% in N370S lymphoblasts at 1 μM and 150% in L444P at 100 μM following a 3 day incubation.

Exploring the Active Conformation of Cyclohexane Carboxylate Positive Allosteric Modulators of the Type 4 Metabotropic Glutamate Receptor

The active conformation of a family of metabotropic glutamate receptor subtype 4 (mGlu4) positive allosteric modulators (PAMs) with the cyclohexane 1,2‐dicarboxylic scaffold present in cis‐2‐(3,5‐dichlorophenylcarbamoyl)cyclohexanecarboxylic acid (VU0155041) was investigated by testing structurally similar six‐membered ring compounds that have a locked conformation. The norbornane and cyclohexane molecules designed as mGlu4 conformational probes and the enantiomers of the trans diastereomer were computationally characterized and tested in mGlu4 pharmacological assays. The results support a VU0155041 active conformation, with the chair cyclohexane having the aromatic amide substituent in an axial position and the carboxylate in an equatorial position. Moreover, the receptor displays enantiomeric discrimination of the chiral PAMs. The constructed pharmacophore characterized a highly constrained mGlu4 allosteric binding site, thus providing a step forward in structure‐based drug design for mGlu4 PAMs.