Hexosaminidase inhibitors as new drug candidates for the therapy of osteoarthritis.

BACKGROUND: Articular cartilage from patients with osteoarthritis is characterized by a decreased concentration and reduced size of glycosaminoglycans. Degeneration of the cartilage matrix is a multifactorial process, which is due in part to accelerated glycosaminoglycan catabolism. Recently, we have demonstrated that hexosaminidase represents the dominant glycosaminoglycan-degrading glycosidase released by chondrocytes into the extracellular compartment and is the dominant glycosidase in synovial fluid from patients with osteoarthritis. Inhibition of hexosaminidase activity may represent a novel approach to the prevention of cartilage matrix glycosaminoglycan degradation and a potentially new strategy to treat osteoarthritis. RESULTS: We have synthesized and investigated a series of iminocyclitols designed as transition-state analog inhibitors of human hexosaminidase, and demonstrated that the five-membered iminocyclitol 4 expresses the strongest inhibitory activity with K(i)=24 nM. Inhibition of hexosaminidase activity in human cultured articular chondrocytes and human chondrosarcoma cells with iminocyclitol 4 resulted in accumulation of hyaluronic acid and sulfated glycosaminoglycans in the cell-associated fraction. Similarly, incubation of human cartilage tissue with iminocyclitol 4 resulted in an accumulation of glycosaminoglycans in the pericellular compartment. CONCLUSIONS: Inhibition of hexosaminidase activity represents a new strategy for preventing or even reversing cartilage degradation in patients with osteoarthritis.     

Enzyme inhibitors as chemical tools to study enzyme catalysis: rational design, synthesis, and applications

Carefully designed molecules that are intimately related to the reaction mechanism of enzymes are often highly selective and potent inhibitors that serve as extremely useful chemical probes for understanding the reaction mechanism and structure of enzymes. This article describes the design, synthesis, and applications of specific inhibitors of two mechanistically distinct groups of enzymes, ATP-dependent amide ligases and Ser- and Thr-hydrolases. Our strategy is based on the premise that stable analogues of the transition state (transition-state analogues) are highly potent inhibitors that serve as good mechanistic probes, and that a key structure of a good inhibitor of one enzyme is also utilized for the inhibitors of other enzymes that share the same chemistry in their catalyzed reactions, irrespective of the degree of structural similarity and evolutionary link between the enzymes. According to these principles, we designed and synthesized a series of phosphinate- and sulfoximine-based transition-state analogue inhibitors of glutathione synthetase, gamma-glutamylcysteine synthetase and asparagine synthetase. For the second group of enzymes, we synthesized a gamma-monofluorophosphono glutamate analogue for mechanism-based affinity labeling of gamma-glutamyltranspeptidase and fluorescent phosphonic acid esters for the active-site titration of lipase. These inhibitors were used successfully as ligands for detailed kinetic analyses, X-ray crystallography, and mass analysis of the enzymes to identify the key amino acid residues responsible for catalysis and substrate recognition in the transition state.     

Substrate-assisted antibody catalysis

A new strategy in transition-state analog design is demonstrated to elicit catalytic antibodies. The strategy is based on substrate-assisted antibody catalysis and utilizes analogs designed to mimic the transition-state for intramolecular catalysis and thereby favor antibodies that can recruit catalytic groups from substrate. The hydrolysis of the benzoyl ester of cocaine provides an illustration. The benzoyl ester of cocaine is distant from the protonated nitrogen in the stable chair conformer but proximate in the strained boat form. An antibody stabilizing the boat form and approximating ester and amine could catalyze ester hydrolysis. To mimic the transition-state for the intramolecular catalysis, we synthesized a cocaine analog that replaces this ester with a methylenephenylphosphinate bridge to the tropane nitrogen. This bridged analog elicited 85 cocaine esterases out of 450 anti-analog antibodies-a performance markedly superior to that of a simple phosphonate ester-based analog with an identical tether. The correspondence of the analog to a "high energy" conformer eliminated product inhibition. For certain polyfunctional targets, substrate assistance can be an effective strategy for eliciting catalytic antibodies.     

Synthesis of a carbohydrate-derived hydroxamic acid inhibitor of the bacterial enzyme (LpxC) involved in lipid A biosynthesis

[reaction: see text] The enzyme LpxC (UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc deacetylase) catalyzes the second step of lipid A biosynthesis and is essential for bacterial growth. A GlcNAc-derived hydroxamic acid inhibitor 8 of this enzyme was synthesized using two different routes. Compound 8 exhibits activity toward LpxC enzymes from a wider spectrum of bacterial species than any of the previously reported hydroxamic acid inhibitors.     

Toward a fragment-based approach to MMPs inhibitors: an expedite and efficient synthesis of N-hydroxylactams

Matrix metalloproteinases (MMPs), a class of zinc-enzymes over-activated in many pathologies, such as arthritis and cancer, can be efficiently inhibited by a variety of molecules bearing zinc-binding groups (ZBGs). The hydroxamic acid moiety represents one of the most potent and widely exploited ZBG but the poor target selectivity and in vivo toxicity have tempered the initial enthusiasm for this class of potential therapeutics. These drawbacks might be circumvented, at least in part, by increasing the structural constraints around the hydroxamic moiety. Following this strategy we designed and prepared N-hydroxylactam molecules of different size through a synthetic protocol based on a ring closing metathesis amenable to a fragment-based approach potentially leading to a large molecular diversity.     

A Convenient Approach to Stereoisomeric Iminocyclitols: Generation of Potent Brain‐Permeable OGA Inhibitors

Pyrrolidine‐based iminocyclitols are a promising class of glycosidase inhibitors. Reported herein is a convenient epimerization strategy that provides direct access to a range of stereoisomeric iminocyclitol inhibitors of O‐GlcNAcase (OGA), the enzyme responsible for catalyzing removal of O‐GlcNAc from nucleocytoplasmic proteins. Structural details regarding the binding of these inhibitors to a bacterial homologue of OGA reveal the basis for potency. These compounds are orally available and permeate into rodent brain to increase O‐GlcNAc, and should prove useful tools for studying the role of OGA in health and disease.     

Synthesis of human renin inhibitory peptides, angiotensinogen transition-state analogs containing a retro-inverso amide bond

The experimental details for the synthesis of human renin inhibitors are described. In order to avoid metabolic degradation of the Phe-His (P3-P2) amide bond in transition-state analogs, structurally modified acyl residues (P4-P3) were incorporated into the inhibitors. Compound 1a, which contained 2-(1-naphthylmethyl)-3-(N-phenethylcarbamoyl)propionyl residue (P4-P3) with a retro-inverso amide bond, L-histidine, and norstatine isoamylamide residue (P1-P1) as a transition-state mimic, had potent human renin inhibitory activity, and it lowered blood pressure when administered orally to common marmosets.     

KNI-102, a novel tripeptide HIV protease inhibitor containing allophenylnorstatine as a transition-state mimic.

HIV-1 protease inhibitors containing allophenylnorstatine [Apns; (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid]-Pro (syn diastereomer) as a transition-state mimic were established to be potent and highly selective. Z-Asn-Apns-Pro-NHBut (KNI-102) is the only tripeptide exhibiting substantial anti-HIV activity and may be of minimum size for potent, selective inhibition of HIV protease. Ready availability due to its simple chemical structure and stability should make it valuable for studies of the development of metabolically stable anti-AIDS drugs.     

Kynostatin (KNI)-227 and -272, highly potent anti-HIV agents: conformationally constrained tripeptide inhibitors of HIV protease containing allophenylnorstatine.

Selective and potent HIV protease inhibitors containing allophenylnorstatine [Apns; (2S, 3S)-3-amino-2-hydroxy-4-phenylbutyric acid] as a transition-state mimic were designed and synthesized. Among them, conformationally constrained tripeptide derivatives, kynostatin (KNI)-227 and -272 (Fig. 1), exhibited highly potent antiviral activities against a wide spectrum of HIV isolates. Ready availability due to the simple synthetic procedure and the excellent antiviral properties indicate that KNI-227 and KNI-272 are promising candidates as selective anti-AIDS drugs.     

Synthesis and evaluation of 1-deoxy-D-xylulose 5-phosphate analogues as chelation-based inhibitors of methylerythritol phosphate synthase

[structures: see text] A series of 1-deoxy-D-xylulose 5-phosphate (DXP) analogues were synthesized and evaluated as inhibitors of E. coli methylerythritol phosphate (MEP) synthase. In analogues 1-4, the methyl group in DXP was replaced by hydroxyl, hydroxylamino, methoxy, and amino moieties, respectively. In analogues 5 and 6, the acetyl moiety in DXP was replaced by hydroxymethyl and aminomethyl groups. These compounds were designed to coordinate to the active site divalent metal in MEP synthase. The carboxylate (1), methyl ester (3), amide (4), and alcohol (5) analogues were inhibitors with IC50's ranging from 0.25 to 1.0 mM. The hydroxamic acid (2) and amino (6) analogues did not inhibit the enzyme.     

Detoxification of VX and Other V‐Type Nerve Agents in Water at 37 °C and pH 7.4 by Substituted Sulfonatocalix[4]arenes

Sulfonatocalix[4]arenes with an appended hydroxamic acid residue can detoxify VX and related V‐type neurotoxic organophosphonates with half‐lives down to 3 min in aqueous buffer at 37 °C and pH 7.4. The detoxification activity is attributed to the millimolar affinity of the calixarene moiety for the positively charged organophosphonates in combination with the correct arrangement of the hydroxamic acid group. The reaction involves phosphonylation of the hydroxamic acid followed by a Lossen rearrangement, thus rendering the mode of action stoichiometric rather than catalytic. Nevertheless, these calixarenes are currently the most efficient low‐molecular‐weight compounds for detoxifying persistent V‐type nerve agents under mild conditions. They thus represent lead structures for novel antidotes that allow treatment of poisonings by these highly toxic chemicals.     

Preparation of sugar derived β,β′-dihydroxy α,α-disubstituted α-amino acids

A novel strategy for the preparation of β,β′-dihydroxy α,α-disubstituted α-amino acids bearing a sugar moiety has been developed. The procedure is based on two Henry reactions: the first Henry reaction involves a sugar aldehyde and nitroethanol to furnish a sugar derived α-hydroxymethyl α-nitroalkanol while the second Henry reaction is between this nitro sugar and formaldehyde. This sequence provided the expected epimers of sugar derived α,α-dihydroxymethyl α -nitroalkanols, from which the corresponding β,β′-dibenzyloxy α-N-benzyloxycarbonylamino esters were easily obtained. All rights reserved.     

Acidity of hydroxamic acids and amides

The relatively strong acidity of hydroxamic acids was analyzed by means of isodesmic reactions in which this acid or its anion is formed from simpler precursors. Acidity of amides was analyzed in the same way. Energies of all compounds involved in the reactions were calculated at the B3LYP/AUG-cc-pVTZ//B3LYP/6-311 + G(d,p) level; at this level a good agreement was reached with the sparse experimental data. Interpretation of the results was the same as in the recent discussion of the acidity of carboxylic acids, and the conclusions were similar: both amides and hydroxamic acids are stabilized with respect to simpler reference molecules of amines or N-alkylhydroxylamines, respectively. However, their anions are stabilized still more and are responsible for the acidity. This effect is stronger in hydroxamic acids or amides than in carboxylic acids. The problem of whether it is due to resonance depends on the definition of this term. Semiquantitative comparison suggests that resonance in hydroxamic acids is more important than in amides and still more than in carboxylic acids. The stronger acidity of hydroxamic acids compared to amides is due to the destabilizing inductive effect of the hydroxyl group in the acid molecule, not to any effect in the anion.     

Direct observation of the protonation state of an imino sugar glycosidase inhibitor upon binding

Glycosidases are some of the most ubiquitous enzyme in nature. Their biological significance, coupled to their enormous catalytic prowess derived from tight binding of the transition state, is reflected in their importance as therapeutic targets. Many glycosidase inhibitors are known. Imino sugars are often potent inhibitors, yet many facets of their mode of action, such as their degree, if any, of transition-state "mimicry" and their protonation state when bound to the target glycosidase remain unclear. Atomic resolution analysis of the endoglucanase, Cel5A, in complex with a cellobio-derived isofagomine in conjunction with the pH dependence of Ki and kcat/KM reveals that this compound binds as a protonated sugar. Surprisingly, both the enzymatic nucleophile and the acid/base are unprotonated in the complex.     

Substrate structural effects in micellar catalysis with perfluorooctanoic acid

Rates of hydrolysis of a series of hydroxamic acids in aqueous acetonitrile, with perfluorooctanoic acid serving as a reactive counterion surfactant, have been determined. The pseudophase ion exchange model satisfactorily explains the surfactant effects. Variation in the structure of the hydrophobic moiety of the hydroxamic acid substrates was obtained by the incorporation of different chain lengths and differently substituted aryl groups into the substrate. The major influence of the change in substrate structure is on the binding constant for binding of the substrate to the surfactant aggregate. The number of substitutions on the aryl ring appears to be more important than the total number of carbon atoms in the substrate. © John Wiley & Sons, Inc.     

Polymers containing hydroxamate groups: nanoreactors for hydrolysis of phosphoryl esters

A polyhydroxamicalkanoate (PHA) polymer containing the functional groups hydroxamic acid and carboxylic acid with the ability to accelerate dephosphorylation reactions is proposed. The methodology used to prepare this polymer favored the position of the two functional groups next to each other, which allows for the cooperativity between these groups. This cooperative effect has an important role when one wants to mimic enzymes. The catalytic effect promoted by the polymer was evaluated on the cleavage of the bis(2,4-dinitrophenyl) phosphate (BDNPP) and diethyl 2,4-dinitrophenyl phosphate (DEDNPP) esters. Indeed, PHA was very efficient and promiscuous because it increased the rate of both reactions by a factor of up to 10(6)-fold. Isothermal titration calorimetry (ITC) experiments showed that the presence of PHA aids the formation of cetyltrimethylammonium bromide (CTABr) micelles. Thus, the effect of the cationic surfactant CTABr on the dephosphorylation of BDNPP by PHA was also investigated, and it was observed that, when CTABr is added to PHA, the reaction is ca. 15-fold faster compared to the reaction when only PHA is present.     

Synthesis of Nicotinamide Adenine Dinucleotide (NAD) Analogues with a Sugar Modified Nicotinamide Moiety

The synthesis of nicotinamide adenine dinucleotide (NAD) analogues in which the ribose unit of the nicotinamide moiety is replaced by a hexitol, altritol, and cyclohexenyl sugar mimic is described.     

Design and evaluation of dihydroxytetrahydro-1H-pyrrolo[2,1-c]- [1,4]benzothiazines as conformationally restricted transition-state inhibitors of beta-ribosidases

[structure: see text] The preparation of three new chiral thiazines from ribose is described. Two of these are dihydroxytetrahydro-1H-pyrrolo[2,1-c][1,4]benzothiazines with iminopentitol substructures corresponding to the L-lyxo and D-ribo configurations. They were designed to present a favorable transition-state mimic for the inhibition of ribosidases. This new thiazine class opens the way to the development of new inhibitors to carbohydrate processing enzymes of therapeutic importance such as nucleoside hydrolases and purine nucleoside phosphorylases.     

Zinc chelation with hydroxamate in histone deacetylases modulated by water access to the linker binding channel

It is of significant biological interest and medical importance to develop class- and isoform-selective histone deacetylase (HDAC) modulators. The impact of the linker component on HDAC inhibition specificity has been revealed but is not understood. Using Born-Oppenheimer ab initio QM/MM MD simulations, a state-of-the-art approach to simulating metallo-enzymes, we have found that the hydroxamic acid remains to be protonated upon its binding to HDAC8, and thus disproved the mechanistic hypothesis that the distinct zinc-hydroxamate chelation modes between two HDAC subclasses come from different protonation states of the hydroxamic acid. Instead, our simulations suggest a novel mechanism in which the chelation mode of hydroxamate with the zinc ion in HDACs is modulated by water access to the linker binding channel. This new insight into the interplay between the linker binding and the zinc chelation emphasizes its importance and gives guidance regarding linker design for the development of new class-IIa-specific HDAC inhibitors.     

Synthesis and evaluation of potential inhibitors of chondroitin AC lyase from Flavobacterium heparinum

Chondroitin AC lyase from Flavobacterium heparinum degrades chondroitin sulfate glycosaminoglycans via an elimination mechanism, resulting in disaccharides or oligosaccharides with Delta4,5-unsaturated uronic acid residues at their nonreducing end. The syntheses and testing of two potential inhibitors of this lyase are described. Methyl O-(2-acetamido-2-deoxy-beta-D-galactopyranosyl)-(1-->4)-alpha-L-threo-hex-4-enopyranoside, 1, has the trigonal geometry at C5 of the uronic acid moiety expected at the transition state, yet retains the "leaving group" sugar moiety. Surprisingly, compound 1 showed no inhibition of the enzyme. The novel 5-nitro sugar, phenyl (5S)-5-nitro-beta-D-xylopyranoside, 2, is a monosaccharide nitro analogue of the natural substrate, with C5 being a carbon acid of pK(a) 8.8. The rate of reprotonation of the anion generated at this center is sufficiently low that the anion of 2 can be used directly in initial steady-state velocity measurements without significant interference from the conjugate carbon acid. The anion of compound 2 was found to be a competitive inhibitor with a K(i) value of 5 mM, whereas the conjugate acid had a K(i) value of 35 mM.