Transition-state Mimetics Research Articles

Catalytic Cycle of Glycoside Hydrolase BglX from Pseudomonas aeruginosa and Its Implications for Biofilm Formation.

BglX is a heretofore uncharacterized periplasmic glycoside hydrolase (GH) of the human pathogen Pseudomonas aeruginosa. X-ray analysis identifies it as a protein homodimer. The two active sites of the homodimer comprise catalytic residues provided by each monomer. This arrangement is seen in <2% of the hydrolases of known structure. In vitro substrate profiling shows BglX is a catalyst for β-(1→2) and β-(1→3) saccharide hydrolysis. Saccharides with β-(1→4) or β-(1→6) bonds, and the β-(1→4) muropeptides from the cell-wall peptidoglycan, are not substrates. Additional structural insights from X-ray analysis (including structures of a mutant enzyme-derived Michaelis complex, two transition-state mimetics, and two enzyme-product complexes) enabled the comprehensive description of BglX catalysis. The half-chair (4H3) conformation of the transition-state oxocarbenium species, the approach of the hydrolytic water molecule to the oxocarbenium species, and the stepwise release of the two reaction products were also visualized. The substrate pattern for BglX aligns with the [β-(1→2)-Glc]x and [β-(1→3)-Glc]x periplasmic osmoregulated periplasmic glucans, and possibly with the Psl exopolysaccharides, of P. aeruginosa. Both polysaccharides are implicated in biofilm formation. Accordingly, we show that inactivation of the bglX gene of P. aeruginosa PAO1 attenuates biofilm formation.

The effect of N-methylation on transition state mimetic inhibitors of the Plasmodium protease, plasmepsin V

An N-methylation strategy has been applied to transition state mimetics that are potent inhibitors of plasmepsin V to improve their physical characteristics and their ability to reduce the viability of Plasmodium parasites in culture.

Transition state mimetics of the Plasmodium export element are potent inhibitors of Plasmepsin V from P. falciparum and P. vivax.

Following erythrocyte invasion, malaria parasites export a catalogue of remodeling proteins into the infected cell that enable parasite development in the human host. Export is dependent on the activity of the aspartyl protease, plasmepsin V (PMV), which cleaves proteins within the Plasmodium export element (PEXEL; RxL↓xE/Q/D) in the parasite's endoplasmic reticulum. Here, we generated transition state mimetics of the native PEXEL substrate that potently inhibit PMV isolated from Plasmodium falciparum and Plasmodium vivax. Through optimization, we identified that the activity of the mimetics was completely dependent on the presence of P1 Leu and P3 Arg. Treatment of P. falciparum-infected erythrocytes with a set of optimized mimetics impaired PEXEL processing and killed the parasites. The striking effect of the compounds provides a clearer understanding of the accessibility of the PMV active site and reaffirms the enzyme as an attractive target for the design of future antimalarials.

Polyoxin and nikkomycin analogs: recent design and synthesis of novel peptidyl nucleosides

Abstract Polyoxins and nikkomycins are a class of naturally occurring peptidyl nucleoside antibiotics that show promise as potential antifungal agents due to their potent ability to inhibit chitin synthase, an enzyme responsible for fungal cell wall biosynthesis. Whole cell assays and in vivo studies have shown that these natural products have poor cellular uptake and are metabolically unstable, and there has been a concerted effort to improve their pharmokinetic properties by synthesizing analogs. These have either been designed as natural substrate analogs, transition state mimetics or mechanistic inhibitors. Recent synthetic efforts and the results of their biological studies are briefly described in this review, and the current trends in the design and construction of polyoxin and nikkomycin analogs are discussed.

Second generation of BACE-1 inhibitors part 3: Towards non hydroxyethylamine transition state mimetics

Our first generation of hydroxyethylamine BACE-1 inhibitors proved unlikely to provide molecules that would lower amyloid in an animal model at low oral doses. This observation led us to the discovery of a second generation of inhibitors having nanomolar activity in a cell-based assay and with the potential for improved pharmacokinetic profiles. In this Letter, we describe our successful strategy for the optimization of oral bioavailability and also give insights into the design of compounds with the potential for improved brain penetration.

3,4-Dihydroxypyrrolidine as Glycosidase Inhibitor

Glycosidases are involved in various important biological processes including digestion of starch in the intestine, oligosaccharide processing inside rough ER and Golgi apparatus, and degradation of glycoconjugates in lysosomes. It is apparent that inhibitors of this class of enzymes are useful in the investigation of biological functions of glycoconjugates. Furthermore, it is believed that these compounds are important as pharmaceuticals. The structures of glycosidase inhibitors can be categorized into two major classes; ground-state mimetics and transition-state mimetics. The former has a chair-shaped six-membered structure that mimics monosaccharides where ring oxygens are often replaced with other elements for an improved binding affinity. On the other hand, the latter possesses a somewhat distorted shape compared with the chair conformation of carbohydrates. One of the ways to derive such distortion is by the introduction of sp2 character into the six-membered ring, and another is by ring contraction to form a five-membered system for the transition-state mimetics. The functions of these transition-state mimetics are often unpredictable regarding inhibitory activity and enzyme specificity. This review focuses on such "difficult to predict" species in an attempt to extract information or common aspects for the future development of inhibitors of glycosidases based on transition-state mimetics.

A theoretical study of anthrax lethal factor inhibition by a set of novel carbamimidolyl-aryl-vinyl-carboxamidines: A possible mechanism involving zinc-ligation by amidine

A congeneric set of carbamimidolyl-aryl-vinyl-carboxamidines from the National Cancer Institute (NCI) open chemical repository were identified as potent inhibitors of anthrax lethal factor (LF), a zinc-dependent metalloprotease that plays a critical role in potentiating Bacillus anthracis infection. Surprisingly, these compounds exhibited no differential change in activity with concentration. Docking studies revealed that the indole-attached amidine substituents of these inhibitors were positioned in close proximity to the biological zinc atom and could potentially function as transition-state mimetics. This broaches the stunning possibility that the dose independence of these inhibitors is linked to zinc-ligation. Because the amidine functionality is highly basic and cationic, it is generally not considered a viable zinc-binding motif. However, quantum chemical calculations on small-molecule models predicted a marked decrease in the p K a of the amidine functionality when it is in close proximity to zinc, thus allowing for the formation of a robust zinc–amidine bond.

Computational inhibitor design against malaria plasmepsins.

Plasmepsins are aspartic proteases involved in the degradation of the host cell hemoglobin that is used as a food source by the malaria parasite. Plasmepsins are highly promising as drug targets, especially when combined with the inhibition of falcipains that are also involved in hemoglobin catabolism. In this review, we discuss the mechanism of plasmepsins I-IV in view of the interest in transition state mimetics as potential compounds for lead development. Inhibitor development against plasmepsin II as well as relevant crystal structures are summarized in order to give an overview of the field. Application of computational techniques, especially binding affinity prediction by the linear interaction energy method, in the development of malarial plasmepsin inhibitors has been highly successful and is discussed in detail. Homology modeling and molecular docking have been useful in the current inhibitor design project, and the combination of such methods with binding free energy calculations is analyzed.

Potent, small-molecule inhibitors of human mast cell tryptase. Antiasthmatic action of a dipeptide-based transition-state analogue containing a benzothiazole ketone.

Inhibitors of human mast cell tryptase (EC 3.4.21.59) have therapeutic potential for treating allergic or inflammatory disorders. We have investigated transition-state mimetics possessing a heterocycle-activated ketone group and identified in particular benzothiazole ketone (2S)-6 (RWJ-56423) as a potent, reversible, low-molecular-weight tryptase inhibitor with a K(i) value of 10 nM. A single-crystal X-ray analysis of the sulfate salt of (2S)-6 confirmed the stereochemistry. Analogues 12 and 15-17 are also potent tryptase inhibitors. Although RWJ-56423 potently inhibits trypsin (K(i) = 8.1 nM), it is selective vs other serine proteases, such as kallikrein, plasmin, and thrombin. We obtained an X-ray structure of (2S)-6 complexed with bovine trypsin (1.9-A resolution), which depicts inter alia a hemiketal involving Ser-189, and hydrogen bonds with His-57 and Gln-192. Aerosol administration of 6 (2R,2S; RWJ-58643) to allergic sheep effectively antagonized antigen-induced asthmatic responses, with 70-75% blockade of the early response and complete ablation of the late response and airway hyperresponsiveness.

Transition-State Mimetics for HIV Protease Inhibitors: Stereocontrolled Synthesis of Hydroxyethylene and Hydroxyethylamine Isosteres by Ester-Derived Titanium Enolate Syn and Anti-Aldol Reactions.

Stereocontrolled syntheses of hydroxyethylene dipeptide isostere and aminoalkyl epoxides for hydroxyethylamine isosteres are described. The stereochemistry of both stereogenic centers of the aminoalkyl epoxides 10 and 15 as well as the gamma-lactone 17 was assembled by our recently developed highly selective ester-derived titanium enolate aldol reactions. The Ti-enolate of 6 reacted with (benzyloxy)acetaldehyde and cinnamaldehyde to provide the syn-aldol product 7 and anti-aldol product 12, respectively. Removal of the chiral template followed by Curtius rearrangement of the resulting acid provided the desired amine functionality. The present syntheses represent practical and enantioselective entry to a range of other dipeptide isosteres, which are not limited to amino acid derived substituents.

Non-peptide renin inhibitors containing 2-(((3-phenylpropyl)phosphoryl)oxy)alkanoic acid moieties as P2-P3 replacements.

A series of novel renin inhibitors containing 2-(((3-phenylpropyl)phosphoryl)oxy)alkanoic acid moieties as P2-P3 surrogates are presented. The P2-P3 mimetics were obtained from (omega-phenylalkyl)-phosphinic acids 1a-c and 2-hydroxyalkanoic acid benzyl esters 2a-f by N,N'-dicyclohexylcarbodiimide-mediated coupling and subsequent oxidation with sodium metaperjodate. Ester cleavage of these derivatives and coupling with P1-P1' transition-state mimetics I-VII provided highly selective compounds with inhibitory potencies in the lower nanomolar range. Small renin inhibitors, such as analogues 8c and 8h with molecular weights of 539 and 537, respectively, could be prepared. These compounds exhibited IC50 values of about 20 nM against human plasma renin. Compound 7i was examined in vivo for its hypotensive effect. In salt-depleted cynomolgus monkeys, 7i inhibited plasma renin activity almost completely and lowered blood pressure after oral administration of a dose of 30 mg/kg.

1,2,4-Triazolo[4,3-a]pyrazine derivatives with human renin inhibitory activity. 3. Synthesis and biological properties of aminodeoxystatine and difluorostatone derivatives

Two series of 1,2,4-triazolo[4,3-a]pyrazine derivatives with human renin inhibitory activity have been synthesized which incorporate the transition-state mimetics (3S,4S)- and (3R,4S)-5-cyclohexyl-3,4-diaminopentanoic acid ((S)- and (R)-CDAPA), and (4S)-4-amino-5-cyclohexyl-2,2-difluoro-3-oxopentanoic acid (ACDFOPA). Several compounds in these series, for example 13a, 19c, and 19f, were highly potent inhibitors of partially purified human renin (IC50 values of 3.9, 1.6, and 1.4 nM, respectively). The ACDFOPA-based compounds 19c and 19f contain no natural amino acid fragments and have molecular weights which compare well with those of previously reported inhibitors of nanomolar in vitro potency. When administered intravenously to anesthetized, sodium-depleted marmosets at doses of 3 mg/kg, compounds 13a and 19c caused a marked reduction in mean arterial pressure, but in the same animal model at 30 mg/kg, oral activity was not seen.

1,2,4-Triazolo[4,3-a]pyrazine derivatives with human renin inhibitory activity. 1. Synthesis and biological properties of alkyl alcohol and statine derivatives

A series of 1,2,4-triazolo[4,3-a]pyrazine derivatives with human renin inhibitory activity, which incorporate (1S,2S)-2-amino-1,3-dicyclohexyl-1-hydroxypropane, statine (Sta), and (3S,4S)-4-amino-5-cyclohexyl-3-hydroxy-pentanoic acid (ACHPA) transition-state mimetics, have been prepared. Structure-activity relationships for renin inhibitory activity in the series are consistent with the 2-[8-isobutyl-6-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3-yl]-3-(3 pyridyl)propionic acid moiety 10b acting as a non-peptidic replacement for the P4-P2 (Pro-Phe-His) residues of the natural substrate angiotensinogen. Compounds 12m, 12o and 12q were potent inhibitors of partially purified human renin (IC50 values 1.7, 6.8, and 3.7 nM, respectively), and also effectively lowered blood pressure in anesthetized, sodium depleted marmosets following intravenous administration. On oral administration however, no blood pressure lowering activity could be detected, and absorption studies in bile duct cannulated rats indicate that this may be due primarily to poor oral absorption, rather than rapid biliary excretion. The reason for the observed poor oral activity is not clear, but it seems unlikely that poor aqueous solubility or metabolic instability to gut enzymes are rate-determining, and other factors such as high molecular weight may also be very important.