Po-Huang Liang

Bisphosphonates target multiple sites in both cis- and trans-prenyltransferases

Bisphosphonate drugs (e.g., Fosamax and Zometa) are thought to act primarily by inhibiting farnesyl diphosphate synthase (FPPS), resulting in decreased prenylation of small GTPases. Here, we show that some bisphosphonates can also inhibit geranylgeranyl diphosphate synthase (GGPPS), as well as undecaprenyl diphosphate synthase (UPPS), a cis-prenyltransferase of interest as a target for antibacterial therapy. Our results on GGPPS (10 structures) show that there are three bisphosphonate-binding sites, consisting of FPP or isopentenyl diphosphate substrate-binding sites together with a GGPP product- or inhibitor-binding site. In UPPS, there are a total of four binding sites (in five structures). These results are of general interest because they provide the first structures of GGPPS- and UPPS-inhibitor complexes, potentially important drug targets, in addition to revealing a remarkably broad spectrum of binding modes not seen in FPPS inhibition.

Structural and functional analysis of three β-glucosidases from bacterium Clostridium cellulovorans, fungus Trichoderma reesei and termite Neotermes koshunensis

β-glucosidases (EC 3.2.1.21) cleave β-glucosidic linkages in disaccharide or glucose-substituted molecules and play important roles in fundamental biological processes. β-Glucosidases have been widely used in agricultural, biotechnological, industrial and medical applications. In this study, a high yield expression (70-250 mg/l) in Escherichia coli of the three functional β-glucosidase genes was obtained from the bacterium Clostridium cellulovorans (CcBglA), the fungus Trichoderma reesei (TrBgl2), and the termite Neotermes koshunensis (NkBgl) with the crystal structures of CcBglA, TrBgl2 and NkBgl, determined at 1.9Å, 1.63Å and 1.34Å resolution, respectively. The overall structures of these enzymes are similar to those belonging to the β-retaining glycosyl hydrolase family 1, which have a classical (α/β)(8)-TIM barrel fold. Each contains a slot-like active site cleft and a more variable outer opening, related to its function in processing different lengths of β-1,4-linked glucose derivatives. The two essential glutamate residues for hydrolysis are spatially conserved in the active site. In both TrBgl2 and NkBgl structures, a Tris molecule was found to bind at the active site, explaining the slight inhibition of hydrolase activity observed in Tris buffer. Manganese ions at 10mM exerted an approximate 2-fold enzyme activity enhancement of all three β-glucosidases, with CcBglA catalyzing the most efficiently in hydrolysis reaction and tolerating Tris as well as some metal inhibition. In summary, our results for the structural and functional properties of these three β-glucosidases from various biological sources open important avenues of exploration for further practical applications.

Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate

Abstract SARS main protease is essential for life cycle of SARS coronavirus and may be a key target for developing anti-SARS drugs. Recently, the enzyme expressed in Escherichia coli was characterized using a HPLC assay to monitor the formation of products from 11 peptide substrates covering the cleavage sites found in the SARS viral genome. This protease easily dissociated into inactive monomer and the deduced K d of the dimer was 100μM. In order to detect enzyme activity, the assay needed to be performed at micromolar enzyme concentration. This makes finding the tight inhibitor (nanomolar range IC50) impossible. In this study, we prepared a peptide with fluorescence quenching pair (Dabcyl and Edans) at both ends of a peptide substrate and used this fluorogenic peptide substrate to characterize SARS main protease and screen inhibitors. The fluorogenic peptide gave extremely sensitive signal upon cleavage catalyzed by the protease. Using this substrate, the protease exhibits a significantly higher activity (k cat=1.9s−1 and K m =17 μ M) compared to the previously reported parameters. Under our assay condition, the enzyme stays as an active dimer without dissociating into monomer and reveals a small K d value (15nM). This enzyme in conjunction with fluorogenic peptide substrate provides us a suitable tool for identifying potent inhibitors of SARS protease.

Crystal Structure of Type-III Geranylgeranyl Pyrophosphate Synthase from Saccharomyces cerevisiae and the Mechanism of Product Chain Length Determination

Geranylgeranyl pyrophosphate synthase (GGPPs) catalyzes a condensation reaction of farnesyl pyrophosphate with isopentenyl pyrophosphate to generate C20 geranylgeranyl pyrophosphate, which is a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaeal ether-linked lipid. For short-chain trans-prenyltransferases that synthesize C10-C25 products, bulky amino acid residues generally occupy the fourth or fifth position upstream from the first DDXXD motif to block further elongation of the final products. However, the short-chain type-III GGPPs in eukaryotes lack any large amino acid at these positions. In this study, the first structure of type-III GGPPs from Saccharomyces cerevisiae has been determined to 1.98 Å resolution. The structure is composed entirely of 15 α-helices joined by connecting loops and is arranged with α-helices around a large central cavity. Distinct from other known structures of trans-prenyltransferases, the N-terminal 17 amino acids (9-amino acid helix A and the following loop) of this GGPPs protrude from the helix core into the other subunit and contribute to the tight dimer formation. Deletion of the first 9 or 17 amino acids caused the dissociation of dimer into monomer, and the Δ(1-17) mutant showed abolished enzyme activity. In each subunit, an elongated hydrophobic crevice surrounded by D, F, G, H, and I α-helices contains two DDXXD motifs at the top for substrate binding with one Mg2+ coordinated by Asp75, Asp79, and four water molecules. It is sealed at the bottom with three large residues of Tyr107, Phe108, and His139. Compared with the major product C30 synthesized by mutant H139A, the products generated by mutant Y107A and F108A are predominantly C40 and C30, respectively, suggesting the most important role of Tyr107 in determining the product chain length.

Stable Benzotriazole Esters as Mechanism-Based Inactivators of the Severe Acute Respiratory Syndrome 3CL Protease

Summary Severe acute respiratory syndrome (SARS) is caused by a newly emerged coronavirus that infected more than 8000 individuals and resulted in more than 800 fatalities in 2003. Currently, there is no effective treatment for this epidemic. SARS-3CLpro has been shown to be essential for replication and is thus a target for drug discovery. Here, a class of stable benzotriazole esters was reported as mechanism-based inactivators of 3CLpro, and the most potent inactivator exhibited a k inact of 0.0011 s−1 and a K i of 7.5 nM. Mechanistic investigation with kinetic and mass spectrometry analyses indicates that the active site Cys145 is acylated, and that no irreversible inactivation was observed with the use of the C145A mutant. In addition, a noncovalent, competitive inhibition became apparent by using benzotriazole ester surrogates in which the bridged ester-oxygen group is replaced with carbon.

Design, synthesis, and evaluation of 3C protease inhibitors as anti-enterovirus 71 agents

Abstract Human enterovirus (EV) belongs to the picornavirus family, which consists of over 200 medically relevant viruses. A peptidomimetic inhibitor AG7088 was developed to inhibit the 3C protease of rhinovirus (a member of the family), a chymotrypsin-like protease required for viral replication, by forming a covalent bond with the active site Cys residue. In this study, we have prepared the recombinant 3C protease from EV71 (TW/2231/98), a particular strain which causes severe outbreaks in Asia, and developed inhibitors against the protease and the viral replication. For inhibitor design, the P3 group of AG7088, which is not interacting with the rhinovirus protease, was replaced with a series of cinnamoyl derivatives directly linked to P2 group through an amide bond to simplify the synthesis. While the replacement caused decreased potency, the activity can be largely improved by substituting the α,β-unsaturated ester with an aldehyde at the P1′ position. The best inhibitor 10b showed EC50 of 18nM without apparent toxicity (CC50 >25μM). Our study provides potent inhibitors of the EV71 3C protease as anti-EV71 agents and facilitates the combinatorial synthesis of derivatives for further improving the inhibitory activity.

Structure of a heterotetrameric geranyl pyrophosphate synthase from mint (Mentha piperita) reveals intersubunit regulation

This work presents the crystal structure of mint heteromeric prenyltransferase, which is responsible for the biosynthesis of geranyl pyrophosphate, a precursor of the monoterpene menthol. By combining biochemical and genetic complementation approaches, the authors show that the molecular mechanism regulating specific product formation is mediated by protein–protein interactions. Terpenes (isoprenoids), derived from isoprenyl pyrophosphates, are versatile natural compounds that act as metabolism mediators, plant volatiles, and ecological communicators. Divergent evolution of homomeric prenyltransferases (PTSs) has allowed PTSs to optimize their active-site pockets to achieve catalytic fidelity and diversity. Little is known about heteromeric PTSs, particularly the mechanisms regulating formation of specific products. Here, we report the crystal structure of the (LSU · SSU)2-type (LSU/SSU = large/small subunit) heterotetrameric geranyl pyrophosphate synthase (GPPS) from mint (Mentha piperita). The LSU and SSU of mint GPPS are responsible for catalysis and regulation, respectively, and this SSU lacks the essential catalytic amino acid residues found in LSU and other PTSs. Whereas no activity was detected for individually expressed LSU or SSU, the intact (LSU · SSU)2 tetramer produced not only C10-GPP at the beginning of the reaction but also C20-GGPP (geranylgeranyl pyrophosphate) at longer reaction times. The activity for synthesizing C10-GPP and C20-GGPP, but not C15-farnesyl pyrophosphate, reflects a conserved active-site structure of the LSU and the closely related mustard (Sinapis alba) homodimeric GGPPS. Furthermore, using a genetic complementation system, we showed that no C20-GGPP is produced by the mint GPPS in vivo. Presumably through protein–protein interactions, the SSU remodels the active-site cavity of LSU for synthesizing C10-GPP, the precursor of volatile C10-monoterpenes.

Structural Basis of Inhibition Specificities of 3C and 3C-like Proteases by Zinc-coordinating and Peptidomimetic Compounds

Human coxsackievirus (CV) belongs to the picornavirus family, which consists of over 200 medically relevant viruses. In picornavirus, a chymotrypsin-like protease (3Cpro) is required for viral replication by processing the polyproteins, and thus it is regarded as an antiviral drug target. A 3C-like protease (3CLpro) also exists in human coronaviruses (CoV) such as 229E and the one causing severe acute respiratory syndrome (SARS). To combat SARS, we previously had developed peptidomimetic and zinc-coordinating inhibitors of 3CLpro. As shown in the present study, some of these compounds were also found to be active against 3Cpro of CV strain B3 (CVB3). Several crystal structures of 3Cpro from CVB3 and 3CLpro from CoV-229E and SARS-CoV in complex with the inhibitors were solved. The zinc-coordinating inhibitor is tetrahedrally coordinated to the His40-Cys147 catalytic dyad of CVB3 3Cpro. The presence of specific binding pockets for the residues of peptidomimetic inhibitors explains the binding specificity. Our results provide a structural basis for inhibitor optimization and development of potential drugs for antiviral therapies.

Inhibition of the severe acute respiratory syndrome 3CL protease by peptidomimetic α,β-unsaturated esters

Abstract The proteolytic processing of polyproteins by the 3CL protease of severe acute respiratory syndrome coronavirus is essential for the viral propagation. A series of tripeptide α,β-unsaturated esters and ketomethylene isosteres, including AG7088, are synthesized and assayed to target the 3CL protease. Though AG7088 is inactive (IC50 >100μM), the ketomethylene isosteres and tripeptide α,β-unsaturated esters containing both P1 and P2 phenylalanine residues show modest inhibitory activity (IC50 =11–39μM). The Phe-Phe dipeptide inhibitors 18a–e are designed on the basis of computer modeling of the enzyme–inhibitor complex. The most potent inhibitor 18c with an inhibition constant of 0.52μM is obtained by condensation of the Phe-Phe dipeptide α,β-unsaturated ester with 4-(dimethylamino)cinnamic acid. The cell-based assays also indicate that 18c is a nontoxic anti-SARS agent with an EC50 value of 0.18μM.

Substrate binding mode and reaction mechanism of undecaprenyl pyrophosphate synthase deduced from crystallographic studies.

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes eight consecutive condensation reactions of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to form a 55‐carbon long‐chain product. We previously reported the crystal structure of the apo‐enzyme from Escherichia coli and the structure of UPPs in complex with sulfate ions (resembling pyrophosphate of substrate), Mg2+, and two Triton molecules (product‐like). In the present study, FPP substrate was soaked into the UPPs crystals, and the complex structure was solved. Based on the crystal structure, the pyrophosphate head group of FPP is bound to the backbone NHs of Gly29 and Arg30 as well as the side chains of Asn28, Arg30, and Arg39 through hydrogen bonds. His43 is close to the C2 carbon of FPP and may stabilize the farnesyl cation intermediate during catalysis. The hydrocarbon moiety of FPP is bound with hydrophobic amino acids including Leu85, Leu88, and Phe89, located on the α3 helix. The binding mode of FPP in cis‐type UPPs is apparently different from that of trans‐type and many other prenyltransferases which utilize Asprich motifs for substrate binding via Mg2+. The new structure provides a plausible mechanism for the catalysis of UPPs.