Heng Zheng

DRAMP: a comprehensive data repository of antimicrobial peptides

The growing problem of antibiotic-resistant microorganisms results in an urgent need for substitutes to conventional antibiotics with novel modes of action and effective activities. Antimicrobial peptides (AMPs), produced by a wide variety of living organisms acting as a defense mechanism against invading pathogenic microbes, are considered to be such promising alternatives. AMPs display a broad spectrum of antimicrobial activity and a low propensity for developing resistance. Therefore, a thorough understanding of AMPs is essential to exploit them as antimicrobial drugs. Considering this, we developed a comprehensive user-friendly data repository of antimicrobial peptides (DRAMP), which holds 17349 antimicrobial sequences, including 4571 general AMPs, 12704 patented sequences and 74 peptides in drug development. Entries in the database have detailed annotations, especially detailed antimicrobial activity data (shown as target organism with MIC value) and structure information. Annotations also include accession numbers crosslinking to Pubmed, Swiss-prot and Protein Data Bank (PDB). The website of the database comes with easy-to-operate browsing as well as searching with sorting and filtering functionalities. Several useful sequence analysis tools are provided, including similarity search, sequence alignment and conserved domain search (CD-Search). DRAMP should be a useful resource for the development of novel antimicrobial peptide drugs.

β-Lactamase inhibitors: an update.

β-lactamase-mediated resistance to β-lactam antibiotics is an increasing threat to clinical antimicrobial chemotherapy. The combinations of β-lactam antibiotics and β-lactamase inhibitors (such as sulbactam, tazobactam and clavulanic acid) have been successfully used for overcoming class A β-lactamase-mediated resistance. However, none of the inhibitors effective against class B, C or D β-lactamases are available in the clinic, which alarms an urgent need to discover/design broad-spectrum β-lactamase inhibitors or new β-lactam antibiotics capable of evading bacterial enzymatic inactivation. In recent years, inhibitors targeted to serine β-lactamases have been developed rapidly with a few of them under clinical trials. In contrast, none promising class B β-lactamase (metallo-β-lactamase) inhibitors with good druggability have been discovered, despite the increasing number of active molecules reported. In this review, we summarized the potential β-lactamase inhibitors reported in recent years and updated the current status of β-lactamase inhibitor discovery.

Bioactive Natural Products from the Antarctic and Arctic Organisms

Polar regions are remote and challenging areas on the earth. In view of the unique environment and the severe competition in polar regions, its considered that the ecological system might be the producer of new compounds with diversity biological activities. This review is an attempt to consolidate the studies about 97 natural products isolated from Antarctic and Arctic organisms including microbes, algae, sponges, bryozoans, and tunicates and so on published in the recent years. The emphasis is mainly about the new compounds, source organisms and biological activities, which signifies the immense competence of Antarctic and Arctic organisms as bioactive natural products producers.

A Tumor-Penetrating Peptide Modification Enhances the Antitumor Activity of Thymosin Alpha 1

A serious limitation of numerous antitumor drugs is the incapacity to penetrate solid tumors. However, addition of an RGD fragment to peptide drugs might solve this problem. In this study, we explored whether the introduction of a permeability-enhancing sequence, such as iRGD (CRGDK/RGPD/EC) fragments, would enhance the activity of thymosin alpha 1 (Tα1). The modified Tα1 (Tα1-iRGD) was successfully expressed and purified, and the in vitro assay showed that Tα1-iRGD presented a similar activity as Tα1 in promoting proliferation of mouse splenocytes. Meanwhile, cell adhesion analysis revealed that Tα1-iRGD exhibited more specific and greater binding with tumor cells compared with Tα1. Furthermore, the iRGD fragment evidently enhanced the basal ability of Tα1 to inhibit proliferation of cancer cells in vitro, particularly of mouse melanoma cell line B16F10 and human lung cancer cell line H460. Our findings indicated that the addition of an iRGD fragment increased the anti-proliferative activity of Tα1 against cancer cells by improving the ability of Tα1 to penetrate the tumor cells. This study highlighted the important roles of an iRGD sequence in the therapeutic strategy of Tα1-iRGD. Thus, Tα1-iRGD could be a novel drug candidate for cancer treatment.

Pimarane diterpenes from the Arctic fungus Eutypella sp. D-1

Two new diterpenes, libertellenone G(1) and libertellenone H(2) were isolated from the fungus Eutypella sp. D-1 isolated from the soil of high latitude of Arctic, together with two known pimarane diterpenes (3–4). The structures of 1 and 2 were elucidated from spectroscopic data (nuclear magnetic resonance, mass spectrometry and infrared). These compounds were evaluated for cytotoxic activity against seven human tumor cell lines. Compound 2 showed a range of cytotoxicity between 3.31 and 44.1 μM. Compound 1 exhibited antibacterial activity against Escherichia coli, Bacillus subtilis and Staphylococcus aureus.

Inhibitor Discovery of Full-Length New Delhi Metallo-β-Lactamase-1 (NDM-1)

New Delhi metallo-β-lactmase-1 (NDM-1) has recently attracted extensive attention for its biological activities to catalyze the hydrolysis of almost all of β-lactam antibiotics. To study the catalytic property of NDM-1, the steady-kinetic parameters of NDM-1 toward several kinds of β-lactam antibiotics have been detected. It could effectively hydrolyze most β-lactams (k cat/K m ratios between 0.03 to 1.28 µmol−1.s−1), except aztreonam. We also found that thiophene-carboxylic acid derivatives could inhibit NDM-1 and have shown synergistic antibacterial activity in combination with meropenem. Flexible docking and quantum mechanics (QM) study revealed electrostatic interactions between the sulfur atom of thiophene-carboxylic acid derivatives and the zinc ion of NDM-1, along with hydrogen bond between inhibitor and His189 of NDM-1. The interaction models proposed here can be used in rational design of NDM-1 inhibitors.

Probing the Effect of the Non-Active-Site Mutation Y229W in New Delhi Metallo-β-lactamase-1 by Site-Directed Mutagenesis, Kinetic Studies, and Molecular Dynamics Simulations

New Delhi metallo-β-lactmase-1 (NDM-1) has attracted extensive attention for its high catalytic activities of hydrolyzing almost all β-lactam antibiotics. NDM-1 shows relatively higher similarity to subclass B1 metallo-β-lactmases (MβLs), but its residue at position 229 is identical to that of B2/B3 MβLs, which is a Tyr instead of a B1-MβL-conserved Trp. To elucidate the possible role of Y229 in the bioactivity of NDM-1, we performed mutagenesis study and molecular dynamics (MD) simulations. Although residue Y229 is spatially distant from the active site and not contacting directly with the substrate or zinc ions, the Y229W mutant was found to have higher kcat and Km values than those of wild-type NDM-1, resulting in 1∼7 fold increases in kcat/Km values against tested antibiotics. In addition, our MD simulations illustrated the enhanced flexibility of Loop 2 upon Y229W mutation, which could increase the kinetics of both substrate entrance (kon) and product egress (koff). The enhanced flexibility of Loop 2 might allow the enzyme to adjust the geometry of its active site to accommodate substrates with different structures, broadening its substrate spectrum. This study indicated the possible role of the residue at position 229 in the evolution of NDM-1.

Dipeptidyl Aminopeptidase IV from Stenotrophomonas maltophilia Exhibits Activity against a Substrate Containing a 4-Hydroxyproline Residue

The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-A resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P4(3)2(1)2, with unit cell parameters a = b = 105.9 A and c = 161.9 A. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal beta-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.

Influencing COX-2 activity by COX related pathways in inflammation and cancer.

Cancer is a major cause of death worldwide despite significant advances in surgery and biology. Several recent studies have suggested that there is a close connection between tumorigenesis and inflammation. Cyclooxygenase-2 (COX-2), a link between inflammation and cancer, was proposed as a prime target for cancer therapy. However, the main available COX-2 inhibitors, nonsteroidal antiinflammatory drugs (NSAIDs), have several side effects that limit their application in cancer treatment. Additional research is needed to identify drugs that can regulate COX-2 or prostaglandin E2 (PGE2) with greater efficacy. Understanding the mechanism of COX-2/PGE2-mediated cancer and inflammation would be very useful for successful drug development. Progress of research and development into the role of COX related signaling both in inflammation and tumorigenesis in recent years has been summarized and introduced in this paper. Novel drugs designed to inhibit COX-2-related cancers are discussed to provide references for further development of novel drugs.

Asp120Asn mutation impairs the catalytic activity of NDM-1 metallo-β-lactamase: experimental and computational study

New Delhi metallo-β-lactamase-1 (NDM-1) has attracted extensive attention in recent years for its high activity for hydrolyzing almost all β-lactam antibiotics. Like other metallo-β-lactamases (MβLs), NDM-1 features an invariant Asp120 that ligates the zinc ion (ZN2) in the active site. Previous studies showed that substitutions of Asp120 with residues such as Ala, Ser, Asn and Glu dramatically impaired the MβL (BcII, IMP-1, L1) activity, but no consensus about the exact role of Asp120 has reached. Here we constructed D120N mutant of NDM-1 by site-directed mutagenesis. The replacement of Asp120 with Asn, which has much weaker metal ligating capabilities than Asp, severely impaired the lactamase activity without abolishing the ZN2 site. Molecular dynamics simulations suggested that the ZN1-ZN2 distance increased because of mutation, leading to a rearrangement of the active site, including the bridging OH(-). Thereby, the Mulliken charges of ZN1 and ZN2 redistributed, especially for ZN2, which might be the major cause of the impaired activity. Reducing the point charges of Asp120 carboxyl oxygens weakened the ionic interactions between Asp120 and ZN2, and the positions of the zinc ions were also changed as a result. It is proposed that Asp120 acts as a strong ZN2 ligand, positioning ZN2 for catalytically important interactions with the substrate, stabilizing the negatively charged amide nitrogen of the hydrolyzed intermediate, and more importantly, orienting the ZN-bound OH(-) for nucleophilic attacks and protonation. These functions are of general importance for catalyzing β-lactam antibiotics by NDM-1 as well as other MβLs.