Hiroaki Suga

Induction and Inhibition of Pseudomonas aeruginosa Quorum Sensing by Synthetic Autoinducer Analogs

We synthesized a library of Pseudomonas aeruginosa autoinducer analogs with variation targeted to the homoserine lactone (HSL) moiety and discovered a new agonist, 3-oxo-C(12)-(2-aminocyclohexanol), capable of activating LasR as a transcription factor. We reconstructed two sets of focused libraries against the quorum-sensing transcription factors LasR and RhlR, respectively. Opposing the prediction that both proteins should have the same binding site for HSL, it was surprising to find that these two related proteins respond to different structural motifs. This suggests that the HSL binding site differs in these proteins. We also found that subtle structural modifications to the agonists yielded compounds with antagonist activity. We performed a series of assays to show that inhibition of quorum sensing by these antagonists significantly reduced the production of virulence factors and biofilm formation.

A highly flexible tRNA acylation method for non-natural polypeptide synthesis.

Here we describe a de novo tRNA acylation system, the flexizyme (Fx) system, for the preparation of acyl tRNAs with nearly unlimited selection of amino and hydroxy acids and tRNAs. The combination of the Fx system with an appropriate cell-free translation system allows us to readily perform mRNA-encoded synthesis of proteins and short polypeptides involving multiple non-natural amino acids.*Note: In the version of this article initially published, the authors did not declare competing financial interests. They have filed a patent covering some of the information described in the paper and now declare competing financial interests. This error has been corrected in the PDF version of the article.

Library Screening for Synthetic Agonists and Antagonists of a Pseudomonas aeruginosa Autoinducer

The autoinducer (AI) that initiates the quorum sensing (QS) signaling cascade in Pseudomonas aeruginosa is an acyl-homoserine lactone (acyl-HSL). We initiated a study of the requirements for binding of the AI to its protein effector LasR by synthesizing a library of analogs with the HSL moiety replaced with different amines and alcohols. We tested each compound for both agonist and antagonist activity using a QS-controlled reporter gene assay and found several new agonists and antagonists. A representative antagonist was further tested for its ability to inhibit virulence factors. This data progresses our understanding of the LasR-AI interaction toward the rational design of therapeutic inhibitors of QS.

Structural basis for the drug extrusion mechanism by a MATE multidrug transporter

Multidrug and toxic compound extrusion (MATE) family transporters are conserved in the three primary domains of life (Archaea, Bacteria and Eukarya), and export xenobiotics using an electrochemical gradient of H+ or Na+ across the membrane. MATE transporters confer multidrug resistance to bacterial pathogens and cancer cells, thus causing critical reductions in the therapeutic efficacies of antibiotics and anti-cancer drugs, respectively. Therefore, the development of MATE inhibitors has long been awaited in the field of clinical medicine. Here we present the crystal structures of the H+-driven MATE transporter from Pyrococcus furiosus in two distinct apo-form conformations, and in complexes with a derivative of the antibacterial drug norfloxacin and three in vitro selected thioether-macrocyclic peptides, at 2.1–3.0 Å resolutions. The structures, combined with functional analyses, show that the protonation of Asp 41 on the amino (N)-terminal lobe induces the bending of TM1, which in turn collapses the N-lobe cavity, thereby extruding the substrate drug to the extracellular space. Moreover, the macrocyclic peptides bind the central cleft in distinct manners, which correlate with their inhibitory activities. The strongest inhibitory peptide that occupies the N-lobe cavity may pave the way towards the development of efficient inhibitors against MATE transporters.

Quorum sensing and the LysR-type transcriptional activator ToxR regulate toxoflavin biosynthesis and transport in Burkholderia glumae.

Burkholderia glumae BGR1 produces a broad‐host range phytotoxin, called toxoflavin, which is a key pathogenicity factor in rice grain rot and wilt in many field crops. Our molecular and genetic analyses of toxoflavin‐deficient mutants demonstrated that gene clusters for toxoflavin production consist of four transcriptional units. The toxoflavin biosynthesis genes were composed of five genes, toxA to toxE, as Suzuki et al. (2004) reported previously. Genes toxF to toxI, which are responsible for toxoflavin transport, were polycistronic and similar to the genes for resistance‐nodulation‐division (RND) efflux systems. Using Tn3‐gusA reporter fusions, we found that ToxR, a LysR‐type regulator, regulates both the toxABCDE and toxFGHI operons in the presence of toxoflavin as a coinducer. In addition, the expression of both operons required a transcriptional activator, ToxJ, whose expression is regulated by quorum sensing. TofI, a LuxI homologue, was responsible for the biosynthesis of both N‐hexanoyl homoserine lactone and N‐octanoyl homoserine lactone (C8‐HSL). C8‐HSL and its cognate receptor TofR, a LuxR homologue, activated toxJ expression. This is the first report that quorum sensing is involved in pathogenicity by the regulation of phytotoxin biosynthesis and its transport in plant pathogenic bacteria.

Flexizymes for genetic code reprogramming

Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to nonproteinogenic ones; thus, specific sequences of nonstandard peptides can be ribosomally expressed according to their mRNA templates. Here we describe a protocol that facilitates genetic code reprogramming using flexizymes integrated with a custom-made in vitro translation apparatus, referred to as the flexible in vitro translation (FIT) system. Flexizymes are flexible tRNA acylation ribozymes that enable the preparation of a diverse array of nonproteinogenic acyl-tRNAs. These acyl-tRNAs read vacant codons created in the FIT system, yielding the desired nonstandard peptides with diverse exotic structures, such as N-methyl amino acids, D-amino acids and physiologically stable macrocyclic scaffolds. The facility of the protocol allows a wide variety of applications in the synthesis of new classes of nonstandard peptides with biological functions. Preparation of flexizymes and tRNA used for genetic code reprogramming, optimization of flexizyme reaction conditions and expression of nonstandard peptides using the FIT system can be completed by one person in ∼1 week. However, once the flexizymes and tRNAs are in hand and reaction conditions are fixed, synthesis of acyl-tRNAs and peptide expression is generally completed in 1 d, and alteration of a peptide sequence can be achieved by simply changing the corresponding mRNA template.

Reprogramming the Translation Initiation for the Synthesis of Physiologically Stable Cyclic Peptides

The initiation codon dictates that the translation initiation event exclusively begins with methionine. We report here a new technology to reprogram the initiation event, where various amino acids and those bearing N (alpha)-acyl groups can be used as an initiator for peptide synthesis. The technology is built upon the concept of genetic code reprogramming, where methionine is depleted from the translation system and the initiation codon is reassigned to the desired amino acid. We have applied this technology to the synthesis of an antitumor cyclic peptide, G7-18NATE, closed by a physiologically stable bond, and it is also extended to the custom synthesis of its analogues with various ring sizes. Significantly, cyclization occurs spontaneously upon translation of the precursor linear peptides. To demonstrate the practicality of this methodology, we also prepared a small cyclic peptide library designated by 160 distinct mRNAs. Thus, this technology offers a new means to prepare a wide array of in vivo compatible cyclic peptide libraries for the discovery of peptidic drug candidates against various therapeutic targets.

Diverse backbone-cyclized peptides via codon reprogramming

We report a methodology for the ribosomal synthesis of backbone-cyclized peptides involving genetic code reprogramming to introduce one or more nonproteinogenic amino acids. Expression of linear peptides bearing a cysteine-proline dipeptide sequence followed by glycolic acid results in self-rearrangement to a C-terminal diketopiperadine-thioester, which non-enzymatically generates a cyclized peptide. We demonstrate the ribosomal synthesis of several naturally occurring backbone-cyclized peptides and a library based on a bicyclic scaffold, and we identify bioactive sequences by screening and deconvolution.

An in vitro evolved precursor tRNA with aminoacylation activity

A set of catalysts for aminoacyl‐tRNA synthesis is an essential component for translation. The RNA world hypothesis postulates that RNA catalysts could have played this role. Here we show an in vitro evolved precursor tRNA consisting of two domains, a catalytic 5′‐leader sequence and an aminoacyl‐acceptor tRNA. The 5′‐leader sequence domain selectively self‐charges phenylalanine on the 3′‐terminus of the tRNA domain. This cis‐acting ribozyme is susceptible to RNase P RNA, generating the corresponding 5′‐leader segment and the mature tRNA. Moreover, the 5′‐leader segment is able to aminoacylate the mature tRNA in trans. Mutational studies have revealed that C74 and C75 at the tRNA aminoacyl‐acceptor end form base pairs with G71 and G70 of the trans‐acting ribozyme. Such Watson–Crick base pairing with tRNA has been observed in RNase P RNA and 23S rRNA, suggesting that all three ribozymes use a similar mechanism for the recognition of the aminoacyl‐acceptor end. Our demonstrations indicate that catalytic precursor tRNAs could have provided the foundations for the genetic coding system in the proto‐translation system.

Discovery of Macrocyclic Peptides Armed with a Mechanism‐Based Warhead: Isoform‐Selective Inhibition of Human Deacetylase SIRT2

Designed to inhibit: by using the random nonstandard peptide integrated discovery (RaPID) system, highly potent isoform-selective inhibitors can be identified from a library of nonstandard macrocyclic peptides. These inhibitors, which contain a mechanism-based warhead residue, are active against the human deacetylase SIRT2, with IC(50) values in the low nanomolar region.