Selective Inhibition of an Apicoplastic Aminoacyl‐tRNA Synthetase from Plasmodium falciparum

The resistance of malaria parasites to available drugs continues to grow, and this makes the need for new antimalarial therapies pressing. Aminoacyl‐tRNA synthetases (ARSs) are essential enzymes and well‐established antibacterial targets and so constitute a promising set of targets for the development of new antimalarials. Despite their potential as drug targets, apicoplastic ARSs remain unexplored. We have characterized the lysylation system of Plasmodium falciparum, and designed, synthesized, and tested a set of inhibitors based on the structure of the natural substrate intermediate: lysyl‐adenylate. Here we demonstrate that selective inhibition of apicoplastic ARSs is feasible and describe new compounds that that specifically inhibit Plasmodium apicoplastic lysyl‐tRNA synthetase and show antimalarial activities in the micromolar range.     

Two‐Tier Screening Platform for Directed Evolution of Aminoacyl–tRNA Synthetases with Enhanced Stop Codon Suppression Efficiency

Genetic code expansion through amber stop codon suppression provides a powerful tool for introducing non‐proteinogenic functionalities into proteins for a broad range of applications. However, ribosomal incorporation of noncanonical amino acids (ncAAs) by means of engineered aminoacyl–tRNA synthetases (aaRSs) often proceeds with significantly reduced efficiency compared to sense codon translation. Here, we report the implementation of a versatile platform for the development of engineered aaRSs with enhanced efficiency in mediating ncAA incorporation by amber stop codon suppression. This system integrates a white/blue colony screen with a plate‐based colorimetric assay, thereby combining high‐throughput capabilities with reliable and quantitative measurement of aaRS‐dependent ncAA incorporation efficiency. This two‐tier functional screening system was successfully applied to obtain a pyrrolysyl–tRNA synthetase (PylRS) variant (CrtK‐RS(4.1)) with significantly improved efficiency (+250–370 %) for mediating the incorporation of N^?‐crotonyl‐lysine and other lysine analogues of relevance for the study of protein post‐translational modifications into a target protein. Interestingly, the beneficial mutations accumulated by CrtK‐RS(4.1) were found to localize within the noncatalytic N‐terminal domain of the enzyme and could be transferred to another PylRS variant, improving the ability of the variant to incorporate its corresponding ncAA substrate. This work introduces an efficient platform for the improvement of aaRSs that could be readily extended to other members of this enzyme family and/or other target ncAAs.     

Evolving the N‐Terminal Domain of Pyrrolysyl‐tRNA Synthetase for Improved Incorporation of Noncanonical Amino Acids

By evolving the N‐terminal domain of Methanosarcina mazei pyrrolysyl‐tRNA synthetase (PylRS) that directly interacts with tRNA^Pyl, a mutant clone displaying improved amber‐suppression efficiency for the genetic incorporation of N^?‐(tert‐butoxycarbonyl)‐l ‐lysine threefold more than the wild type was identified. The identified mutations were R19H/H29R/T122S. Direct transfer of these mutations to two other PylRS mutants that were previously evolved for the genetic incorporation of N^?‐acetyl‐l ‐lysine and N^?‐(4‐azidobenzoxycarbonyl)‐l ‐δ,?‐dehydrolysine also improved the incorporation efficiency of these two noncanonical amino acids. As the three identified mutations were found in the N‐terminal domain of PylRS that was separated from its catalytic domain for charging tRNA^Pyl with a noncanonical amino acid, they could potentially be introduced to all other PylRS mutants to improve the incorporation efficiency of their corresponding noncanonical amino acids. Therefore, it represents a general strategy to optimize the pyrrolysine incorporation system‐based noncanonical amino‐acid mutagenesis.     

Design of S‐Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl‐tRNA Synthetase Variant

The noncanonical amino acid S‐allyl cysteine (Sac) is one of the major compounds of garlic extract and exhibits a range of biological activities. It is also a small bioorthogonal alkene tag capable of undergoing controlled chemical modifications, such as photoinduced thiol‐ene coupling or Pd‐mediated deprotection. Its small size guarantees minimal interference with protein structure and function. Here, we report a simple protocol efficiently to couple in‐situ semisynthetic biosynthesis of Sac and its incorporation into proteins in response to amber (UAG) stop codons. We exploited the exceptional malleability of pyrrolysyl‐tRNA synthetase (PylRS) and evolved an S‐allylcysteinyl‐tRNA synthetase (SacRS) capable of specifically accepting the small, polar amino acid instead of its long and bulky aliphatic natural substrate. We succeeded in generating a novel and inexpensive strategy for the incorporation of a functionally versatile amino acid. This will help in the conversion of orthogonal translation from a standard technique in academic research to industrial biotechnology.     

Synthesis of 3′‐Fluoro‐tRNA Analogues for Exploring Non‐ribosomal Peptide Synthesis in Bacteria

Aminoacyl‐tRNAs (aa‐tRNAs) participate in a vast repertoire of metabolic pathways, including the synthesis of the peptidoglycan network in the cell walls of bacterial pathogens. Synthesis of aminoacyl‐tRNA analogues is critical for further understanding the mechanisms of these reactions. Here we report the semi‐synthesis of 3′‐fluoro analogues of Ala‐tRNA^Ala. The presence of fluorine in the 3′‐position blocks Ala at the 2′‐position by preventing spontaneous migration of the residue between positions 2′ and 3′. NMR analyses showed that substitution of the 3′‐hydroxy group by fluorine in the ribo configuration favours the S‐type conformation of the furanose ring of terminal adenosine A76. In contrast, the N‐type conformation is favoured by the presence of fluorine in the xylo configuration. Thus, introduction of fluorine in the ribo and xylo configurations affects the conformation of the furanose ring in reciprocal ways. These compounds should provide insight into substrate recognition by Fem transferases and the Ala‐tRNA synthetases.     

Dual Unnatural Amino Acid Incorporation and Click‐Chemistry Labeling to Enable Single‐Molecule FRET Studies of p97 Folding

Many cellular functions are critically dependent on the folding of complex multimeric proteins, such as p97, a hexameric multidomain AAA+ chaperone. Given the complex architecture of p97, single‐molecule (sm) FRET would be a powerful tool for studying folding while avoiding ensemble averaging. However, dual site‐specific labeling of such a large protein for smFRET is a significant challenge. Here, we address this issue by using bioorthogonal azide–alkyne chemistry to attach an smFRET dye pair to site‐specifically incorporated unnatural amino acids, allowing us to generate p97 variants reporting on inter‐ or intradomain structural features. An initial proof‐of‐principle set of smFRET results demonstrated the strengths of this labeling method. Our results highlight this as a powerful tool for structural studies of p97 and other large protein machines.     

Directed Evolution of Scanning Unnatural‐Protease‐Resistant (SUPR) Peptides for in Vivo Applications

Peptides typically have poor biostabilities, and natural sequences cannot easily be converted into drug‐like molecules without extensive medicinal chemistry. We have adapted mRNA display to drive the evolution of highly stable cyclic peptides while preserving target affinity. To do this, we incorporated an unnatural amino acid in an mRNA display library that was subjected to proteolysis prior to selection for function. The resulting “SUPR (scanning unnatural protease resistant) peptide” showed ≈500‐fold improvement in serum stability (t =160 h) and up to 3700‐fold improvement in protease resistance versus the parent sequence. We extended this approach by carrying out SUPR peptide selections against Her2‐positive cells in culture. The resulting SUPR4 peptide showed low‐nanomolar affinity toward Her2, excellent specificity, and selective tumor uptake in vivo. These results argue that this is a general method to design potent and stable peptides for in vivo imaging and therapy.     

Genetic Encoding of Photocaged Tyrosines with Improved Light‐Activation Properties for the Optical Control of Protease Function

We genetically encoded three new caged tyrosine analogues with improved photochemical properties by using an engineered pyrrolysyl‐tRNA synthetase/tRNA_CUA pair in bacterial and mammalian cells. We applied the new tyrosine analogues to the photoregulation of firefly luciferase by caging its key tyrosine residue, Tyr340, and observed excellent off‐to‐on light switching. This reporter was then used to evaluate the activation rates of the different light‐removable protecting groups in live cells. We identified the nitropiperonyl caging group as an excellent compromise between incorporation efficiency and photoactivation properties. To demonstrate applicability of the new caged tyrosines, an important proteolytic enzyme, tobacco etch virus (TEV) protease, was engineered for optical control. The ability to incorporate differently caged tyrosine analogues into proteins in live cells further expands the unnatural amino acid and optogenetic toolbox.     

Synthetic biology approach for the development of conditionally replicating HIV‐1 vaccine

While the combined antiretroviral therapy has resulted in a significant decrease in HIV‐1 related morbidity and mortality, the HIV‐1 pandemic has not been substantially averted. To curtail the 2.4 million new infections each year, a prophylactic HIV‐1 vaccine is urgently needed. This review first summarizes four major completed clinical efficacy trials of prophylactic HIV‐1 vaccine and their outcomes. Next, it discusses several other approaches that have not yet advanced to clinical efficacy trials, but provided valuable insights into vaccine design. Among them, live‐attenuated vaccines (LAVs) provided excellent protection in a non‐human primate model. However, safety concerns have precluded the current version of LAVs from clinical application. As the major component of this review, two synthetic biology approaches for improving the safety of HIV‐1 LAVs through controlling HIV‐1 replication are discussed. Particular focus is on a novel approach that uses unnatural amino acid‐mediated suppression of amber nonsense codon to generate conditionally replicating HIV‐1 variants. The objective is to attract more attention towards this promising research field and to provoke creative designs and innovative utilization of the two control strategies. © 2016 Society of Chemical Industry     

Selection,Addiction and Catalysis: Emerging Trends for the Incorporation of Noncanonical Amino Acids into Peptides and Proteins in Vivo

Expanding the genetic code of organisms by incorporating noncanonical amino acids (ncAAs) into target proteins through the suppression of stop codons in vivo has profoundly impacted how we perform protein modification or detect proteins and their interaction partners in their native environment. Yet, with genetic code expansion strategies maturing over the past 15 years, new applications that make use—or indeed repurpose—these techniques are beginning to emerge. This Concept article highlights three of these developments: 1) The incorporation of ncAAs for the biosynthesis and selection of bioactive macrocyclic peptides with novel ring architectures, 2) synthetic biocontainment strategies based on the addiction of microorganisms to ncAAs, and 3) enzyme design strategies, in which ncAAs with unique functionalities enable the catalysis of new-to-nature reactions. Key advances in all three areas are presented and potential future applications discussed.     

Genetic Encoding of a Bicyclo[6.1.0]nonyne‐Charged Amino Acid Enables Fast Cellular Protein Imaging by Metal‐Free Ligation

Visualizing biomolecules by fluorescent tagging is a powerful method for studying their behaviour and function inside cells. We prepared and genetically encoded an unnatural amino acid (UAA) that features a bicyclononyne moiety. This UAA offered exceptional reactivity in strain‐promoted azide–alkyne cycloadditions. Kinetic measurements revealed that the UAA reacted also remarkably fast in the inverse‐electron‐demand Diels–Alder cycloaddition with tetrazine‐conjugated dyes. Genetic encoding of the new UAA inside mammalian cells and its subsequent selective labeling at low dye concentrations demonstrate the usefulness of the new amino acid for future imaging studies.     

Structural Insights into the Recovery of Aldolase Activity in N‐Acetylneuraminic Acid Lyase by Replacement of the Catalytically Active Lysine with γ‐Thialysine by Using a Chemical Mutagenesis Strategy

Chemical modification has been used to introduce the unnatural amino acid γ‐thialysine in place of the catalytically important Lys165 in the enzyme N‐acetylneuraminic acid lyase (NAL). The Staphylococcus aureus nanA gene, encoding NAL, was cloned and expressed in E. coli. The protein, purified in high yield, has all the properties expected of a class I NAL. The S. aureus NAL which contains no natural cysteine residues was subjected to site‐directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site. Subsequently chemical mutagenesis completely converted the cysteine into γ‐thialysine through dehydroalanine (Dha) as demonstrated by ESI‐MS. Initial kinetic characterisation showed that the protein containing γ‐thialysine regained 17 % of the wild‐type activity. To understand the reason for this lower activity, we solved X‐ray crystal structures of the wild‐type S. aureus NAL, both in the absence of, and in complex with, pyruvate. We also report the structures of the K165C variant, and the K165‐γ‐thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that γ‐thialysine in NAL is an excellent structural mimic of lysine. Measurement of the pH‐activity profile of the thialysine modified enzyme revealed that its pH optimum is shifted from 7.4 to 6.8. At its optimum pH, the thialysine‐containing enzyme showed almost 30 % of the activity of the wild‐type enzyme at its pH optimum. The lowered activity and altered pH profile of the unnatural amino acid‐containing enzyme can be rationalised by imbalances of the ionisation states of residues within the active site when the pK_a of the residue at position 165 is perturbed by replacement with γ‐thialysine. The results reveal the utility of chemical mutagenesis for the modification of enzyme active sites and the exquisite sensitivity of catalysis to the local structural and electrostatic environment in NAL.