Computational Methods for Molecular Understanding of the Antibiotic-Aminoacyl tRNA Synthetase Interaction.

Developing an understanding of the interactions between an antibiotic and its binding site in a pathogen cell is the key to antibiotic design-an important cost-saving methodology compared to the costly and time-consuming random trial-and-error approach. The rapid development of antibiotic resistance provides an impetus for such studies. Recent years have witnessed the beginning of the use of combined computational techniques, including computer simulations and quantum mechanical computations, to understand how antibiotics bind at the active site of aminoacyl tRNA synthetases (aaRSs) from pathogens. Such computational protocols assist the knowledge-based design of antibiotics targeting aaRSs, which are their validated targets. After the ideas behind the protocols and their strategic planning are discussed, the protocols are described along with their major outcomes. This is followed by an integration of results from the different basic protocols. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Analysis of active-site residues from primary sequence of synthetase and transfer RNAs Basic Protocol 2: Molecular dynamics simulation-based protocol to study the structure and dynamics of the aaRS active site:antibiotic complex Basic Protocol 3: Quantum mechanical method-based protocol to study the structure and dynamics of the aaRS active site:antibiotic complex.

The tRNA identity landscape for aminoacylation and beyond.

tRNAs are key partners in ribosome-dependent protein synthesis. This process is highly dependent on the fidelity of tRNA aminoacylation by aminoacyl-tRNA synthetases and relies primarily on sets of identities within tRNA molecules composed of determinants and antideterminants preventing mischarging by non-cognate synthetases. Such identity sets were discovered in the tRNAs of a few model organisms, and their properties were generalized as universal identity rules. Since then, the panel of identity elements governing the accuracy of tRNA aminoacylation has expanded considerably, but the increasing number of reported functional idiosyncrasies has led to some confusion. In parallel, the description of other processes involving tRNAs, often well beyond aminoacylation, has progressed considerably, greatly expanding their interactome and uncovering multiple novel identities on the same tRNA molecule. This review highlights key findings on the mechanistics and evolution of tRNA and tRNA-like identities. In addition, new methods and their results for searching sets of multiple identities on a single tRNA are discussed. Taken together, this knowledge shows that a comprehensive understanding of the functional role of individual and collective nucleotide identity sets in tRNA molecules is needed for medical, biotechnological and other applications.

ThrRS-Mediated Translation Regulation of the RNA Polymerase III Subunit RPC10 Occurs through an Element with Similarity to Cognate tRNA ASL and Affects tRNA Levels.

Aminoacyl tRNA synthetases (aaRSs) are a well-studied family of enzymes with a canonical role in charging tRNAs with a specific amino acid. These proteins appear to also have non-canonical roles, including post-transcriptional regulation of mRNA expression. Many aaRSs were found to bind mRNAs and regulate their translation into proteins. However, the mRNA targets, mechanism of interaction, and regulatory consequences of this binding are not fully resolved. Here, we focused on yeast cytosolic threonine tRNA synthetase (ThrRS) to decipher its impact on mRNA binding. Affinity purification of ThrRS with its associated mRNAs followed by transcriptome analysis revealed a preference for mRNAs encoding RNA polymerase subunits. An mRNA that was significantly bound compared to all others was the mRNA encoding RPC10, a small subunit of RNA polymerase III. Structural modeling suggested that this mRNA includes a stem-loop element that is similar to the anti-codon stem loop (ASL) structure of ThrRS cognate tRNA (tRNAThr). We introduced random mutations within this element and found that almost every change from the normal sequence leads to reduced binding by ThrRS. Furthermore, point mutations at six key positions that abolish the predicted ASL-like structure showed a significant decrease in ThrRS binding with a decrease in RPC10 protein levels. Concomitantly, tRNAThr levels were reduced in the mutated strain. These data suggest a novel regulatory mechanism in which cellular tRNA levels are regulated through a mimicking element within an RNA polymerase III subunit in a manner that involves the tRNA cognate aaRS.

HIV-1 Gag Binds the Multi-Aminoacyl-tRNA Synthetase Complex via the EPRS Subunit.

Host factor tRNAs facilitate the replication of retroviruses such as human immunodeficiency virus type 1 (HIV-1). HIV-1 uses human tRNALys3 as the primer for reverse transcription, and the assembly of HIV-1 structural protein Gag at the plasma membrane (PM) is regulated by matrix (MA) domain-tRNA interactions. A large, dynamic multi-aminoacyl-tRNA synthetase complex (MSC) exists in the cytosol and consists of eight aminoacyl-tRNA synthetases (ARSs) and three other cellular proteins. Proteomic studies to identify HIV-host interactions have identified the MSC as part of the HIV-1 Gag and MA interactomes. Here, we confirmed that the MA domain of HIV-1 Gag forms a stable complex with the MSC, mapped the primary interaction site to the linker domain of bi-functional human glutamyl-prolyl-tRNA synthetase (EPRS), and showed that the MA-EPRS interaction was RNA dependent. MA mutations that significantly reduced the EPRS interaction reduced viral infectivity and mapped to MA residues that also interact with phosphatidylinositol-(4,5)-bisphosphate. Overexpression of EPRS or EPRS fragments did not affect susceptibility to HIV-1 infection, and knockdown of EPRS reduced both a control reporter gene and HIV-1 protein translation. EPRS knockdown resulted in decreased progeny virion production, but the decrease could not be attributed to selective effects on virus gene expression, and the specific infectivity of the virions remained unchanged. While the precise function of the Gag-EPRS interaction remains uncertain, we discuss possible effects of the interaction on either virus or host activities.

Targeting prolyl-tRNA synthetase via a series of ATP-mimetics to accelerate drug discovery against toxoplasmosis.

The prolyl-tRNA synthetase (PRS) is a validated drug target for febrifugine and its synthetic analog halofuginone (HFG) against multiple apicomplexan parasites including Plasmodium falciparum and Toxoplasma gondii. Here, a novel ATP-mimetic centered on 1-(pyridin-4-yl) pyrrolidin-2-one (PPL) scaffold has been validated to bind to Toxoplasma gondii PRS and kill toxoplasma parasites. PPL series exhibited potent inhibition at the cellular (T. gondii parasites) and enzymatic (TgPRS) levels compared to the human counterparts. Cell-based chemical mutagenesis was employed to determine the mechanism of action via a forward genetic screen. Tg-resistant parasites were analyzed with wild-type strain by RNA-seq to identify mutations in the coding sequence conferring drug resistance by computational analysis of variants. DNA sequencing established two mutations, T477A and T592S, proximal to terminals of the PPL scaffold and not directly in the ATP, tRNA, or L-pro sites, as supported by the structural data from high-resolution crystal structures of drug-bound enzyme complexes. These data provide an avenue for structure-based activity enhancement of this chemical series as anti-infectives.

Noncanonical amino acids as doubly bio-orthogonal handles for one-pot preparation of protein multiconjugates.

Genetic encoding of noncanonical amino acid (ncAA) for site-specific protein modification has been widely applied for many biological and therapeutic applications. To efficiently prepare homogeneous protein multiconjugates, we design two encodable noncanonical amino acids (ncAAs), 4-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (pTAF) and 3-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (mTAF), containing mutually orthogonal and bioorthogonal azide and tetrazine reaction handles. Recombinant proteins and antibody fragments containing the TAFs can easily be functionalized in one-pot reactions with combinations of commercially available fluorophores, radioisotopes, PEGs, and drugs in a plug-and-play manner to afford protein dual conjugates to assess combinations of tumor diagnosis, image-guided surgery, and targeted therapy in mouse models. Furthermore, we demonstrate that simultaneously incorporating mTAF and a ketone-containing ncAA into one protein via two non-sense codons allows preparation of a site-specific protein triconjugate. Our results demonstrate that TAFs are doubly bio-orthogonal handles for efficient and scalable preparation of homogeneous protein multiconjugates.

Design, Synthesis and Antimicrobial Evaluation of New N-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)(hetero)aryl-2-carboxamides as Potential Inhibitors of Mycobacterial Leucyl-tRNA Synthetase.

Tuberculosis remains a serious killer among infectious diseases due to its incidence, mortality, and occurrence of resistant mycobacterial strains. The challenge to discover new antimycobacterial agents forced us to prepare a series of N-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)(hetero)aryl-2-carboxamides 1-19 via the acylation of 6-aminobenzo[c][1,2]oxaborol-1(3H)-ol with various activated (hetero)arylcarboxylic acids. These novel compounds have been tested in vitro against a panel of clinically important fungi and bacteria, including mycobacteria. Some of the compounds inhibited the growth of mycobacteria in the range of micromolar concentrations and retained this activity also against multidrug-resistant clinical isolates. Half the maximal inhibitory concentrations against the HepG2 cell line indicated an acceptable toxicological profile. No growth inhibition of other bacteria and fungi demonstrated selectivity of the compounds against mycobacteria. The structure-activity relationships have been derived and supported with a molecular docking study, which confirmed a selectivity toward the potential target leucyl-tRNA synthetase without an impact on the human enzyme. The presented compounds can become important materials in antimycobacterial research.

Decreased expression of mitochondrial aminoacyl-tRNA synthetases causes downregulation of OXPHOS subunits in type 2 diabetic muscle.

Type 2 diabetes mellitus (T2D) affects millions of people worldwide and is one of the leading causes of morbidity and mortality. The skeletal muscle (SKM) is one of the most important tissues involved in maintaining glucose homeostasis and substrate oxidation, and it undergoes insulin resistance in T2D. In this study, we identify the existence of alterations in the expression of mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) in skeletal muscle from two different forms of T2D: early-onset type 2 diabetes (YT2) (onset of the disease before 30 years of age) and the classical form of the disease (OT2). GSEA analysis from microarray studies revealed the repression of mitochondrial mt-aaRSs independently of age, which was validated by real-time PCR assays. In agreement with this, a reduced expression of several encoding mt-aaRSs was also detected in skeletal muscle from diabetic (db/db) mice but not in obese ob/ob mice. In addition, the expression of the mt-aaRSs proteins most relevant in the synthesis of mitochondrial proteins, threonyl-tRNA, and leucyl-tRNA synthetases (TARS2 and LARS2) were also repressed in muscle from db/db mice. It is likely that these alterations participate in the reduced expression of proteins synthesized in the mitochondria detected in db/db mice. We also document an increased iNOS abundance in mitochondrial-enriched muscle fractions from diabetic mice that may inhibit aminoacylation of TARS2 and LARS2 by nitrosative stress. Our results indicate a reduced expression of mt-aaRSs in skeletal muscle from T2D patients, which may participate in the reduced expression of proteins synthesized in mitochondria. An enhanced mitochondrial iNOS could play a regulatory role in diabetes.

The binding mode of orphan glycyl-tRNA synthetase with tRNA supports the synthetase classification and reveals large domain movements.

As a class of essential enzymes in protein translation, aminoacyl-transfer RNA (tRNA) synthetases (aaRSs) are organized into two classes of 10 enzymes each, based on two conserved active site architectures. The (αß)2 glycyl-tRNA synthetase (GlyRS) in many bacteria is an orphan aaRS whose sequence and unprecedented X-shaped structure are distinct from those of all other aaRSs, including many other bacterial and all eukaryotic GlyRSs. Here, we report a cocrystal structure to elucidate how the orphan GlyRS kingdom specifically recognizes its substrate tRNA. This structure is sharply different from those of other aaRS-tRNA complexes but conforms to the clash-free, cross-class aaRS-tRNA docking found with conventional structures and reinforces the class-reconstruction paradigm. In addition, noteworthy, the X shape of orphan GlyRS is condensed with the largest known spatial rearrangement needed by aaRSs to capture tRNAs, which suggests potential nonactive site targets for aaRS-directed antibiotics, instead of less differentiated hard-to-drug active site locations.

Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression.

Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expression at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells. Using chemically induced dimerization domains, we developed split o-aaRSs that mediate gene expression by conditionally suppressing stop codons in the presence of the small molecules rapamycin and abscisic acid. By activating o-aaRSs, these molecular switches induce stop codon suppression, and in their absence stop codon suppression is turned off. We demonstrate, in Escherichia coli and in human cells, that split o-aaRSs function as genetically encoded AND gates where stop codon suppression is controlled by two distinct molecular inputs. In addition, we show that split o-aaRSs can be used as versatile biosensors to detect therapeutically relevant protein-protein interactions, including those involved in cancer, and those that mediate severe acute respiratory syndrome-coronavirus-2 infection.

A streamlined process for discovery and characterization of inhibitors against phenylalanyl-tRNA synthetase of Mycobacterium tuberculosis.

Aminoacyl-tRNA synthetases (aaRSs) catalyze aminoacylation of tRNAs to produce aminoacyl-tRNAs for protein synthesis. Bacterial aaRSs have distinctive features, play an essential role in channeling amino acids into biomolecular assembly, and are vulnerable to inhibition by small molecules. The aaRSs continue to be targets for potential antibacterial drug development. The first step of aaRS reaction is the activation of amino acid by hydrolyzing ATP to form an acyladenylate intermediate with the concomitant release of pyrophosphate. None-radioactive assays usually measure the rate of ATP consumption or phosphate generation, offering advantages in high-throughput drug screening. These simple aaRS enzyme assays can be adapted to study the mode of inhibition of natural or synthetic aaRS inhibitors. Taking phenylalanyl-tRNA synthetase (PheRS) of Mycobacterium tuberculosis (Mtb) as an example, we describe a process for identification and characterization of Mtb PheRS inhibitor.

Photo-regulated genetic encoding of dibenzo[c,g][1,2]diazocine on proteins via configuration switching.

We report two evolved Methanosarcina mazei pyrrolysine tRNA synthetases to genetically incorporate the isomers of dibenzo[c,g][1,2]diazocine-alanine (DBDAA) into proteins either in the dark or under regulation of 405 nm photo-stimulation. The genetic-encoded DBDAA realizes photo-tuning of enzymatic activity via the host-guest recognition of cucurbit[7]uril.

Tyrosine-targeted covalent inhibition of a tRNA synthetase aided by zinc ion.

Aminoacyl-tRNA synthetases (AARSs), a family of essential protein synthesis enzymes, are attractive targets for drug development. Although several different types of AARS inhibitors have been identified, AARS covalent inhibitors have not been reported. Here we present five unusual crystal structures showing that threonyl-tRNA synthetase (ThrRS) is covalently inhibited by a natural product, obafluorin (OB). The residue forming a covalent bond with OB is a tyrosine in ThrRS active center, which is not commonly modified by covalent inhibitors. The two hydroxyl groups on the o-diphenol moiety of OB form two coordination bonds with the conserved zinc ion in the active center of ThrRS. Therefore, the ß-lactone structure of OB can undergo ester exchange reaction with the phenolic group of the adjacent tyrosine to form a covalent bond between the compound and the enzyme, and allow its nitrobenzene structure to occupy the binding site of tRNA. In addition, when this tyrosine was replaced by a lysine or even a weakly nucleophilic arginine, similar bonds could also be formed. Our report of the mechanism of a class of AARS covalent inhibitor targeting multiple amino acid residues could facilitate approaches to drug discovery for cancer and infectious diseases.

Prolyl-tRNA synthetase as a novel therapeutic target in multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy characterised by aberrant production of immunoglobulins requiring survival mechanisms to adapt to proteotoxic stress. We here show that glutamyl-prolyl-tRNA synthetase (GluProRS) inhibition constitutes a novel therapeutic target. Genomic data suggest that GluProRS promotes disease progression and is associated with poor prognosis, while downregulation in MM cells triggers apoptosis. We developed NCP26, a novel ATP-competitive ProRS inhibitor that demonstrates significant anti-tumour activity in multiple in vitro and in vivo systems and overcomes metabolic adaptation observed with other inhibitor chemotypes. We demonstrate a complex phenotypic response involving protein quality control mechanisms that centers around the ribosome as an integrating hub. Using systems approaches, we identified multiple downregulated proline-rich motif-containing proteins as downstream effectors. These include CD138, transcription factors such as MYC, and transcription factor 3 (TCF3), which we establish as a novel determinant in MM pathobiology through functional and genomic validation. Our preclinical data therefore provide evidence that blockade of prolyl-aminoacylation evokes a complex pro-apoptotic response beyond the canonical integrated stress response and establish a framework for its evaluation in a clinical setting.

Chemically Acylated tRNAs are Functional in Zebrafish Embryos.

Genetic code expansion has pushed protein chemistry past the canonical 22 amino acids. The key enzymes that make this possible are engineered aminoacyl tRNA synthetases. However, as the number of genetically encoded amino acids has increased over the years, obvious limits in the type and size of novel side chains that can be accommodated by the synthetase enzyme become apparent. Here, we show that chemically acylating tRNAs allow for robust, site-specific incorporation of unnatural amino acids into proteins in zebrafish embryos, an important model organism for human health and development. We apply this approach to incorporate a unique photocaged histidine analogue for which synthetase engineering efforts have failed. Additionally, we demonstrate optical control over different enzymes in live embryos by installing photocaged histidine into their active sites.

Tuning phenylalanine fluorination to assess aromatic contributions to protein function and stability in cells.

The aromatic side-chains of phenylalanine, tyrosine, and tryptophan interact with their environments via both hydrophobic and electrostatic interactions. Determining the extent to which these contribute to protein function and stability is not possible with conventional mutagenesis. Serial fluorination of a given aromatic is a validated method in vitro and in silico to specifically alter electrostatic characteristics, but this approach is restricted to a select few experimental systems. Here, we report a group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA pairs (tRNA/RS pairs) that enable the site-specific encoding of a varied spectrum of fluorinated phenylalanine amino acids in E. coli and mammalian (HEK 293T) cells. By allowing the cross-kingdom expression of proteins bearing these unnatural amino acids at biochemical scale, these tools may potentially enable the study of biological mechanisms which utilize aromatic interactions in structural and cellular contexts.

Update of the Pyrrolysyl-tRNA Synthetase/tRNAPyl Pair and Derivatives for Genetic Code Expansion.

The cotranslational incorporation of pyrrolysine (Pyl), the 22nd proteinogenic amino acid, into proteins in response to the UAG stop codon represents an outstanding example of natural genetic code expansion. Genetic encoding of Pyl is conducted by the pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA, tRNAPyl. Owing to the high tolerance of PylRS toward diverse amino acid substrates and great orthogonality in various model organisms, the PylRS/tRNAPyl-derived pairs are ideal for genetic code expansion to insert noncanonical amino acids (ncAAs) into proteins of interest. Since the discovery of cellular components involved in the biosynthesis and genetic encoding of Pyl, synthetic biologists have been enthusiastic about engineering PylRS/tRNAPyl-derived pairs to rewrite the genetic code of living cells. Recently, considerable progress has been made in understanding the molecular phylogeny, biochemical properties, and structural features of the PylRS/tRNAPyl pair, guiding its further engineering and optimization. In this review, we cover the basic and updated knowledge of the PylRS/tRNAPyl pair's unique characteristics that make it an outstanding tool for reprogramming the genetic code. In addition, we summarize the recent efforts to create efficient and (mutually) orthogonal PylRS/tRNAPyl-derived pairs for incorporation of diverse ncAAs by genome mining, rational design, and advanced directed evolution methods.

Enabling Genetic Code Expansion and Peptide Macrocyclization in mRNA Display via a Promiscuous Orthogonal Aminoacyl-tRNA Synthetase.

mRNA display is revolutionizing peptide drug discovery through its ability to quickly identify potent peptide binders of therapeutic protein targets. Methods to expand the chemical diversity of display libraries are continually needed to increase the likelihood of identifying clinically relevant peptide ligands. Orthogonal aminoacyl-tRNA synthetases (ORSs) have proven utility in cellular genetic code expansion, but are relatively underexplored for in vitro translation (IVT) and mRNA display. Herein, we demonstrate that the promiscuous ORS p-CNF-RS can incorporate noncanonical amino acids at amber codons in IVT, including the novel substrate p-cyanopyridylalanine (p-CNpyrA), to enable a pyridine-thiazoline (pyr-thn) macrocyclization in mRNA display. Pyr-thn-based selections against the deubiquitinase USP15 yielded a potent macrocyclic binder that exhibits good selectivity for USP15 and close homologues over other ubiquitin-specific proteases (USPs). Overall, this work exemplifies how promiscuous ORSs can both expand side chain diversity and provide structural novelty in mRNA display libraries through a heterocycle forming macrocyclization.

Peptidyl tRNA Hydrolase Is Required for Robust Prolyl-tRNA Turnover in Mycobacterium tuberculosis.

Enzymes involved in rescuing stalled ribosomes and recycling translation machinery are ubiquitous in bacteria and required for growth. Peptidyl tRNA drop-off is a type of abortive translation that results in the release of a truncated peptide that is still bound to tRNA (peptidyl tRNA) into the cytoplasm. Peptidyl tRNA hydrolase (Pth) recycles the released tRNA by cleaving off the unfinished peptide and is essential in most bacteria. We developed a sequencing-based strategy called copper sulfate-based tRNA sequencing (Cu-tRNAseq) to study the physiological role of Pth in Mycobacterium tuberculosis (Mtb). While most peptidyl tRNA species accumulated in a strain with impaired Pth expression, peptidyl prolyl-tRNA was particularly enriched, suggesting that Pth is required for robust peptidyl prolyl-tRNA turnover. Reducing Pth levels increased Mtb's susceptibility to tRNA synthetase inhibitors that are in development to treat tuberculosis (TB) and rendered this pathogen highly susceptible to macrolides, drugs that are ordinarily ineffective against Mtb. Collectively, our findings reveal the potency of Cu-tRNAseq for profiling peptidyl tRNAs and suggest that targeting Pth would open new therapeutic approaches for TB. IMPORTANCE Peptidyl tRNA hydrolase (Pth) is an enzyme that cuts unfinished peptides off tRNA that has been prematurely released from a stalled ribosome. Pth is essential in nearly all bacteria, including the pathogen Mycobacterium tuberculosis (Mtb), but it has not been clear why. We have used genetic and novel biochemical approaches to show that when Pth levels decline in Mtb, peptidyl tRNA accumulates to such an extent that usable tRNA pools drop. Thus, Pth is needed to maintain normal tRNA levels, most strikingly for prolyl-tRNAs. Many antibiotics act on protein synthesis and could be affected by altering the availability of tRNA. This is certainly true for tRNA synthetase inhibitors, several of which are drug candidates for tuberculosis. We find that their action is potentiated by Pth depletion. Furthermore, Pth depletion results in hypersensitivity to macrolides, drugs that are not active enough under ordinary circumstances to be useful for tuberculosis.

Adenosine-Dependent Activation Mechanism of Prodrugs Targeting an Aminoacyl-tRNA Synthetase.

Prodrugs have little or no pharmacological activity and are converted to active drugs in the body by enzymes, metabolic reactions, or through human-controlled actions. However, prodrugs promoting their chemical bioconversion without any of these processes have not been reported before. Here, we present an enzyme-independent prodrug activation mechanism by boron-based compounds (benzoxaboroles) targeting leucyl-tRNA synthetase (LeuRS), including an antibiotic that recently has completed phase II clinical trials to cure tuberculosis. We combine nuclear magnetic resonance spectroscopy and X-ray crystallography with isothermal titration calorimetry to show that these benzoxaboroles do not bind directly to their drug target LeuRS, instead they are prodrugs that activate their bioconversion by forming a highly specific and reversible LeuRS inhibition adduct with ATP, AMP, or the terminal adenosine of the tRNALeu. We demonstrate how the oxaborole group of the prodrugs cyclizes with the adenosine ribose at physiological concentrations to form the active molecule. This bioconversion mechanism explains the remarkably good druglike properties of benzoxaboroles showing efficacy against radically different human pathogens and fully explains the mechanism of action of these compounds. Thus, this adenosine-dependent activation mechanism represents a novel concept in prodrug chemistry that can be applied to improve the solubility, permeability and metabolic stability of challenging drugs.