Design Of Transition-state Analogs Research Articles

The catalytic mechanism of the RNA methyltransferase METTL3.

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on messenger RNA (mRNA) in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a BA representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release, and suggests that the latter step is rate-limiting for METTL3. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors.

The catalytic mechanism of the RNA methyltransferase METTL3

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on messenger RNA (mRNA) in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a BA representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release, and suggests that the latter step is rate-limiting for METTL3. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors.

New insights into the catalytic mechanism of the SARS-CoV-2 main protease: an ONIOM QM/MM approach.

SARS-CoV-2 Mpro, also known as the main protease or 3C-like protease, is a key enzyme involved in the replication process of the virus that is causing the COVID-19 pandemic. It is also the most promising antiviral drug target targeting SARS-CoV-2 virus. In this work, the catalytic mechanism of Mpro was studied using the full model of the enzyme and a computational QM/MM methodology with a 69/72-atoms QM region treated at DLPNO-CCSD(T)/CBS//B3LYP/6-31G(d,p):AMBER level and including the catalytic important oxyanion-hole residues. The transition state of each step was fully characterized and described together with the related reactants and products. The rate-limiting step of the catalytic process is the hydrolysis of the thioester-enzyme adduct, and the calculated barrier closely agrees with the available kinetic data. The calculated Gibbs free energy profile, together with the full atomistic detail of the structures involved in catalysis, can now serve as valuable models for the rational drug design of transition state analogs as new inhibitors targeting the SARS-CoV-2 virus.Graphic abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s11030-021-10259-7.

Characterization and development of new anti‐toxins targeting Clostridium difficile

Clostridium difficile is a bacterial pathogen that causes serious and potentially fatal inflammatory disease of the colon. One strategy for targeting C. difficile infection is to development treatments that target bacterial virulence. Anti-virulence strategies to treat C. difficile infection are attractive options to antibiotic therapy because the human microbiota is spared and damage to host tissue is minimized. The Tcd toxins, TcdA and TcdB, are the major virulence factors produced by C. difficile and mediate host cytotoxicity by glucosylating and inactivating Rho family GTPases using UDP-glucose as a glucosyl donor. Glucosylation of Rho GTPases leads to cell rounding and eventually cell death. We used kinetic isotope effects (KIEs) and transition state theory to solve the transition state structure of the glycosyltransferase domain of TcdB (TcdB-GTD). This permits the design of transition state analogues which are potent enzyme inhibitors. KIEs compare the chemical steps of enzymatic reaction rates with isotopically labeled substrates and unlabeled substrates and provide critical information on the catalytic mechanism, bond lengths and geometry at the transition state. KIE analysis on the glycohydrolase reaction of TcdB-GTD supported the formation of a late, dissociative glucocation-like transition state where positive charge develops on the anomeric carbon. Glucocationic transition states are best mimicked by cationic nitrogen groups at or near the anomeric carbon. Iminosugars contain a nitrogen atom in the sugar ring in place of oxygen and mimic glucocationic transition states. We tested iminosugars for inhibition of TcdB-GTD and TcdA-GTD. Isofagomine (Ki TcdB-GTD = 1.4 µM and Ki TcdA-GTD = 0.24 µM) and noeuromycin (Ki TcdB-GTD = 10.6 µM and Ki TcdA-GTD = 4.7 µM) were the most potent iminosugars identified. Isofagomine and noeuromycin were shown to exhibit an uncompetitive mechanism of inhibition suggesting that both iminosugars act by binding to the enzyme-UDP complex. Isothermal titration calorimetry experiments with TcdB-GTD supported this finding where isofagomine and noeuromycin could only bind to TcdB-GTD in the presence of UDP with Kd = 0.133 µM and Kd = 0.4 µM respectively. In addition, the X-ray crystal structure of TcdB-GTD in complex with either Isofagomine or noeuromycin with UDP revealed the formation of an ion-pair interaction between the cationic nitrogen atom of the iminosugar and the β-phosphate of UDP. Finally, we confirmed that isofagomine and noeuromycin could prevent Tcd toxin-induced cytotoxicity of IMR90 cells. Firstly, both iminosugars were able to prevent Tcd toxin-induced cell rounding as visualized by light microscopy. Secondly, Western blot analysis of Tcd toxin treated IMR90 cell lysates revealed that both isofagomine and noeuromycin could prevent glucosylation of Rac1 by Tcd toxin. Isofagomine and noeuromycin are effective in preventing Tcd toxin action. Transition state analogues against TcdA and TcdB have the potential to be used as treatments for C. difficile infections.

The transition to magic bullets - transition state analogue drug design.

In the absence of industry partnerships, most academic groups lack the infrastructure to rationally design and build drugs via methods used in industry. Instead, academia needs to work smarter using mechanism-based design. Working smarter can mean the development of new drug discovery paradigms and then demonstrating their utility and reproducibility to industry. The collaboration between Vern Schramm's group at the Albert Einstein College of Medicine, USA and Peter Tyler at the Ferrier Research Institute at The Victoria University of Wellington, NZ has refined a drug discovery process called transition state analogue design. This process has been applied to several biomedically relevant nucleoside processing enzymes. In 2017, Mundesine®, conceived using transition state analogue design, received market approval for the treatment of peripheral T-cell lymphoma in Japan. This short review looks at a brief history of transition state analogue design, the fundamentals behind the development of this process, and the success of enzyme inhibitors produced using this drug design methodology.

Transition State Structure of RNA Depurination by Saporin L3.

Saporin L3 from the leaves of the common soapwort is a catalyst for hydrolytic depurination of adenine from RNA. Saporin L3 is a type 1 ribosome inactivating protein (RIP) composed only of a catalytic domain. Other RIPs have been used in immunotoxin cancer therapy, but off-target effects have limited their development. In the current study, we use transition state theory to understand the chemical mechanism and transition state structure of saporin L3. In favorable cases, transition state structures guide the design of transition state analogues as inhibitors. Kinetic isotope effects (KIEs) were determined for an A14C mutant of saporin L3. To permit KIE measurements, small stem-loop RNAs that contain an AGGG tetraloop structure were enzymatically synthesized with the single adenylate bearing specific isotopic substitutions. KIEs were measured and corrected for forward commitment to obtain intrinsic values. A model of the transition state structure for depurination of stem-loop RNA (5'-GGGAGGGCCC-3') by saporin L3 was determined by matching KIE values predicted via quantum chemical calculations to a family of intrinsic KIEs. This model indicates saporin L3 displays a late transition state with the N-ribosidic bond to the adenine nearly cleaved, and the attacking water nucleophile weakly bonded to the ribosyl anomeric carbon. The transition state retains partial ribocation character, a feature common to most N-ribosyl transferases. However, the transition state geometry for saporin L3 is distinct from ricin A-chain, the only other RIP whose transition state is known.

Quantum Chemical Approaches: Semiempirical Molecular Orbital and Hybrid Quantum Mechanical/Molecular Mechanical Techniques

The use of computational quantum chemical methods to aid drug discovery is surveyed. An overview of the various computational models spanning ab initio, density function theory, semiempirical molecular orbital (MO), and hybrid quantum mechanical (QM)/molecular mechanical (MM) methods is given and their strengths and weaknesses are highlighted, focussing on the challenge of obtaining the accuracy essential for them to make a meaningful contribution to drug discovery. Particular attention is given to hybrid QM/MM and semiempirical MO methods which have the potential to yield the necessary accurate predictions of macromolecular structure and reactivity. These methods are shown to have advanced the study of many aspects of substrate-ligand interactions relevant to drug discovery. Thus, the successful parametrization of semiempirical MO methods and QM/MM methods can be used to model noncovalent substrate-protein interactions, and to lead to improved scoring functions. QM/MM methods can be used in crystal structure refinement and are particularly valuable for modelling covalent protein-ligand interactions and can thus aid the design of transition state analogues. An extensive collection of examples from the areas of metalloenzyme structure, enzyme inhibition, and ligand binding affinities and scoring functions are used to illustrate the power of these techniques.

Transition-State-Guided Drug Design for Treatment of Parasitic Neglected Tropical Diseases

Many of the deadliest neglected tropical diseases are caused by protozoan and helminthic parasites. These organisms have evolved several enzymes to exploit their host's metabolic resources and evade immune responses. Because these essential proteins are absent in humans, they are targets for antiparasitic drug development. Despite decades of investigation, no therapy has been successful in the eradication of these diseases, so new approaches are desired. Chemically stable analogues of the transition states of enzymatic reactions are often potent inhibitors, and several examples of clinically effective compounds are known for other diseases. The design of transition-state analogues is aided by structural models of the transition state, which are obtained by complementing experimental measurement of kinetic isotope effects with theoretical calculations. Such transition-state-guided inhibitor design has been demonstrated for human, bovine, malarial, and trypanosomal enzymes of the purine salvage pathway, including purine nucleoside phosphorylase, nucleoside hydrolases, and adenosine deaminase. Cysteine proteases, trans-sialidase, 1-deoxy-d-xylulose-5-phosphate reductoisomerase, and trypanothione synthetase are presented as additional candidates for application of transition-state analysis with the goal of identifying new leads for the treatment of parasitic neglected tropical diseases.

Transition-state inhibitors of purine salvage and other prospective enzyme targets in malaria

Malaria is a leading cause of human death within the tropics. The gradual generation of drug resistance imposes an urgent need for the development of new and selective antimalarial agents. Kinetic isotope effects coupled to computational chemistry have provided the relevant details on geometry and charge of enzymatic transition states to facilitate the design of transition-state analogs. These features have been reproduced into chemically stable mimics through synthetic chemistry, generating inhibitors with dissociation constants in the pico- to femto-molar range. Transition-state analogs are expected to contribute to the control of malaria.

Purine and Pyrimidine Pathways as Targets in Plasmodium falciparum

Malaria is a leading cause of morbidity and mortality in the tropics. Chemotherapeutic and vector control strategies have been applied for more than a century but have not been efficient in disease eradication. Increased resistance of malaria parasites to drug treatment and of mosquito vectors to insecticides requires the development of novel chemotherapeutic agents. Malaria parasites exhibit rapid nucleic acid synthesis during their intraerythrocytic growth phase. Plasmodium purine and pyrimidine metabolic pathways are distinct from those of their human hosts. Thus, targeting purine and pyrimidine metabolic pathways provides a promising route for novel drug development. Recent developments in enzymatic transition state analysis have provided an improved route to inhibitor design targeted to specific enzymes, including those of purine and pyrimidine metabolism. Modern transition state analogue drug discovery has resulted in transition state analogues capable of binding to target enzymes with unprecedented affinity and specificity. These agents can provide specific blocks in essential pathways. The combination of tight binding with the high specificity of these logically designed inhibitors, results in low toxicity and minor side effects. These features reduce two of the major problems with the current antimalarials. Transition state analogue design is being applied to generate new lead compounds to treat malaria by targeting purine and pyrimidine pathways.

Enzymatic Transition States, Transition-State Analogs, Dynamics, Thermodynamics, and Lifetimes

Experimental analysis of enzymatic transition-state structures uses kinetic isotope effects (KIEs) to report on bonding and geometry differences between reactants and the transition state. Computational correlation of experimental values with chemical models permits three-dimensional geometric and electrostatic assignment of transition states formed at enzymatic catalytic sites. The combination of experimental and computational access to transition-state information permits (a) the design of transition-state analogs as powerful enzymatic inhibitors, (b) exploration of protein features linked to transition-state structure, (c) analysis of ensemble atomic motions involved in achieving the transition state, (d) transition-state lifetimes, and (e) separation of ground-state (Michaelis complexes) from transition-state effects. Transition-state analogs with picomolar dissociation constants have been achieved for several enzymatic targets. Transition states of closely related isozymes indicate that the protein's dynamic architecture is linked to transition-state structure. Fast dynamic motions in catalytic sites are linked to transition-state generation. Enzymatic transition states have lifetimes of femtoseconds, the lifetime of bond vibrations. Binding isotope effects (BIEs) reveal relative reactant and transition-state analog binding distortion for comparison with actual transition states.

Catalysis: transition-state molecular recognition?

The key to understanding the fundamental processes of catalysis is the transition state (TS): indeed, catalysis is a transition-state molecular recognition event. Practical objectives, such as the design of TS analogues as potential drugs, or the design of synthetic catalysts (including catalytic antibodies), require prior knowledge of the TS structure to be mimicked. Examples, both old and new, of computational modelling studies are discussed, which illustrate this fundamental concept. It is shown that reactant binding is intrinsically inhibitory, and that attempts to design catalysts that focus simply upon attractive interactions in a binding site may fail. Free-energy changes along the reaction coordinate for SN2 methyl transfer catalysed by the enzyme catechol-O-methyl transferase are described and compared with those for a model reaction in water, as computed by hybrid quantum-mechanical/molecular-mechanical molecular dynamics simulations. The case is discussed of molecular recognition in a xylanase enzyme that stabilises its sugar substrate in a (normally unfavourable) boat conformation and in which a single-atom mutation affects the free-energy of activation dramatically.

Four generations of transition-state analogues for human purine nucleoside phosphorylase

Inhibition of human purine nucleoside phosphorylase (PNP) stops growth of activated T-cells and the formation of 6-oxypurine bases, making it a target for leukemia, autoimmune disorders, and gout. Four generations of ribocation transition-state mimics bound to PNP are structurally characterized. Immucillin-H (K*i(1/4) 58 pM, first generation)contains an iminoribitol cation with four asymmetric carbons. DADMe-Immucillin-H (K*i(1/4) 9 pM, second-generation),uses a methylene-bridged dihydroxypyrrolidine cation with twoasymmetric centers.DATMe-Immucillin-H (K*i(1/4)9 pM, third-generation) contains an open-chain amino alcohol cation with two asymmetric carbons. SerMe-ImmH (K*i(1/4) 5 pM, fourth-generation) uses achiral dihydroxyaminoalcohol seramide as the ribocation mimic. Crystal structures of PNPs establish features of tight binding to be; 1) ion-pair formation between bound phosphate (or its mimic) and inhibitor cation, 2) leaving-group interactions to N1, O6, and N7 of 9-deazahypoxanthine, 3) interaction between phosphate and inhibitor hydroxyl groups, and 4) His257 interacting with the 5'-hydroxyl group. The first generation analogue is an imperfect fit to the catalytic site with a long ion pair distance between the iminoribitol and bound phosphate and weaker interactions to the leaving group. Increasing the ribocation to leaving-group distance in the second- to fourth-generation analogues provides powerful binding interactions and a facile synthetic route to powerful inhibitors. Despite chemical diversity in the four generations of transition-state analogues, the catalytic site geometry is almost the same for all analogues. Multiple solutions in transition-state analogue design are available to convert the energy of catalytic rate enhancement to binding energy in human PNP.

Design of transition-state analogues for gaba-transaminase

Compounds which inhibit the mitochondrial enzyme GABA-transaminase have considerable potential as anticonvulsant drugs. An approach which may provide potent and specific enzyme inhibitors involves the use of the transition-state structure of the reaction catalyzed by the enzyme as a template for the design of transition-state analogues. Molecular orbital calculations have been used to calculate the potential energy surface and identify the transition-state structure for the reaction catalyzed by GABA-transaminase. A series of transition-state analogues based on this structure have been synthesized and were found to be biologically active. An active site model has also been developed on the basis of the calculated reaction pathway.

Rational Design of Transition-State Analogues as Potent Enzyme Inhibitors with Therapeutic Applications

The structures of the transition states for a variety of enzyme-catalyzed ribosyl group transfer reactions, determined by computational evaluation of multiple tritium and heavy atom kinetic isotope effects on these enzymatic reactions, have been found to show a considerable variation in the extent of bond cleavage at the ribosyl anomeric carbon. The calculated transition-state structures have been used to guide the design of high-affinity transition-state analogue inhibitors for 5'-methylthioadenosine nucleosidases with potential as therapeutic agents.

Enzymatic Transition State Theory and Transition State Analogue Design

Enzymatic Transition State Theory and Transition State Analogue Design

Deacylation Transition States of a Bacterial DD-Peptidase

Beta-lactam antibiotics restrict bacterial growth by inhibiting DD-peptidases. These enzymes catalyze the final transpeptidation step in bacterial cell wall biosynthesis. Although much structural information is now available for these enzymes, the mechanism of the actual transpeptidation reaction has not been studied in detail. The reaction is known to involve a double-displacement mechanism with an acyl-enzyme intermediate, which can be attacked by water, specific amino acids, peptides, and other acyl acceptors. We describe in this paper an investigation of acyl acceptor specificity and assess the need for general base catalysis in the deacylation transition state of the Streptomyces R61 DD-peptidase. We show, by the criterion of solvent deuterium kinetic isotope effect measurements and proton inventories, that the transition states of specific and nonspecific substrates are very similar, at least with respect to proton motion. The transition states for attack (tetrahedral intermediate formation) by d-amino acids and Gly-l-Xaa dipeptides do not include a general base catalyst, while such catalysis is essential for reaction with water and d-alpha-hydroxy acids. D-Alpha-hydroxy acids act as acyl acceptors for glycyl substrates but not for more specific d-alanyl substrates; hydroxy acids actually behave, more generally, as mixed inhibitors of the DD-peptidase. The structural and mechanistic bases of these observations are discussed; they should inform transition state analogue design.

Femtomolar Transition State Analogue Inhibitors of 5′-Methylthioadenosine/S-Adenosylhomocysteine Nucleosidase from Escherichia coli

Escherichia coli 5'-methylthioadenosine/S-adenosyl-homocysteine nucleosidase (MTAN) hydrolyzes its substrates to form adenine and 5-methylthioribose (MTR) or S-ribosylhomocysteine (SRH). 5'-Methylthioadenosine (MTA) is a by-product of polyamine synthesis and SRH is a precursor to the biosynthesis of one or more quorum sensing autoinducer molecules. MTAN is therefore involved in quorum sensing, recycling MTA from the polyamine pathway via adenine phosphoribosyltransferase and recycling MTR to methionine. Hydrolysis of MTA by E. coli MTAN involves a highly dissociative transition state with ribooxacarbenium ion character. Iminoribitol mimics of MTA at the transition state of MTAN were synthesized and tested as inhibitors. 5'-Methylthio-Immucillin-A (MT-ImmA) is a slow-onset tight-binding inhibitor giving a dissociation constant (K(i)(*)) of 77 pm. Substitution of the methylthio group with a p-Cl-phenylthio group gives a more powerful inhibitor with a dissociation constant of 2 pm. DADMe-Immucillins are better inhibitors of E. coli MTAN, since they are more closely related to the highly dissociative nature of the transition state. MT-DADMe-Immucillin-A binds with a K(i)(*) value of 2 pm. Replacing the 5'-methyl group with other hydrophobic groups gave 17 transition state analogue inhibitors with dissociation constants from 10(-12) to 10(-14) m. The most powerful inhibitor was 5'-p-Cl-phenylthio-DADMe-Immucillin-A (pClPhT-DADMe-ImmA) with a K(i)(*) value of 47 fm (47 x 10(-15) m). These are among the most powerful non-covalent inhibitors reported for any enzyme, binding 9-91 million times tighter than the MTA and SAH substrates, respectively. The inhibitory potential of these transition state analogue inhibitors supports a transition state structure closely resembling a fully dissociated ribooxacarbenium ion. Powerful inhibitors of MTAN are candidates to disrupt key bacterial pathways including methylation, polyamine synthesis, methionine salvage, and quorum sensing. The accompanying article reports crystal structures of MTAN with these analogues.

The Synthesis of N‐Ribosyl Transferase Inhibitors Based on a Transition State Blueprint.

AbstractFor Abstract see ChemInform Abstract in Full Text.

The Synthesis of N-Ribosyl Transferase Inhibitors Based on a Transition State Blueprint

A quarter of a century ago transition state analysis and transition state analogue design promised the prospect of extraordinarily potent enzyme inhibitors. The present overview describes the transition state analysis of a variety of N-ribosyl transferases, the design and synthesis of extremely powerful transition state analogue inhibitors of these nucleoside processing enzymes, and their current therapeutic uses and potentials.