Halo-1,2,3-triazoles: Valuable Compounds to Access Biologically Relevant Molecules.

1,2,3-triazole is an important building block in organic chemistry. It is now well known as a bioisostere for various functions, such as the amide or the ester bond, positioning it as a key pharmacophore in medicinal chemistry and it has found applications in various fields including life sciences. Attention was first focused on the synthesis of 1,4-disubstituted 1,2,3-triazole molecules however 1,4,5-trisubstituted 1,2,3-triazoles have now emerged as valuable molecules due to the possibility to expand the structural modularity. In the last decade, methods mainly derived from the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction have been developed to access halo-triazole compounds and have been applied to nucleosides, carbohydrates, peptides and proteins. In addition, late-stage modification of halo-triazole derivatives by metal-mediated cross-coupling or halo-exchange reactions offer the possibility to access highly functionalized molecules that can be used as tools for chemical biology. This review summarizes the synthesis, the functionalization, and the applications of 1,4,5-trisubstituted halo-1,2,3-triazoles in biologically relevant molecules.

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 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 bisubstrate analogue 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 catalysed by METTL3, and suggests that the latter step is rate-limiting. 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.

Synthesis of Bisubstrate Analogues for RNA Methylation Studies using two Transition-Metal-Catalyzed Reactions.

RNA methyltransferases (RNA MTases) are a family of enzymes that catalyze the methylation of RNA using the cofactor S-adenosyl-L-methionine. While RNA MTases are promising drug targets, new molecules are needed to fully understand their roles in disease and to develop effective drugs that can modulate their activity. Since RNA MTases are suitable for bisubstrate binding, we report an original strategy for the synthesis of a new family of m6A MTases bisubstrate analogues. Six compounds containing a S-adenosyl-L-methionine (SAM) analogue unit covalently tethered by a triazole ring to the N-6 position of an adenosine were synthesized. A procedure using two transition-metal-catalyzed reactions was used to introduce the α-amino acid motif mimicking the methionine chain of the cofactor SAM. First, a copper(I)-catalyzed alkyne-azide iodo-cycloaddition (iCuAAC) reaction afforded the 5-iodo-1,4-disubstituted-1,2,3-triazole which was functionalized by palladium-catalyzed cross-coupling to connect the α-amino acid substituent. Docking studies of our molecules in the active site of the m6A ribosomal MTase RlmJ show that the use of triazole as a linker provides additional interactions and the presence of the α-amino acid chain stabilizes the bisubstrate. The synthetic method developed here enhances the structural diversity of bisubstrate analogues to explore the active site of RNA modification enzymes and to develop new inhibitors.

Modulation of the Specificity of Carbapenems and Diazabicyclooctanes for Selective Activity against Mycobacterium tuberculosis.

Treatment of multidrug-resistant tuberculosis with combinations of carbapenems and ß-lactamase inhibitors carries risks for dysbiosis and for the development of resistances in the intestinal microbiota. Using Escherichia coli producing carbapenemase KPC-2 as a model, we show that carbapenems can be modified to obtain drugs that are inactive against E. coli but retain antitubercular activity. Furthermore, functionalization of the diazabicyclooctanes scaffold provided drugs that did not effectively inactivate KPC-2 but retained activity against Mycobacterium tuberculosis targets.

Synthesis of RNA-cofactor conjugates and structural exploration of RNA recognition by an m6A RNA methyltransferase.

Chemical synthesis of RNA conjugates has opened new strategies to study enzymatic mechanisms in RNA biology. To gain insights into poorly understood RNA nucleotide methylation processes, we developed a new method to synthesize RNA-conjugates for the study of RNA recognition and methyl-transfer mechanisms of SAM-dependent m6A RNA methyltransferases. These RNA conjugates contain a SAM cofactor analogue connected at the N6-atom of an adenosine within dinucleotides, a trinucleotide or a 13mer RNA. Our chemical route is chemo- and regio-selective and allows flexible modification of the RNA length and sequence. These compounds were used in crystallization assays with RlmJ, a bacterial m6A rRNA methyltransferase. Two crystal structures of RlmJ in complex with RNA-SAM conjugates were solved and revealed the RNA-specific recognition elements used by RlmJ to clamp the RNA substrate in its active site. From these structures, a model of a trinucleotide bound in the RlmJ active site could be built and validated by methyltransferase assays on RlmJ mutants. The methyl transfer by RlmJ could also be deduced. This study therefore shows that RNA-cofactor conjugates are potent molecular tools to explore the active site of RNA modification enzymes.

Click and Release Chemistry for Activity-Based Purification of ß-Lactam Targets.

ß-Lactams, the cornerstone of antibiotherapy, inhibit multiple and partially redundant targets referred to as transpeptidases or penicillin-binding proteins. These enzymes catalyze the essential cross-linking step of the polymerization of cell wall peptidoglycan. The understanding of the mechanisms of action of ß-lactams and of resistance to these drugs requires the development of reliable methods to characterize their targets. Here, we describe an activity-based purification method of ß-lactam targets based on click and release chemistry. We synthesized alkyne-carbapenems with suitable properties with respect to the kinetics of acylation of a model target, the Ldtfm L,D-transpeptidase, the stability of the resulting acylenzyme, and the reactivity of the alkyne for the cycloaddition of an azido probe containing a biotin moiety for affinity purification and a bioorthogonal cleavable linker. The probe provided access to the fluorescent target in a single click and release step.

Synthesis of Carbapenems Containing Peptidoglycan Mimetics and Inhibition of the Cross-Linking Activity of a Transpeptidase of l,d Specificity.

The carbapenem class of ß-lactams has been optimized against Gram-negative bacteria producing extended-spectrum ß-lactamases by introducing substituents at position C2. Carbapenems are currently investigated for the treatment of tuberculosis as these drugs are potent covalent inhibitors of l,d-transpeptidases involved in mycobacterial cell wall assembly. The optimization of carbapenems for inactivation of these unusual targets is sought herein by exploiting the nucleophilicity of the C8 hydroxyl group to introduce chemical diversity. As ß-lactams are structure analogs of peptidoglycan precursors, the substituents were chosen to increase similarity between the drug and the substrate. Fourteen peptido-carbapenems were efficiently synthesized. They were more effective than the reference drug, meropenem, owing to the positive impact of a phenethylthio substituent introduced at position C2 but the peptidomimetics added at position C8 did not further improve the activity. Thus, position C8 can be modified to modulate the pharmacokinetic properties of highly efficient carbapenems.

Synthesis of Triazole-Linked SAM-Adenosine Conjugates: Functionalization of Adenosine at N-1 or N-6 Position without Protecting Groups.

More than 150 RNA chemical modifications have been identified to date. Among them, methylation of adenosine at the N-6 position (m6A) is crucial for RNA metabolism, stability and other important biological events. In particular, this is the most abundant mark found in mRNA in mammalian cells. The presence of a methyl group at the N-1 position of adenosine (m1A) is mostly found in ncRNA and mRNA and is mainly responsible for stability and translation fidelity. These modifications are installed by m6A and m1A RNA methyltransferases (RNA MTases), respectively. In human, deregulation of m6A RNA MTases activity is associated with many diseases including cancer. To date, the molecular mechanism involved in the methyl transfer, in particular substrate recognition, remains unclear. We report the synthesis of new SAM-adenosine conjugates containing a triazole linker branched at the N-1 or N-6 position of adenosine. Our methodology does not require protecting groups for the functionalization of adenosine at these two positions. The molecules described here were designed as potential bisubstrate analogues for m6A and m1A RNA MTases that could be further employed for structural studies. This is the first report of compounds mimicking the transition state of the methylation reaction catalyzed by m1A RNA MTases.

Bisubstrate analogues as structural tools to investigate m6A methyltransferase active sites.

RNA methyltransferases (MTases) catalyse the transfer of a methyl group to their RNA substrates using most-often S-adenosyl-L-methionine (SAM) as cofactor. Only few RNA-bound MTases structures are currently available due to the difficulties in crystallising RNA:protein complexes. The lack of complex structures results in poorly understood RNA recognition patterns and methylation reaction mechanisms. On the contrary, many cofactor-bound MTase structures are available, resulting in well-understood protein:cofactor recognition, that can guide the design of bisubstrate analogues that mimic the state at which both the substrate and the cofactor is bound. Such bisubstrate analogues were recently synthesized for proteins monomethylating the N6-atom of adenine (m6A). These proteins include, amongst others, RlmJ in E. coli and METLL3:METT14 and METTL16 in human. As a proof-of-concept, we here test the ability of the bisubstrate analogues to mimic the substrate:cofactor bound state during catalysis by studying their binding to RlmJ using differential scanning fluorimetry, isothermal titration calorimetry and X-ray crystallography. We find that the methylated adenine base binds in the correct pocket, and thus these analogues could potentially be used broadly to study the RNA recognition and catalytic mechanism of m6A MTases. Two bisubstrate analogues bind RlmJ with micro-molar affinity, and could serve as starting scaffolds for inhibitor design against m6A RNA MTases. The same analogues cause changes in the melting temperature of the m1A RNA MTase, TrmK, indicating non-selective protein:compound complex formation. Thus, optimization of these molecular scaffolds for m6A RNA MTase inhibition should aim to increase selectivity, as well as affinity.

Critical Impact of Peptidoglycan Precursor Amidation on the Activity of l,d-Transpeptidases from Enterococcus faecium and Mycobacterium tuberculosis.

The bacterial cell wall peptidoglycan contains unusual l- and d-amino acids assembled as branched peptides. Insight into the biosynthesis of the polymer has been hampered by limited access to substrates and to suitable polymerization assays. Here we report the full synthesis of the peptide stem of peptidoglycan precursors from two pathogenic bacteria, Enterococcus faecium and Mycobacterium tuberculosis, and the development of a sensitive post-derivatization assay for their cross-linking by l,d-transpeptidases. Access to series of stem peptides showed that amidation of free carboxyl groups is essential for optimal enzyme activity, in particular the amidation of diaminopimelate (DAP) residues for the cross-linking activity of the l,d-transpeptidase LdtMt2 from M. tuberculosis. Accordingly, construction of a conditional mutant established the essential role of AsnB indicating that this DAP amidotransferase is an attractive target for the development of anti-mycobacterial drugs.

Synthesis of tRNA analogues containing a terminal ribose locked in the South conformation to study tRNA-dependent enzymes.

We report here the synthetic route of two constrained dinucleotides and the determination of the sugar puckering by NMR analyses of the starting nucleosides. Enzymatic ligation to microhelix-RNAs provide access to tRNA analogues containing a 3' terminal A76 locked in South conformation. Biological evaluation of our tRNA analogues has been performed using amino-acyl tRNA-dependent transferase FemXWv, which mediates non-ribosomal incorporation of amino acids into the bacterial cell wall. We have shown that our tRNA analogues inhibited the aminoacyl transfer reaction catalyzed by FemXWv with IC50s of 10 and 8 µM. These results indicate that FemXWv displays a moderate preference for tRNAs containing a terminal A76 locked in the South conformation and that a South to North switch in the conformation of the terminal ribose might contribute to the release of the uncharged tRNAAla product of the aminoacyl transfer reaction catalyzed by FemXwv.

Insights into the interaction of high potency inhibitor IRC-083864 with phosphatase CDC25.

CDC25 phosphatases play a crucial role in cell cycle regulation. They have been found to be over-expressed in various human tumours and to be valuable targets for cancer treatment. Here, we report the first model of binding of the most potent CDC25 inhibitor to date, the bis-quinone IRC-083864, into CDC25B obtained by combining molecular modeling and NMR studies. Our study provides new insights into key interactions of the catalytic site inhibitor and CDC25B in the absence of any available experimental structure of CDC25 with a bound catalytic site inhibitor. The docking model reveals that IRC-083864 occupies both the active site and the inhibitor binding pocket of the CDC25B catalytic domain. NMR saturation transfer difference and WaterLOGSY data indicate the binding zones of the inhibitor and support the docking model. Probing interactions of analogues of the two quinone units of IRC-083864 with CDC25B demonstrate that IRC-083864 competes with each monomer. Proteins 2017; 85:593-601. © 2016 Wiley Periodicals, Inc.

Electrophilic RNA for Peptidyl-RNA Synthesis and Site-Specific Cross-Linking with tRNA-Binding Enzymes.

RNA functionalization is challenging due to the instability of RNA and the limited range of available enzymatic reactions. We developed a strategy based on solid phase synthesis and post-functionalization to introduce an electrophilic site at the 3' end of tRNA analogues. The squarate diester used as an electrophile enabled sequential amidation and provided asymmetric squaramides with high selectivity. The squaramate-RNAs specifically reacted with the lysine of UDP-MurNAc-pentapeptide, a peptidoglycan precursor used by the aminoacyl-transferase FemXWv for synthesis of the bacterial cell wall. The peptidyl-RNA obtained with squaramate-RNA and unprotected UDP-MurNAc-pentapeptide efficiently inhibited FemXWv . The squaramate unit also promoted specific cross-linking of RNA to the catalytic Lys of FemXWv but not to related transferases recognizing different aminoacyl-tRNAs. Thus, squaramate-RNAs provide specificity for cross-linking with defined groups in complex biomolecules due to its unique reactivity.

Routes of Synthesis of Carbapenems for Optimizing Both the Inactivation of L,D-Transpeptidase LdtMt1 of Mycobacterium tuberculosis and the Stability toward Hydrolysis by ß-Lactamase BlaC.

Combinations of ß-lactams of the carbapenem class, such as meropenem, with clavulanate, a ß-lactamase inhibitor, are being evaluated for the treatment of drug-resistant tuberculosis. However, carbapenems approved for human use have never been optimized for inactivation of the unusual ß-lactam targets of Mycobacterium tuberculosis or for escaping to hydrolysis by broad-spectrum ß-lactamase BlaC. Here, we report three routes of synthesis for modification of the two side chains carried by the ß-lactam and the five-membered rings of the carbapenem core. In particular, we show that the azide-alkyne Huisgen cycloaddition reaction catalyzed by copper(I) is fully compatible with the highly unstable ß-lactam ring of carbapenems and that the triazole ring generated by this reaction is well tolerated for inactivation of the L,D-transpeptidase LdtMt1 target. Several of our new carbapenems are superior to meropenem both with respect to the efficiency of in vitro inactivation of LdtMt1 and reduced hydrolysis by BlaC.

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.

Synthesis of 3'-triazoyl-dinucleotides as precursors of stable Phe-tRNA(Phe) and Leu-tRNA(Leu) analogues.

We report here the synthesis of stable Phe-tRNA(Phe) and Leu-tRNA(Leu) analogues containing a 1,2,3-triazole ring instead of the ribose-amino acid ester bond. The 1,2,3-triazole ring is generated by dipolar cycloaddition of alkyne Phe and Leu analogues to 3'-azido-3'-deoxyadenosine via the Cu(I)-catalysed Huisgen, Meldal, Sharpless 1,3-cycloaddition. The corresponding triazoyl pdCpA dinucleotides, obtained by classical phosphoramidite chemistry, were enzymatically ligated to 22-nt or 74-nt RNA generating stable Phe-tRNA(Phe) analogues containing the acceptor stem or full tRNA moieties, respectively. These molecules represent useful tools to study the contribution of the RNA and amino acid moieties in stabilization of aminoacyl-tRNA/protein complexes.

Synthesis and biological evaluation of non-isomerizable analogues of Ala-tRNA(Ala).

Aminoacyl-tRNAs serve as amino acid donors in many reactions in addition to protein synthesis by the ribosome, including synthesis of the peptidoglycan network in the cell wall of bacterial pathogens. Synthesis of analogs of aminoacylated tRNAs is critical to further improve the mechanism of these reactions. Here we have described the synthesis of two non-isomerizable analogues of Ala-tRNA(Ala) containing an amide bond instead of the isomerizable ester that connects the amino acid with the terminal adenosine in the natural substrate. The non-isomerizable 2' and 3' regioisomers were not used as substrates by FemX(Wv), an alanyl-transferase essential for peptidoglycan synthesis, but inhibited this enzyme with IC50 of 5.8 and 5.5 µM, respectively.

Design and synthesis of novel bis-thiazolone derivatives as micromolar CDC25 phosphatase inhibitors: effect of dimerisation on phosphatase inhibition.

CDC25 phosphatases are involved in deregulated cell cycle progression and tumor development with poor prognosis. Among the most potent CDC25 inhibitors, quinonoid-based derivatives have been extensively studied. Dimerisation of heterocyclic quinones has led to IRC-083864, a bis-quinone compound with increased CDC25B inhibitory activity. Thirty-one bis-thiazolone derivatives were synthesized and assayed for CDC25 inhibitory activity. Most of the dimers displayed enhanced inhibitory activities with micromolar IC(50) values lower than that observed for each thiazolone scaffold separately. Moreover, most of these compounds were selective CDC25 inhibitors. Dimer 40 showed an IC(50) value of 2.9 µM and could inhibit CDC25 activity without generating reactive oxygen species which is likely to occur with quinone-based inhibitors. Molecular docking studies suggested that the dimers could bind simultaneously to the active site and the inhibitor binding pocket.

Study of the docking-dependent PLK1 phosphorylation of the CDC25B phosphatase.

CDC25 (A, B and C) phosphatases control cell cycle progression through the timely dephosphorylation and activation of cyclin-dependent kinases (CDK). At mitosis the CDC25B phosphatase activity is dependent on its phosphorylation by multiple kinases impinging on its localisation, stability and catalytic activity. Here we report that prior phosphorylation of CDC25B by CDK1 enhances its substrate properties for PLK1 in vitro, and we also show that phosphorylated S50 serves as a docking site for PLK1. Using a sophisticated strategy based on the sequential phosphorylation of CDC25B with (16)O and (18)O ATP prior to nanoLC-MS/MS analysis we identified 13 sites phosphorylated by PLK1. This study illustrates the complexity of the phosphorylation pattern and of the subsequent regulation of CDC25B activity.