Guillaume Bec

kinITC: A New Method for Obtaining Joint Thermodynamic and Kinetic Data by Isothermal Titration Calorimetry

Isothermal titration calorimetry (ITC) is the method of choice for obtaining thermodynamic data on a great variety of systems. Here we show that modern ITC apparatus and new processing methods allow researchers to obtain a complete kinetic description of systems more diverse than previously thought, ranging from simple ligand binding to complex RNA folding. We illustrate these new features with a simple case (HIV-1 reverse transcriptase/inhibitor interaction) and with the more complex case of the folding of a riboswitch triggered by the binding of its ligand. The originality of the new kinITC method lies in its ability to dissect, both thermodynamically and kinetically, the two components: primary ligand binding and subsequent RNA folding. We are not aware of another single method that can yield, in a simple way, such deep insight into a composite process. Our study also rationalizes common observations from daily ITC use.

HIV-1 Vif binds to APOBEC3G mRNA and inhibits its translation

The HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells (including most natural HIV-1 targets) by counteracting the cellular cytosine deaminases APOBEC-3G (hA3G) and hA3F. The Vif-induced degradation of these restriction factors by the proteasome has been extensively studied, but little is known about the translational repression of hA3G and hA3F by Vif, which has also been proposed to participate in Vif function. Here, we studied Vif binding to hA3G mRNA and its role in translational repression. Filter binding assays and fluorescence titration curves revealed that Vif tightly binds to hA3G mRNA. Vif overall binding affinity was higher for the 3′UTR than for the 5′UTR, even though this region contained at least one high affinity Vif binding site (apparent Kd = 27 ± 6 nM). Several Vif binding sites were identified in 5′ and 3′UTRs using RNase footprinting. In vitro translation evidenced that Vif inhibited hA3G translation by two mechanisms: a main time-independent process requiring the 5′UTR and an additional time-dependent, UTR-independent process. Results using a Vif protein mutated in the multimerization domain suggested that the molecular mechanism of translational control is more complicated than a simple physical blockage of scanning ribosomes.

Identification of modifications in microbial, native tRNA that suppress immunostimulatory activity

2′-O-methylation of guanosine 18 is a naturally occurring tRNA modification that can suppress immune TLR7 responses.

Discovery of Chiral Cyclopropyl Dihydro-Alkylthio-Benzyl-Oxopyrimidine (S-DABO) Derivatives as Potent HIV-1 Reverse Transcriptase Inhibitors with High Activity Against Clinically Relevant Mutants

The role played by stereochemistry in the C2-substituent (left part) on the S-DABO scaffold for anti-HIV-1 activity has been investigated for the first time. A series of S-DABO analogues, where the double bond in the C2-substituent is replaced by an enantiopure isosteric cyclopropyl moiety, has been synthesized, leading to the identification of a potent lead compound endowed with picomolar activity against RT (wt) and nanomolar activity against selected drug-resistant mutants. Molecular modeling calculation, enzymatic studies, and surface plasmon resonance experiments allowed us to rationalize the biological behavior of the synthesized compounds, which act as mixed-type inhibitors of HIV-1 RT K103N, with a preferential association to the enzyme-substrate complex. Taken together, our data show that the right combination of stereochemistry on the left and right parts (C6-substituent) of the S-DABO scaffold plays a key role in the inhibition of both wild-type and drug-resistant enzymes, especially the K103N mutant.

Crystal Structure of HIV-1 Reverse Transcriptase Bound to a Non-Nucleoside Inhibitor with a Novel Mechanism of Action

HIV-1 reverse transcriptase (RT) is a heterodimeric enzyme that converts the genomic viral RNA into proviral DNA. HIV-1 RT inhibitors used in antiretroviral therapy are divided into two classes: 1) nucleoside-analogue RT inhibitors (NRTIs), which compete with the natural nucleoside substrate and terminate proviral DNA synthesis, and 2) nonnucleoside RT inhibitors (NNRTIs), which are a family of structurally diverse compounds that bind to a hydrophobic pocket (non-nucleoside inhibitor binding pocket, NNIBP) adjacent to the polymerase active site. In spite of the efficiency of NRTIs and NNRTIs, the rapid emergence of multidrug-resistant mutations requires the development of new RT inhibitors with an alternative mechanism of action. Recently, two families of compounds forming a new class of HIV-1 RT inhibitors with a novel inhibition mechanism have been reported: the indolopyridones VRX-329747 and INDOPY-1 (or VRX-413638), and the 4-dimethylamino-6vinylpyrimidines (DAVPs, Scheme 1). Unlike classical NNRTIs, these non-nucleoside RT inhibitors compete with the nucleotide substrate and remain unaffected by most mutations that alter NNRTIs binding and activity. Consequently, it was proposed to refer to this class of compounds as “nucleotide-competing RT inhibitors” (NcRTIs). In contrast to common NNRTIs, the antiviral activity of INDOPY-1 is not restricted to HIV-1 but is extended to HIV-2 and SIV, likely reflecting the binding of the inhibitor close to the polymerase active site. Unfortunately, attempts to obtain a crystal structure of the RT/INDOPY-1 complex were unsuccessful, probably because of the specificity of this NcRTI for the RT/template primer complex. Here we present the first crystal structure at 2.5 resolution of the HIV-1 RT bound to a NcRTI, DAVP-1. The structure reveals a novel binding site, distinct from the NNIBP and close to the RT polymerase catalytic site. The structural requirements for binding of this NcRTI to the RT disclosed by this structure provide essential information for the rational development of new NcRTIs. Because of their structural similarity with the nonnucleosidic reference compound TNK-651 (Scheme 1) and the loss of activity observed against the two most frequent mutations associated with NNRTIs resistance (Lys103Asn and Tyr181Ile), it was initially expected that the DAVPs would also behave as NNRTIs. However, enzymological studies on DAVP-1, DAVP-2, and DAVP-3 revealed a competitive inhibition mechanism with the nucleotide substrate, similar that of the indolopyridone INDOPY-1. But in contrast to INDOPY-1, the binding kinetics of DAVP-1 did not indicate a specificity for the RT/template primer complex: kinetic analysis revealed that DAVP-1 binds the free RT with the same equilibrium dissociation constant as the RT/DNA complex (Ki = 8 nm). [5] In addition, a marked decrease of the association rate (kon) was observed only for the RT/DNA/ dNTP complex (compared to the free enzyme and to the RT/ DNA complex), with no significant change in the dissociation rate (koff). These data are therefore consistent with the classification of DAVP-1 in the newly identified NcRTI class of RT inhibitors. Considering the peculiar behavior of this Scheme 1. Chemical structures of the DAVP derivatives, the reference NNRTI (TNK-651), and the indolopyridones VRX-329747 and INDOPY-1.

Thermodynamics of HIV-1 reverse transcriptase in action elucidates the mechanism of action of non-nucleoside inhibitors.

HIV-1 reverse transcriptase (RT) is a heterodimeric enzyme that converts the genomic viral RNA into proviral DNA. Despite intensive biochemical and structural studies, direct thermodynamic data regarding RT interactions with its substrates are still lacking. Here we addressed the mechanism of action of RT and of non-nucleoside RT inhibitors (NNRTIs) by isothermal titration calorimetry (ITC). Using a new incremental-ITC approach, a step-by-step thermodynamic dissection of the RT polymerization activity showed that most of the driving force for DNA synthesis is provided by initial dNTP binding. Surprisingly, thermodynamic and kinetic data led to a reinterpretation of the mechanism of inhibition of NNRTIs. Binding of NNRTIs to preformed RT/DNA complexes is hindered by a kinetic barrier and NNRTIs mostly interact with free RT. Once formed, RT/NNRTI complexes bind DNA either in a seemingly polymerase-competent orientation or form high-affinity dead-end complexes, both RT/NNRTI/DNA complexes being unable to bind the incoming nucleotide substrate.

Quantitative and predictive model of kinetic regulation by E. coli TPP riboswitches.

ABSTRACT Riboswitches are non-coding elements upstream or downstream of mRNAs that, upon binding of a specific ligand, regulate transcription and/or translation initiation in bacteria, or alternative splicing in plants and fungi. We have studied thiamine pyrophosphate (TPP) riboswitches regulating translation of thiM operon and transcription and translation of thiC operon in E. coli, and that of THIC in the plant A. thaliana. For all, we ascertained an induced-fit mechanism involving initial binding of the TPP followed by a conformational change leading to a higher-affinity complex. The experimental values obtained for all kinetic and thermodynamic parameters of TPP binding imply that the regulation by A. thaliana riboswitch is governed by mass-action law, whereas it is of kinetic nature for the two bacterial riboswitches. Kinetic regulation requires that the RNA polymerase pauses after synthesis of each riboswitch aptamer to leave time for TPP binding, but only when its concentration is sufficient. A quantitative model of regulation highlighted how the pausing time has to be linked to the kinetic rates of initial TPP binding to obtain an ON/OFF switch in the correct concentration range of TPP. We verified the existence of these pauses and the model prediction on their duration. Our analysis also led to quantitative estimates of the respective efficiency of kinetic and thermodynamic regulations, which shows that kinetically regulated riboswitches react more sharply to concentration variation of their ligand than thermodynamically regulated riboswitches. This rationalizes the interest of kinetic regulation and confirms empirical observations that were obtained by numerical simulations.

Double methylation of tRNA-U54 to 2′-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7

Abstract Sensing of nucleic acids for molecular discrimination between self and non-self is a challenging task for the innate immune system. RNA acts as a potent stimulus for pattern recognition receptors including in particular human Toll-like receptor 7 (TLR7). Certain RNA modifications limit potentially harmful self-recognition of endogenous RNA. Previous studies had identified the 2′-O-methylation of guanosine 18 (Gm18) within tRNAs as an antagonist of TLR7 leading to an impaired immune response. However, human tRNALys3 was non-stimulatory despite lacking Gm18. To identify the underlying molecular principle, interferon responses of human peripheral blood mononuclear cells to differentially modified tRNALys3 were determined. The investigation of synthetic modivariants allowed attributing a significant part of the immunosilencing effect to the 2′-O-methylthymidine (m5Um) modification at position 54. The effect was contingent upon the synergistic presence of both methyl groups at positions C5 and 2’O, as shown by the fact that neither Um54 nor m5U54 produced any effect alone. Testing permutations of the nucleobase at ribose-methylated position 54 suggested that the extent of silencing and antagonism of the TLR7 response was governed by hydrogen patterns and lipophilic interactions of the nucleobase. The results identify a new immune-modulatory endogenous RNA modification that limits TLR7 activation by RNA.