The Role of Water Networks in Phosphodiesterase Inhibitor Dissociation and Kinetic Selectivity.

In search of new opportunities to develop Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) inhibitors that have selectivity over the off-target human PDE4 (hPDE4), different stages of a fragment-growing campaign were studied using a variety of biochemical, structural, thermodynamic, and kinetic binding assays. Remarkable differences in binding kinetics were identified and this kinetic selectivity was explored with computational methods, including molecular dynamics and interaction fingerprint analyses. These studies indicate that a key hydrogen bond between GlnQ.50 and the inhibitors is exposed to a water channel in TbrPDEB1, leading to fast unbinding. This water channel is not present in hPDE4, leading to inhibitors with a longer residence time. The computer-aided drug design protocols were applied to a recently disclosed TbrPDEB1 inhibitor with a different scaffold and our results confirm that shielding this key hydrogen bond through disruption of the water channel represents a viable design strategy to develop more selective inhibitors of TbrPDEB1. Our work shows how computational protocols can be used to understand the contribution of solvent dynamics to inhibitor binding, and our results can be applied in the design of selective inhibitors for homologous PDEs found in related parasites.

Exploring the Activity Profile of TbrPDEB1 and hPDE4 Inhibitors Using Free Energy Perturbation.

Human African trypanosomiasis (HAT) is a neglected tropical disease caused by the parasite Trypanosoma brucei (T.b.). A validated target for the treatment of HAT is the parasitic T.b. cyclic nucleotide phosphodiesterase B1 (TbrPDEB1). Although nanomolar TbrPDEB1 inhibitors have been obtained, their activity against the off-target human PDE4 (hPDE4) is likely to lead to undesirable clinical side effects, such as nausea, emesis, and immune suppression. Thus, new and more selective TbrPDEB1 inhibitors are still needed. This retrospective study evaluated the free energy perturbation (FEP+) method to predict the affinity profiles of TbrPDEB1 inhibitors against hPDE4. We demonstrate that FEP+ can be used to accurately predict the activity profiles of these homologous proteins. Moreover, we show how FEP+ can overcome challenges like protein flexibility and high sequence conservation. This also implies that the method can be applied prospectively for the lead optimization campaigns to design new and more selective TbrPDEB1 inhibitors.

Progress in Free Energy Perturbation: Options for Evolving Fragments.

One of the remaining bottlenecks in fragment-based drug design (FBDD) is the initial exploration and optimization of the identified hit fragments. There is a growing interest in computational approaches that can guide these efforts by predicting the binding affinity of newly designed analogues. Among others, alchemical free energy (AFE) calculations promise high accuracy at a computational cost that allows their application during lead optimization campaigns. In this review, we discuss how AFE could have a strong impact in fragment evolution, and we raise awareness on the challenges that could be encountered applying this methodology in FBDD studies.

A malachite green light-up aptasensor for the detection of theophylline.

Biosensors are of interest for the quantitative detection of small molecules (metabolites, drugs and contaminants for instance). To this end, fluorescence is a widely used technique that is easily associated to aptamers. Light-up aptamers constitute a particular class of oligonucleotides that, specifically induce fluorescence emission when binding to cognate fluorogenic ligands such as malachite green (MG). We engineered a dual aptasensor for theophylline (Th) based on the combination of switching hairpin aptamers specific for MG on the one hand and for Th on the other hand, hence their names: malaswitch (Msw) and theoswitch (Thsw). The two aptaswitches form a loop-loop or kissing Msw-Thsw complex only in the presence of theophylline, allowing binding of MG, subsequently generating a fluorescent signal. The combination of the best Msw and Thsw variants, MswG12 and Thsw19.1, results in a 20-fold fluorescence enhancement of MG at saturating theophylline concentration. This aptasensor discriminates between theophylline and its analogues caffeine and theobromine. Kissing aptaswitches derived from light-up aptamers constitute a novel sensing device.

Anti-pesticide DNA aptamers fail to recognize their targets with asserted micromolar dissociation constants.

Herein, we originally aimed at developing fluorescence anisotropy biosensor platforms devoted to the homogeneous-phase detection of isocarbophos and phorate pesticides by using previously isolated DNA aptamers. To achieve this, two reporting approaches displaying very high generalizability features were implemented, based on either the complementary strand or the SYBR green intercalator displacement strategies. Unfortunately, none of the transduction methods led to phorate-dependent signals. Only the SYBR green displacement method provided a small output in the presence of isocarbophos, but at an analyte concentration greater than 100 µM. In order to identify the origin of such data, isothermal titration calorimetry (ITC) experiments were subsequently performed. It was shown that aptamers bind neither isocarbophos nor phorate in free solution with the claimed micromolar dissociation constants. This work puts forward some doubts about the previously described aptasensors that rely on the use of these functional DNA molecules. It also highlights the need to carefully investigate the binding capabilities of aptamers after their isolation and to include appropriate control experiments with scrambled or mutated oligonucleotides.

Engineering Light-Up Aptamers for the Detection of RNA Hairpins through Kissing Interaction.

Aptasensors are biosensors that include aptamers for detecting a target of interest. We engineered signaling aptasensors for the detection of RNA hairpins from the previously described malachite green (MG) RNA aptamer. The top part of this imperfect hairpin aptamer was modified in such a way that it can engage loop-loop (so-called kissing) interactions with RNA hairpins displaying partly complementary apical loops. These newly derived oligonucleotides named malaswitches bind their cognate fluorogenic ligand (MG) exclusively when RNA-RNA kissing complexes are formed, whereas MG does not bind to malaswitches alone. Consequently, the formation of the ternary target RNA-malaswitch RNA-MG complex results in fluorescence emission, and malaswitches constitute sensors for detecting RNA hairpins. Malaswitches were designed that specifically detect precursors of microRNAs let7b and miR-206.

Functionalized C-nucleosides as remarkable RNA binders: targeting of prokaryotic ribosomal A-site RNA.

RNA represents an extremely promising and yet challenging therapeutic target. Here, we report the design of a series of C-nucleosides as original RNA binders. Some of them bind strongly and selectively to A-site prokaryotic ribosomal RNA.

Development of an impedimetric aptasensor for the determination of aflatoxin M1 in milk.

An aptasensor was designed for the determination of aflatoxin M1 (AFM1) in milk based on DNA-aptamer recognition and electrochemical impedance spectroscopy detection. A hexaethyleneglycol-modified 21-mer oligonucleotide was immobilized on a carbon screen-printed electrode through carbodiimide immobilization, after diazonium activation of the sensing surface. Cyclic voltammetry and electrochemical impedance spectroscopy in the presence of ferri/ferrocyanide redox probe were used to characterize each step of the aptasensor development. Aptamer-AFM1 interaction induced an increase in electron-transfer resistance, allowing the determination of AFM1 in buffer in the range 2-150 ng/L (LOD=1.15 ng/L). Application to milk analysis showed that a preliminary treatment was mandatory. A simple filtration through a 0.2 µm PTFE membrane allowed determination of AFM1 in milk for concentrations ranging from 20 to 1000 ng/kg. These performances are compatible with the AFM1 levels set in European Union for milk and dairy products for adults (50 ng/kg) and infants (25 ng/kg).