Targeting the phosphoserine phosphatase MtSerB2 for tuberculosis drug discovery, an hybrid knowledge based /fragment based approach.

Tuberculosis is currently still one of the leading causes of death from a treatable pathogen. The proportion of cases of resistance to common antibiotics is frequently increasing and the development of new drugs with new therapeutic targets is becoming necessary. The Mycobacterium tuberculosis phosphoserine phosphatase MtSerB2 is an interesting enzyme to target in drug design because of its ability to allow immune evasion of the bacteria. Research has already been carried out on this protein both from a mechanistic point of view and from the point of view of its inhibition by trisubstituted harmine derivatives. Based on this work, a new approach based on virtual screening is presented in the selection of fragment-sized harmine-derived compounds as well as chelators to target the catalytic magnesium of MtSerB2. The selection of a minimum list of fragments is explained as well as the screening cascade (DSF, Ligand-based NMR, High concentration enzymatic assay) to characterise their affinity for MtSerB2. Crystallogenesis assays have provided structural information on some promising fragments and the development of a pharmacophore model with the structural elements necessary for the development of more complex inhibitors. Ultimately, this work on fragment growth would allow the development of antimycobacterial molecules inhibiting MtSerB2 as well as the growth of the pathogen.

Investigation of bound and unbound phosphoserine phosphatase conformations through elastic network models and molecular dynamics simulations.

The human phosphoserine phosphatase (hPSP) catalyses the last step in the biosynthesis of L-serine. It involves conformational changes of the enzyme lid once the substrate, phosphoserine (PSer), is bound in the active site. Here, Elastic Network Model (ENM) is applied to the crystal structure of hPSP to probe the transition between open and closed conformations of hPSP. Molecular Dynamics (MD) simulations are carried out on several PSer-hPSP systems to characterise the intermolecular interactions and their effect on the dynamics of the enzyme lid. Systems involving either Ca++ or Mg++ are considered. The first ENM normal mode shows that an open-closed transition can be explained from a simple description of the enzyme in terms of harmonic potentials. Principal Component Analyses applied to the MD trajectories also highlight a trend for a closing/opening motion. Different PSer orientations inside the enzyme cavity are identified, i.e. either the carboxylate, the phosphate group of PSer, or both, are oriented towards the cation. The interaction patterns are analysed in terms of hydrogen bonds, electrostatics, and bond critical points of the electron density distributions. The latter approach yields a global description of the bonding intermolecular interactions. The PSer orientation determines the content of the cation coordination shell and the mobility of the substrate, while Lys158 and Thr182, involved in the reaction mechanism, are always in interaction with the substrate. Closed enzyme conformations involve Met52-Gln204, Arg49-Glu29, and Arg50-Glu29 interactions. Met52, as well as Arg49 and Arg50, also stabilize PSer inside the cavity. Communicated by Ramaswamy H. Sarma.

Identification and Repurposing of Trisubstituted Harmine Derivatives as Novel Inhibitors of Mycobacterium tuberculosis Phosphoserine Phosphatase.

Mycobacterium tuberculosis is still the deadliest bacterial pathogen worldwide and the increasing number of multidrug-resistant tuberculosis cases further complicates this global health issue. M. tuberculosis phosphoserine phosphatase SerB2 is a promising target for drug design. Besides being a key essential metabolic enzyme of the pathogen's serine pathway, it appears to be involved in immune evasion mechanisms. In this work, a malachite green-based phosphatase assay has been used to screen 122 compounds from an internal chemolibrary. Trisubstituted harmine derivatives were found among the best hits that inhibited SerB2 activity. Synthesis of an original compound helped to discuss a brief structure activity relationship evaluation. Kinetics experiments showed that the most potent derivatives inhibit the phosphatase in a parabolic competitive fashion with apparent inhibition constants ( K i ) values in the micromolar range. Their interaction modes with the enzyme were investigated through induced fit docking experiments, leading to results consistent with the experimental data. Cellular assays showed that the selected compounds also inhibited M. tuberculosis growth in vitro. Those promising results may provide a basis for the development of new antimycobacterial agents targeting SerB2.

Crystal structures and snapshots along the reaction pathway of human phosphoserine phosphatase.

The equilibrium between phosphorylation and dephosphorylation is one of the most important processes that takes place in living cells. Human phosphoserine phosphatase (hPSP) is a key enzyme in the production of serine by the dephosphorylation of phospho-L-serine. It is directly involved in the biosynthesis of other important metabolites such as glycine and D-serine (a neuromodulator). hPSP is involved in the survival mechanism of cancer cells and has recently been found to be an essential biomarker. Here, three new high-resolution crystal structures of hPSP (1.5-2.0 Å) in complexes with phosphoserine and with serine, which are the substrate and the product of the reaction, respectively, and in complex with a noncleavable substrate analogue (homocysteic acid) are presented. New types of interactions take place between the enzyme and its ligands. Moreover, the loop involved in the open/closed state of the enzyme is fully refined in a totally unfolded conformation. This loop is further studied through molecular-dynamics simulations. Finally, all of these analyses allow a more complete reaction mechanism for this enzyme to be proposed which is consistent with previous publications on the subject.

Targeting the Serine Pathway: A Promising Approach against Tuberculosis?

Tuberculosis is still the leading cause of death by a single infectious agent. Effective chemotherapy has been used and improved since the 1950s, but strains resistant to this therapy and most antibacterial drugs on the market are emerging. Only 10 new drugs are in clinical trials, and two of them have already demonstrated resistance. This paper gives an overview of current treatment options against tuberculosis and points out a promising approach of discovering new effective drugs. The serine production pathway is composed of three enzymes (SerA1, SerC and SerB2), which are considered essential for bacterial growth, and all of them are considered as a therapeutic drug target. Their crystal structure are described and essential regulatory domains pointed out. Sequence alignment with similar enzymes in other host would help to identify key residues to target in order to achieve selective inhibition. Currently, only inhibitors of SerB2 are described in the literature. However, inhibitors of human enzymes are discussed, and could be used as a good starting point for a drug discovery program. The aim of this paper is to give some guidance for the design of new hits for every enzyme in this pathway.

Synthesis of 4- and 5-arylthiazolinethiones as inhibitors of indoleamine 2,3-dioxygenase.

Docking studies of 4-phenylthiazolinethione on human IDO1 suggest complexation of the heme iron by the exocyclic sulfur atom further reinforced by hydrophobic interactions of the phenyl ring within pocket A of the enzyme. On this basis, chemical modifications were proposed to increase inhibition activity. Synthetic routes had to be adapted and optimized to yield the desired substituted 4- and 5-arylthiazolinethiones. Their biological evaluation shows that 5-aryl regioisomers are systematically less potent than the corresponding 4-aryl analogs. Substitution on the phenyl ring does not significantly increase inhibition potency, except for 4-Br and 4-Cl derivatives.