Exploring the dark side of tertiary and quaternary structure dynamics in MtbFBPaseII.

Tuberculosis (TB) is a major global cause of mortality, primarily stemming from latent tuberculosis infection (LTBI). Failure to fully treat LTBI can result in drug-resistant forms of TB. Therefore, it is essential to develop novel drugs with unique mechanisms of action to combat TB effectively. One crucial metabolic pathway in Mycobacterium tuberculosis (Mtb), which contributes to TB infection and persistence, is gluconeogenesis. Within this pathway, the enzyme fructose bisphosphatase (FBPase) plays a significant role and is considered a promising target for drug development. By targeting MtbFBPaseII, a specific class of FBPase, researchers have employed molecular dynamics simulations to identify regions capable of binding new drugs, thereby inhibiting the enzyme's activity and potentially paving the way for the development of effective treatments.Communicated by Ramaswamy H. Sarma.

New structures of Class II Fructose-1,6-Bisphosphatase from Francisella tularensis provide a framework for a novel catalytic mechanism for the entire class.

Class II Fructose-1,6-bisphosphatases (FBPaseII) (EC: 3.1.3.11) are highly conserved essential enzymes in the gluconeogenic pathway of microorganisms. Previous crystallographic studies of FBPasesII provided insights into various inactivated states of the enzyme in different species. Presented here is the first crystal structure of FBPaseII in an active state, solved for the enzyme from Francisella tularensis (FtFBPaseII), containing native metal cofactor Mn2+ and complexed with catalytic product fructose-6-phosphate (F6P). Another crystal structure of the same enzyme complex is presented in the inactivated state due to the structural changes introduced by crystal packing. Analysis of the interatomic distances among the substrate, product, and divalent metal cations in the catalytic centers of the enzyme led to a revision of the catalytic mechanism suggested previously for class II FBPases. We propose that phosphate-1 is cleaved from the substrate fructose-1,6-bisphosphate (F1,6BP) by T89 in a proximal α-helix backbone (G88-T89-T90-I91-T92-S93-K94) in which the substrate transition state is stabilized by the positive dipole of the 〈-helix backbone. Once cleaved a water molecule found in the active site liberates the inorganic phosphate from T89 completing the catalytic mechanism. Additionally, a crystal structure of Mycobacterium tuberculosis FBPaseII (MtFBPaseII) containing a bound F1,6BP is presented to further support the substrate binding and novel catalytic mechanism suggested for this class of enzymes.

Notes of a protein crystallographer: the advantages of combining new integrated methods of structure solution with traditional data visuals.

The suggestion is made that combining analysis using the most advanced crystallographic software with the integrated visual tools of the field will result in more knowledgeable and better trained future generations of structural biologists. The use of integrated visuals could also expedite the structure solution of some recalcitrant and complex macromolecular crystal structures that resist automatic workflows.

Notes of a protein crystallographer: the legacy of J.-B. J. Fourier - crystallography, time and beyond.

The importance of the Fourier transform as a fundamental tool for crystallography is well known in the field. However, the complete legacy of Jean-Baptiste Joseph Fourier (1768-1830) as a pioneer Egyptologist and premier mathematician and physicist of his time, and the implications of his work in other scientific fields, is less well known. Significantly, his theoretical and experimental work on phenomena related to the transmission of heat founded the mathematical study of irreversible phenomena and introduced the flow of time in physico-chemical processes and geology, with its implications for biological evolution. Fourier's insights are discussed in contrast to the prevalent notion of reversible dynamic time in the early 20th century, which was dominated by Albert Einstein's (1875-1953) theory of general relativity versus the philosophical notion of durée proposed by the French philosopher Henri-Louis Bergson (1859-1941). The current status of the mathematical description of irreversible processes by Ilya Romanovich Prigogine (1917-2003) is briefly discussed as part of the enduring legacy of the pioneering work of J.-B. J. Fourier, first established nearly two centuries ago, in numerous scientific endeavors.

Ligand efficiency indices for effective drug discovery: a unifying vector formulation.

INTRODUCTION: The area of ligand efficiency indices (LEIs) in drug discovery has developed significantly since the initial publications nearly 20 years ago. A large number of different LEIs have been defined and applied with certain degrees of success and acceptance in the community. An overall view emphasizing more the common elements than the differences is needed. AREAS COVERED: In this review, the author accentuates the numerical and algebraic relationships among the different LEIs and proposes the notion of 'ligand efficiency index' (LEI) as a vector variable comprising two interrelated components that provide 'direction' and 'distance' along the drug discovery process. The same concept had been suggested before relating to the graphical representation of the content of Structure-Activity Databases (SAR-Databases). EXPERT OPINION: The extension of the concept of ligand efficiency from a scalar to a vector will help to unify the different formulations by emphasizing the relationship among the different variables. It should also provide an algebraically robust framework to critically assess the value of LEIs, and to incorporate them routinely in various workflows and protocols. Only cautious and rigorous testing by the community could provide a definitive proof of their possible value as reliable optimization variables in drug discovery.

Structural and biochemical characterization of the class II fructose-1,6-bisphosphatase from Francisella tularensis.

The crystal structure of the class II fructose-1,6-bisphosphatase (FBPaseII) from the important pathogen Francisella tularensis is presented at 2.4 Šresolution. Its structural and functional relationships to the closely related phosphatases from Mycobacterium tuberculosis (MtFBPaseII) and Escherichia coli (EcFBPaseII) and to the dual phosphatase from Synechocystis strain 6803 are discussed. FBPaseII from F. tularensis (FtFBPaseII) was crystallized in a monoclinic crystal form (space group P21, unit-cell parameters a = 76.30, b = 100.17, c = 92.02 Å, ß = 90.003°) with four chains in the asymmetric unit. Chain A had two coordinated Mg2+ ions in its active center, which is distinct from previous findings, and is presumably deactivated by their presence. The structure revealed an approximate 222 (D2) symmetry homotetramer analogous to that previously described for MtFBPaseII, which is formed by a crystallographic dyad and which differs from the exact tetramer found in EcFBPaseII at a 222 symmetry site in the crystal. Instead, the approximate homotetramer is very similar to that found in the dual phosphatase from Synechocystis, even though no allosteric effector was found in FtFBPase. The amino-acid sequence and folding of the active site of FtFBPaseII result in structural characteristics that are more similar to those of the previously published EcFBPaseII than to those of MtFBPaseII. The kinetic parameters of native FtFBPaseII were found to be in agreement with published studies. Kinetic analyses of the Thr89Ser and Thr89Ala mutations in the active site of the enzyme are consistent with the previously proposed mechanism for other class II bisphosphatases. The Thr89Ala variant enzyme was inactive but the Thr89Ser variant was partially active, with an approximately fourfold lower Km and Vmax than the native enzyme. The structural and functional insights derived from the structure of FtFBPaseII will provide valuable information for the design of specific inhibitors.

Structure of the N-terminal domain of ClpC1 in complex with the antituberculosis natural product ecumicin reveals unique binding interactions.

The biological processes related to protein homeostasis in Mycobacterium tuberculosis, the etiologic agent of tuberculosis, have recently been established as critical pathways for therapeutic intervention. Proteins of particular interest are ClpC1 and the ClpC1-ClpP1-ClpP2 proteasome complex. The structure of the potent antituberculosis macrocyclic depsipeptide ecumicin complexed with the N-terminal domain of ClpC1 (ClpC1-NTD) is presented here. Crystals of the ClpC1-NTD-ecumicin complex were monoclinic (unit-cell parameters a = 80.0, b = 130.0, c = 112.0 Å, ß = 90.07°; space group P21; 12 complexes per asymmetric unit) and diffracted to 2.5 Šresolution. The structure was solved by molecular replacement using the self-rotation function to resolve space-group ambiguities. The new structure of the ecumicin complex showed a unique 1:2 (target:ligand) stoichiometry exploiting the intramolecular dyad in the α-helical fold of the target N-terminal domain. The structure of the ecumicin complex unveiled extensive interactions in the uniquely extended N-terminus, a critical binding site for the known cyclopeptide complexes. This structure, in comparison with the previously reported rufomycin I complex, revealed unique features that could be relevant for understanding the mechanism of action of these potential antituberculosis drug leads. Comparison of the ecumicin complex and the ClpC1-NTD-L92S/L96P double-mutant structure with the available structures of rufomycin I and cyclomarin A complexes revealed a range of conformational changes available to this small N-terminal helical domain and the minor helical alterations involved in the antibiotic-resistance mechanism. The different modes of binding and structural alterations could be related to distinct modes of action.

High-Resolution Structure of ClpC1-Rufomycin and Ligand Binding Studies Provide a Framework to Design and Optimize Anti-Tuberculosis Leads.

Addressing the urgent need to develop novel drugs against drug-resistant Mycobacterium tuberculosis ( M. tb) strains, ecumicin (ECU) and rufomycin I (RUFI) are being explored as promising new leads targeting cellular proteostasis via the caseinolytic protein ClpC1. Details of the binding topology and chemical mode of (inter)action of these cyclopeptides help drive further development of novel potency-optimized entities as tuberculosis drugs. ClpC1 M. tb protein constructs with mutations driving resistance to ECU and RUFI show reduced binding affinity by surface plasmon resonance (SPR). Despite certain structural similarities, ECU and RUFI resistant mutation sites did not overlap in their SPR binding patterns. SPR competition experiments show ECU prevents RUFI binding, whereas RUFI partially inhibits ECU binding. The X-ray structure of the ClpC1-NTD-RUFI complex reveals distinct differences compared to the previously reported ClpC1-NTD-cyclomarin A structure. Surprisingly, the complex structure revealed that the epoxide moiety of RUFI opened and covalently bound to ClpC1-NTD via the sulfur atom of Met1. Furthermore, RUFI analogues indicate that the epoxy group of RUFI is critical for binding and bactericidal activity. The outcomes demonstrate the significance of ClpC1 as a novel target and the importance of SAR analysis of identified macrocyclic peptides for drug discovery.

Structures of the Mycobacterium tuberculosis GlpX protein (class II fructose-1,6-bisphosphatase): implications for the active oligomeric state, catalytic mechanism and citrate inhibition.

The crystal structures of native class II fructose-1,6-bisphosphatase (FBPaseII) from Mycobacterium tuberculosis at 2.6 Šresolution and two active-site protein variants are presented. The variants were complexed with the reaction product fructose 6-phosphate (F6P). The Thr84Ala mutant is inactive, while the Thr84Ser mutant has a lower catalytic activity. The structures reveal the presence of a 222 tetramer, similar to those described for fructose-1,6/sedoheptulose-1,7-bisphosphatase from Synechocystis (strain 6803) as well as the equivalent enzyme from Thermosynechococcus elongatus. This homotetramer corresponds to a homologous oligomer that is present but not described in the crystal structure of FBPaseII from Escherichia coli and is probably conserved in all FBPaseIIs. The constellation of amino-acid residues in the active site of FBPaseII from M. tuberculosis (MtFBPaseII) is conserved and is analogous to that described previously for the E. coli enzyme. Moreover, the structure of the active site of the partially active (Thr84Ser) variant and the analysis of the kinetics are consistent with the previously proposed catalytic mechanism. The presence of metabolites in the crystallization medium (for example citrate and malonate) and in the corresponding crystal structures of MtFBPaseII, combined with their observed inhibitory effect, could suggest the existence of an uncharacterized inhibition of this class of enzymes besides the allosteric inhibition by adenosine monophosphate observed for the Synechocystis enzyme. The structural and functional insights derived from the structure of MtFBPaseII will provide critical information for the design of lead inhibitors, which will be used to validate this target for future chemical intervention.

Mutagenesis of threonine to serine in the active site of Mycobacterium tuberculosis fructose-1,6-bisphosphatase (Class II) retains partial enzyme activity.

The glpX gene encodes for the Class II fructose-1,6-bisphosphatase enzyme in Mycobacterium tuberculosis (Mt), an essential enzyme for pathogenesis. We have performed site directed mutagenesis to introduce two mutations at residue Thr84, T84A and T84S, to explore the binding affinity of the substrate and the catalytic mechanism. The T84A mutant fully abolishes enzyme activity while retaining substrate binding affinity. In contrast, the T84S mutant retains some activity having a 10 times reduction in Vmax and exhibited similar sensitivity to lithium when compared to the wildtype. Homology modeling using the Escherichia coli enzyme structure suggests that the replacement of the critical nucleophile OH- in the Thr84 residue of the wildtype of MtFBPase by Ser84 results in subtle alterations of the position and orientation that reduce the catalytic efficiency. This mutant could be used to trap reaction intermediates, through crystallographic methods, facilitating the design of potent inhibitors via structure-based drug design.

Structures of SAICAR synthetase (PurC) from Streptococcus pneumoniae with ADP, Mg2+, AIR and Asp.

Streptococcus pneumoniae is a multidrug-resistant pathogen that is a target of considerable interest for antibacterial drug development. One strategy for drug discovery is to inhibit an essential metabolic enzyme. The seventh step of the de novo purine-biosynthesis pathway converts carboxyaminoimidazoleribonucleotide (CAIR) and L-aspartic acid (Asp) to 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) in the presence of adenosine 5'-triphosphate (ATP) using the enzyme PurC. PurC has been shown to be conditionally essential for bacterial replication. Two crystal structures of this essential enzyme from Streptococcus pneumoniae (spPurC) in the presence of adenosine 5'-diphosphate (ADP), Mg(2+), aminoimidazoleribonucleotide (AIR) and/or Asp have been obtained. This is the first structural study of spPurC, as well as the first of PurC from any species with Asp in the active site. Based on these findings, two model structures are proposed for the active site with all of the essential ligands (ATP, Mg(2+), Asp and CAIR) present, and a relay mechanism for the formation of the product SAICAR is suggested.

Ask the experts: past, present and future of the rule of five.

Coined in 1997, by Christopher Lipinki et al., the rule of five (Ro5) comprises a set of parameters that determine drug-likeness for oral delivery. The parameters are as follows: no more than five hydrogen bond donors (nitrogen or oxygen atoms with one or more hydrogen atoms); no more than ten hydrogen bond acceptors (nitrogen or oxygen atoms); a molecular mass less than 500 Da; and an octanol-water partition coefficient log P no greater than 5. Future Medicinal Chemistry invited a selection of leading researchers to express their views on Lipinski's Ro5, which has influenced drug design for over a decade. Their enlightening responses provide an insight into the current and future role of Ro5, and other rules of thumb, in the evolving world of medicinal chemistry.

AtlasCBS: a web server to map and explore chemico-biological space.

New approaches are needed that can help decrease the unsustainable failure in small-molecule drug discovery. Ligand Efficiency Indices (LEI) are making a great impact on early-stage compound selection and prioritization. Given a target-ligand database with chemical structures and associated biological affinities/activities for a target, the AtlasCBS server generates two-dimensional, dynamical representations of its contents in terms of LEI. These variables allow an effective decoupling of the chemical (angular) and biological (radial) components. BindingDB, PDBBind and ChEMBL databases are currently implemented. Proprietary datasets can also be uploaded and compared. The utility of this atlas-like representation in the future of drug design is highlighted with some examples. The web server can be accessed at http://ub.cbm.uam.es/atlascbs and https://www.ebi.ac.uk/chembl/atlascbs.

Benzoxazole benzenesulfonamides as allosteric inhibitors of fructose-1,6-bisphosphatase.

A series of novel benzoxazole benzenesulfonamides was synthesized as inhibitors of fructose-1,6-bisphosphatase (FBPase-1). Extensive SAR studies led to a potent inhibitor, 53, with an IC(50) of 0.57microM. Compound 17 exhibited excellent bioavailability and a good pharmacokinetic profile in rats.

Benzoxazole benzenesulfonamides are novel allosteric inhibitors of fructose-1,6-bisphosphatase with a distinct binding mode.

We have identified benzoxazole benzenesulfonamide 1 as a novel allosteric inhibitor of fructose-1,6-bisphosphatase (FBPase-1). X-ray crystallographic and biological studies of 1 indicate a distinct binding mode that recapitulates features of several previously reported FBPase-1 inhibitor classes.