Frederik R. Ehrmann

Six Biophysical Screening Methods Miss a Large Proportion of Crystallographically Discovered Fragment Hits: A Case Study

Fragment-based lead discovery (FBLD) has become a pillar in drug development. Typical applications of this method comprise at least two biophysical screens as prefilter and a follow-up crystallographic experiment on a subset of fragments. Clearly, structural information is pivotal in FBLD, but a key question is whether such a screening cascade strategy will retrieve the majority of fragment-bound structures. We therefore set out to screen 361 fragments for binding to endothiapepsin, a representative of the challenging group of aspartic proteases, employing six screening techniques and crystallography in parallel. Crystallography resulted in the very high number of 71 structures. Yet alarmingly, 44% of these hits were not detected by any biophysical screening approach. Moreover, any screening cascade, building on the results from two or more screening methods, would have failed to predict at least 73% of these hits. We thus conclude that, at least in the present case, the frequently applied biophysical prescreening filters deteriorate the number of possible X-ray hits while only the immediate use of crystallography enables exhaustive retrieval of a maximum of fragment structures, which represent a rich source guiding hit-to-lead-to-drug evolution.

Experimental Active-Site Mapping by Fragments - Hot Spots Remote from the Catalytic Center of Endothiapepsin

Successful optimization of a given lead scaffold requires thorough binding-site mapping of the target protein particular in regions remote from the catalytic center where high conservation across protein families is given. We screened a 361-entry fragment library for binding to the aspartic protease endothiapepsin by crystallography. This enzyme is frequently used as a surrogate for the design of renin and β-secretase inhibitors. A hit rate of 20% was achieved, providing 71 crystal structures. Here, we discuss 45 binding poses of fragments accommodated in pockets remote from the catalytic dyad. Three major hot spots are discovered in remote binding areas: Asp81, Asp119, and Phe291. Compared to the dyad binders, bulkier fragments occupy these regions. Many of the discovered fragments suggest an optimization concept on how to grow them into larger ligands occupying adjacent binding pockets that will possibly endow them with the desired selectivity for one given member of a protein family.

Replacement of Water Molecules in a Phosphate Binding Site by Furanoside‐Appended lin‐Benzoguanine Ligands of tRNA‐Guanine Transglycosylase (TGT)

The enzyme tRNA-guanine transglycosylase has been identified as a drug target for the foodborne illness shigellosis. A key challenge in structure-based design for this enzyme is the filling of the polar ribose-34 pocket. Herein, we describe a novel series of ligands consisting of furanoside-appended lin-benzoguanines. They were designed to replace a conserved water cluster and differ by the functional groups at C(2) and C(3) of the furanosyl moiety being either OH or OMe. The unfavorable desolvation of Asp102 and Asp280, which are located close to the ribose-34 pocket, had a significant impact on binding affinity. While the enzyme has tRNA as its natural substrate, X-ray co-crystal structures revealed that the furanosyl moieties of the ligands are not accommodated in the tRNA ribose-34 site, but at the location of the adjacent phosphate group. A remarkable similarity of the position of the oxygen atoms in these two structures suggests furanosides as a potential phosphate isoster.

Occupying a flat subpocket in a tRNA-modifying enzyme with ordered or disordered side chains: Favorable or unfavorable for binding?

Small-molecule ligands binding with partial disorder or enhanced residual mobility are usually assumed as unfavorable with respect to their binding properties. Considering thermodynamics, disorder or residual mobility is entropically favorable and contributes to the Gibbs energy of binding. In the present study, we analyzed a series of congeneric ligands inhibiting the tRNA-modifying enzyme TGT. Attached to the parent lin-benzoguanine scaffold, substituents in position 2 accommodate in a flat solvent-exposed pocket and exhibit varying degree of residual mobility. This is indicated in the crystal structures by enhanced B-factors, reduced occupancies, or distributions over split conformers. MD simulations of the complexes suggest an even larger scatter over several conformational families. Introduction of a terminal acidic group fixes the substituent by a salt-bridge to an Arg residue. Overall, all substituted derivatives show the same affinity underpinning that neither order nor disorder is a determinant factor for binding affinity. The additional salt bridge remains strongly solvent-exposed and thus does not contribute to affinity. MD suggests temporary fluctuation of this contact.

Soaking suggests "alternative facts": Only cocrystallization discloses major ligand-induced interface rearrangements of a homodimeric tRNA-binding protein indicating a novel mode-of-inhibition

For the efficient pathogenesis of Shigella, the causative agent of bacillary dysentery, full functionality of tRNA-guanine transglycosylase (TGT) is mandatory. TGT performs post-transcriptional modifications of tRNAs in the anticodon loop taking impact on virulence development. This suggests TGT as a putative target for selective anti-shigellosis drug therapy. Since bacterial TGT is only functional as homodimer, its activity can be inhibited either by blocking its active site or by preventing dimerization. Recently, we discovered that in some crystal structures obtained by soaking the full conformational adaptation most likely induced in solution upon ligand binding is not displayed. Thus, soaked structures may be misleading and suggest irrelevant binding modes. Accordingly, we re-investigated these complexes by co-crystallization. The obtained structures revealed large conformational rearrangements not visible in the soaked complexes. They result from spatial perturbations in the ribose-34/phosphate-35 recognition pocket and, consequently, an extended loop-helix motif required to prevent access of water molecules into the dimer interface loses its geometric integrity. Thermodynamic profiles of ligand binding in solution indicate favorable entropic contributions to complex formation when large conformational adaptations in the dimer interface are involved. Native MS titration experiments reveal the extent to which the homodimer is destabilized in the presence of each inhibitor. Unexpectedly, one ligand causes a complete rearrangement of subunit packing within the homodimer, never observed in any other TGT crystal structure before. Likely, this novel twisted dimer is catalytically inactive and, therefore, suggests that stabilizing this non-productive subunit arrangement may be used as a further strategy for TGT inhibition.

A False-Positive Screening Hit in Fragment-Based Lead Discovery: Watch out for the Red Herring

With the rising popularity of fragment-based approaches in drug development, more and more attention has to be devoted to the detection of false-positive screening results. In particular, the small size and low affinity of fragments drives screening techniques to their limit. The pursuit of a false-positive hit can cause significant loss of time and resources. Here, we present an instructive and intriguing investigation into the origin of misleading assay results for a fragment that emerged as the most potent binder for the aspartic protease endothiapepsin (EP) across multiple screening assays. This molecule shows its biological effect mainly after conversion into another entity through a reaction cascade that involves major rearrangements of its heterocyclic scaffold. The formed ligand binds EP through an induced-fit mechanism involving remarkable electrostatic interactions. Structural information in the initial screening proved to be crucial for the identification of this false-positive hit.

An Immucillin-Based Transition-State-Analogous Inhibitor of tRNA-Guanine Transglycosylase (TGT).

Shigellosis is one of the most severe diarrheal diseases worldwide without any efficient treatment so far. The enzyme tRNA-guanine transglycosylase (TGT) has been identified as a promising target for small-molecule drug design. Herein, we report a transition-state analogue, a small, immucillin-derived inhibitor, as a new lead structure with a novel mode of action. The complex inhibitor synthesis was accomplished in 18 steps with an overall yield of 3 %. A co-crystal structure of the inhibitor bound to Z. mobilis TGT confirmed the predicted conformation of the immucillin derivative in the enzyme active site.

Sugar Acetonides are a Superior Motif for Addressing the Large, Solvent-Exposed Ribose-33 Pocket of tRNA-Guanine Transglycosylase

The intestinal disease shigellosis caused by Shigella bacteria affects over 120 million people annually. There is an urgent demand for new drugs as resistance against common antibiotics emerges. Bacterial tRNA-guanine transglycosylase (TGT) is a druggable target and controls the pathogenicity of Shigella flexneri. We report the synthesis of sugar-functionalized lin-benzoguanines addressing the ribose-33 pocket of TGT from Zymomonas mobilis. Ligand binding was analyzed by isothermal titration calorimetry and X-ray crystallography. Pocket occupancy was optimized by variation of size and protective groups of the sugars. The participation of a polycyclic water-cluster in the recognition of the sugar moiety was revealed. Acetonide-protected ribo- and psicofuranosyl derivatives are highly potent, benefiting from structural rigidity, good solubility, and metabolic stability. We conclude that sugar acetonides have a significant but not yet broadly recognized value in drug development.

Swapping Interface Contacts in the Homodimeric tRNA‐Guanine Transglycosylase: An Option for Functional Regulation

The enzyme tRNA-guanine transglycosylase, a target to fight Shigellosis, recognizes tRNA only as a homodimer and performs full nucleobase exchange at the wobble position. Active-site inhibitors block the enzyme function by competitively replacing tRNA. In solution, the wild-type homodimer dissociates only marginally, whereas mutated variants show substantial monomerization in solution. Surprisingly, one inhibitor transforms the protein into a twisted state, whereby one monomer unit rotates by approximately 130°. In this altered geometry, the enzyme is no longer capable of binding and processing tRNA. Three sugar-type inhibitors have been designed and synthesized, which bind to the protein in either the functionally competent or twisted inactive state. They crystallize with the enzyme side-by-side under identical conditions from the same crystallization well. Possibly, the twisted inactive form corresponds to a resting state of the enzyme, important for its functional regulation.