Fragment-Based Identification of an Inducible Binding Site on Cell Surface Receptor CD44 for the Design of Protein–Carbohydrate Interaction Inhibitors

Selective inhibitors of hyaluronan (HA) binding to the cell surface receptor CD44 will have value as probes of CD44-mediated signaling and have potential as therapeutic agents in chronic inflammation, cardiovascular disease, and cancer. Using biophysical binding assays, fragment screening, and crystallographic characterization of complexes with the CD44 HA binding domain, we have discovered an inducible pocket adjacent to the HA binding groove into which small molecules may bind. Iterations of fragment combination and structure-driven design have allowed identification of a series of 1,2,3,4-tetrahydroisoquinolines as the first nonglycosidic inhibitors of the CD44–HA interaction. The affinity of these molecules for the CD44 HA binding domain parallels their ability to interfere with CD44 binding to polymeric HA in vitro. X-ray crystallographic complexes of lead compounds are described and compared to a new complex with a short HA tetrasaccharide, to establish the tetrahydroisoquinoline pharmacophore as an attractive starting point for lead optimization

Searching for Likeness in a Database of Macromolecular Complexes

A software tool and workflow based on distance geometry is presented that can be used to search for local similarity in substructures in a comprehensive database of experimentally derived macromolecular structure. The method does not rely on fold annotation, specific secondary structure assignments, or sequence homology and may be used to locate compound substructures of multiple segments spanning different macromolecules that share a queried backbone geometry. This generalized substructure searching capability is intended to allow users to play an active part in exploring the role specific substructures play in larger protein domains, quaternary assemblies of proteins, and macromolecular complexes of proteins and polynucleotides. The user may select any portion or portions of an existing structure or complex to serve as a template for searching, and other structures that share the same structural features are identified, retrieved and overlaid to emphasize substructural likeness. Matching structures may be compared using a variety of integrated tools including molecular graphics for structure visualization and matching substructure sequence logos. A number of examples are provided that illustrate how generalized substructure searching may be used to understand both the similarity, and individuality of specific macromolecular structures. Web-based access to our substructure searching services is freely available at https://drugsite.msi.umn.edu

Caught before Released: Structural Mapping of the Reaction Trajectory for the Sofosbuvir Activating Enzyme, Human Histidine Triad Nucleotide Binding Protein 1 (hHint1)

Human histidine triad nucleotide binding protein 1 (hHint1) is classified as an efficient nucleoside phosphoramidase and acyl-adenosine monophosphate hydrolase. Human Hint1 has been shown to be essential for the metabolic activation of nucleotide antiviral pronucleotides (i.e., proTides), such as the FDA approved hepatitis C drug, sofosbuvir. The active site of hHint1 comprises an ensemble of strictly conserved histidines, including nucleophilic His112. To structurally investigate the mechanism of hHint1 catalysis, we have designed and prepared nucleoside thiophosphoramidate substrates that are able to capture the transiently formed nucleotidylated-His112 intermediate (<b>E*</b>) using time-dependent crystallography. Utilizing a catalytically inactive hHint1 His112Asn enzyme variant and wild-type enzyme, the enzyme–substrate (<b>ES</b>^<b>1</b>) and product (<b>EP</b>^<b>2</b>) complexes were also cocrystallized, respectively, thus providing a structural map of the reaction trajectory. On the basis of these observations and the mechanistic necessity of proton transfers, proton inventory studies were carried out. Although we cannot completely exclude the possibility of more than one proton in flight, the results of these studies were consistent with the transfer of a single proton during the formation of the intermediate. Interestingly, structural analysis revealed that the critical proton transfers required for intermediate formation and hydrolysis may be mediated by a conserved active site water channel. Taken together, our results provide mechanistic insights underpinning histidine nucleophilic catalysis in general and hHint1 catalysis, in particular, thus aiding the design of future proTides and the elucidation of the natural function of the Hint family of enzymes

Rational Optimization of Mechanism-Based Inhibitors through Determination of the Microscopic Rate Constants of Inactivation

Mechanism-based inhibitors (MBIs) are widely employed in chemistry, biology, and medicine because of their exquisite specificity and sustained duration of inhibition. Optimization of MBIs is complicated because of time-dependent inhibition resulting from multistep inactivation mechanisms. The global kinetic parameters <i>k</i>_inact and <i>K</i>_I have been used to characterize MBIs, but they provide far less information than is commonly assumed, as shown by derivation and simulation of these parameters. We illustrate an alternative and more rigorous approach for MBI characterization through determination of the individual microscopic rate constants. Kinetic analysis revealed the rate-limiting step of inactivation of the PLP-dependent enzyme BioA by dihydro-(1,4)-pyridone <b>1</b>. This knowledge was subsequently applied to rationally design a second-generation inhibitor scaffold with a nearly optimal maximum inactivation rate (0.48 min^–1)

Design, Synthesis, and Characterization of Sulfamide and Sulfamate Nucleotidomimetic Inhibitors of hHint1

Hint1 has recently emerged to be an important target of interest due to its involvement in the regulation of a broad range of CNS functions including opioid signaling, tolerance, neuropathic pain, and nicotine dependence. A series of inhibitors were rationally designed, synthesized, and tested for their inhibitory activity against hHint1 using isothermal titration calorimetry (ITC). The studies resulted in the development of the first small-molecule inhibitors of hHint1 with submicromolar binding affinities. A combination of thermodynamic and high-resolution X-ray crystallographic studies provides an insight into the biomolecular recognition of ligands by hHint1. These novel inhibitors have potential utility as molecular probes to better understand the role and function of hHint1 in the CNS

Fragment-Based Exploration of Binding Site Flexibility in Mycobacterium tuberculosis BioA

The PLP-dependent transaminase (BioA) of Mycobacterium tuberculosis and other pathogens that catalyzes the second step of biotin biosynthesis is a now well-validated target for antibacterial development. Fragment screening by differential scanning fluorimetry has been performed to discover new chemical scaffolds and promote optimization of existing inhibitors. Calorimetry confirms binding of six molecules with high ligand efficiency. Thermodynamic data identifies which molecules bind with the enthalpy driven stabilization preferred in compounds that represent attractive starting points for future optimization. Crystallographic characterization of complexes with these molecules reveals the dynamic nature of the BioA active site. Different side chain conformational states are stabilized in response to binding by different molecules. A detailed analysis of conformational diversity in available BioA structures is presented, resulting in the identification of two states that might be targeted with molecular scaffolds incorporating well-defined conformational attributes. This new structural data can be used as part of a scaffold hopping strategy to further optimize existing inhibitors or create new small molecules with improved therapeutic potential

A Crystal Structure Based Guide to the Design of Human Histidine Triad Nucleotide Binding Protein 1 (hHint1) Activated ProTides

Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTide) have found utility as prodrugs. In humans, ProTides can be cleaved by human histidine triad nucleotide binding protein 1 (hHint1) to expose the nucleotide monophosphate. Activation by this route circumvents highly selective nucleoside kinases that limit the use of nucleosides as prodrugs. To better understand the diversity of potential substrates of hHint1, we created and studied a series of phosphoramidate nucleosides. Using a combination of enzyme kinetics, X-ray crystallography, and isothermal titration calorimetry with both wild-type and inactive mutant enzymes, we have been able to explore the energetics of substrate binding and establish a structural basis for catalytic efficiency. Diverse nucleobases are well tolerated, but portions of the ribose are needed to position substrates for catalysis. Beneficial characteristics of the amine leaving group are also revealed. Structural principles revealed by these results may be exploited to tune the rate of substrate hydrolysis to strategically alter the intracellular release of the product nucleoside monophosphate from the ProTide

Structure-Based Optimization of Pyridoxal 5′-Phosphate-Dependent Transaminase Enzyme (BioA) Inhibitors that Target Biotin Biosynthesis in <i>Mycobacterium tuberculosis</i>

The pyridoxal 5′-phosphate (PLP)-dependent transaminase BioA catalyzes the second step in the biosynthesis of biotin in <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) and is an essential enzyme for bacterial survival and persistence in vivo. A promising BioA inhibitor <b>6</b> containing an <i>N</i>-aryl, <i>N</i>′-benzoylpiperazine scaffold was previously identified by target-based whole-cell screening. Here, we explore the structure–activity relationships (SAR) through the design, synthesis, and biological evaluation of a systematic series of analogues of the original hit using a structure-based drug design strategy, which was enabled by cocrystallization of several analogues with BioA. To confirm target engagement and discern analogues with off-target activity, each compound was evaluated against wild-type (WT) <i>Mtb</i> in biotin-free and -containing medium as well as BioA under- and overexpressing <i>Mtb</i> strains. Conformationally constrained derivative <b>36</b> emerged as the most potent analogue with a <i>K</i>_D of 76 nM against BioA and a minimum inhibitory concentration of 1.7 μM (0.6 μg/mL) against <i>Mtb</i> in biotin-free medium

Structure-Based Optimization of Pyridoxal 5′-Phosphate-Dependent Transaminase Enzyme (BioA) Inhibitors that Target Biotin Biosynthesis in <i>Mycobacterium tuberculosis</i>

The pyridoxal 5′-phosphate (PLP)-dependent transaminase BioA catalyzes the second step in the biosynthesis of biotin in <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) and is an essential enzyme for bacterial survival and persistence in vivo. A promising BioA inhibitor <b>6</b> containing an <i>N</i>-aryl, <i>N</i>′-benzoylpiperazine scaffold was previously identified by target-based whole-cell screening. Here, we explore the structure–activity relationships (SAR) through the design, synthesis, and biological evaluation of a systematic series of analogues of the original hit using a structure-based drug design strategy, which was enabled by cocrystallization of several analogues with BioA. To confirm target engagement and discern analogues with off-target activity, each compound was evaluated against wild-type (WT) <i>Mtb</i> in biotin-free and -containing medium as well as BioA under- and overexpressing <i>Mtb</i> strains. Conformationally constrained derivative <b>36</b> emerged as the most potent analogue with a <i>K</i>_D of 76 nM against BioA and a minimum inhibitory concentration of 1.7 μM (0.6 μg/mL) against <i>Mtb</i> in biotin-free medium

Probing the S2′ Subsite of the Anthrax Toxin Lethal Factor Using Novel N‑Alkylated Hydroxamates

The lethal factor (LF) enzyme secreted by <i>Bacillus anthracis</i> is a zinc hydrolase that is chiefly responsible for anthrax-related cell death. Although many studies of the design of small molecule LF inhibitors have been conducted, no LF inhibitor is yet available as a therapeutic agent. Inhibitors with considerable chemical diversity have been developed and investigated; however, the LF S2′ subsite has not yet been systematically explored as a potential target for lead optimization. Here we present synthesis, experimental evaluation, modeling, and structural biology for a novel series of sulfonamide hydroxamate LF inhibitor analogues specifically designed to extend into, and probe chemical preferences of, this S2′ subsite. We discovered that this region accommodates a wide variety of chemical functionalities and that a broad selection of ligand structural modifications directed to this area can be incorporated without significant deleterious alterations in biological activity. We also identified key residues in this subsite that can potentially be targeted to improve inhibitor binding