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Prion disease is a rapidly progressive neurodegenerative disorder caused by misfolding and aggregation of the prion protein (PrP), and there are currently no therapeutic options. PrP ligands could theoretically antagonize prion formation by protecting the native protein from misfolding or by targeting it for degradation, but no validated small-molecule binders have been discovered to date. We deployed a variety of screening methods in an effort to discover binders of PrP, including 19F-observed and saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, differential scanning fluorimetry (DSF), DNA-encoded library selection, and in silico screening. A single benzimidazole compound was confirmed in concentration-response, but affinity was very weak (Kd > 1 mM), and it could not be advanced further. The exceptionally low hit rate observed here suggests that PrP is a difficult target for small-molecule binders. While orthogonal binder discovery methods could yield high affinity compounds, non-small-molecule modalities may offer independent paths forward against prion disease.

MELK kinase has been implicated in playing an important role in tumorigenesis. Our previous studies suggested that MELK is involved in the regulation of cell cycle and its genetic depletion leads to growth inhibition in a subset of high MELK-expressing basal-like breast cancer cell lines. Herein we describe the discovery and optimization of novel MELK inhibitors 8a and 8b that recapitulate the cellular effects observed by short hairpin ribonucleic acid (shRNA)-mediated MELK knockdown in cellular models. We also discovered a novel fluorine-induced hydrophobic collapse that locked the ligand in its bioactive conformation and led to a 20-fold gain in potency. These novel pharmacological inhibitors achieved high exposure in vivo and were well tolerated, which may allow further in vivo evaluation.

We performed a fragment screen on the dengue virus serotype 3 RNA-dependent RNA polymerase using x-ray crystallography. A screen of 1,400 fragments in pools of eight identified a single hit that bound in a novel pocket in the protein. This pocket is located in the polymerase palm subdomain and conserved across the four serotypes of dengue virus. The compound binds to the polymerase in solution as evidenced by surface plasmon resonance and isothermal titration calorimetry analyses. Related compounds where a phenyl is replaced by a thiophene show higher affinity binding, indicating the potential for rational design. Importantly, inhibition of enzyme activity correlated with the binding affinity, showing that the pocket is functionally important for polymerase activity. This fragment is an excellent starting point for optimization through rational structure-based design.

Over the past 30 years, drug discovery has evolved from a pure phenotypic approach to an integrated target-based strategy. The implementation of high-throughput biochemical and cellular assays has enabled the screening of large compound libraries which has become an important and often times the main source of new chemical matter that serve as starting point for medicinal chemistry efforts. In addition, biophysical methods measuring the physical interaction (affinity) between a low molecular weight ligand and a target protein became an integral part of hit validation/optimization to rule out false positives due to assay artifacts. Recent advances in throughput, robustness, and sensitivity of biophysical affinity screening methods have broadened their application in hit identification and validation such that they can now complement classical functional readouts. As a result, new target classes can be accessed that have not been amenable to functional assays. In this chapter, two affinity screening methods, differential scanning fluorimetry and surface plasmon resonance, which are broadly utilized in both academia and pharmaceutical industry are discussed in respect to their use in hit identification and validation. These methods exemplify how assays which differ in complexity, throughput, and information content can support the hit identification/validation process. This chapter focuses on the practical aspects and caveats of these techniques in order to enable the reader to establish their own affinity-based screens in both formats.

Over the past 30 years, drug discovery has evolved from a pure phenotypic approach to an integrated target-based strategy. The implementation of high-throughput biochemical and cellular assays has enabled the screening of large compound libraries which has become an important and often times the main source of new chemical matter that serve as starting point for medicinal chemistry efforts. In addition, biophysical methods measuring the physical interaction (affinity) between a low molecular weight ligand and a target protein became an integral part of hit validation/optimization to rule out false positives due to assay artifacts. Recent advances in throughput, robustness, and sensitivity of biophysical affinity screening methods have broadened their application in hit identification and validation such that they can now complement classical functional readouts. As a result, new target classes can be accessed that have not been amenable to functional assays. In this chapter, two affinity screening methods, differential scanning fluorimetry and surface plasmon resonance, which are broadly utilized in both academia and pharmaceutical industry are discussed in respect to their use in hit identification and validation. These methods exemplify how assays which differ in complexity, throughput, and information content can support the hit identification/validation process. This chapter focuses on the practical aspects and caveats of these techniques in order to enable the reader to establish their own affinity-based screens in both formats.

There are many challenges to the drug discovery process, including the complexity of the target, its interactions, and how these factors play a role in causing the disease. Traditionally, biophysics has been used for hit validation and chemical lead optimization. With its increased throughput and sensitivity, biophysics is now being applied earlier in this process to empower target characterization and hit finding. Areas covered: In this article, the authors provide an overview of how biophysics can be utilized to assess the quality of the reagents used in screening assays, to validate potential tool compounds, to test the integrity of screening assays, and to create follow-up strategies for compound characterization. They also briefly discuss the utilization of different biophysical methods in hit validation to help avoid the resource consuming pitfalls caused by the lack of hit overlap between biophysical methods. Expert opinion: The use of biophysics early on in the drug discovery process has proven crucial to identifying and characterizing targets of complex nature. It also has enabled the identification and classification of small molecules which interact in an allosteric or covalent manner with the target. By applying biophysics in this manner and at the early stages of this process, the chances of finding chemical leads with novel mechanisms of action are increased. In the future, focused screens with biophysics as a primary readout will become increasingly common.

Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1R132H mutation destabilizes an IDH1 “regulatory segment,” which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

The inhibitor of apoptosis protein (IAP) family of molecules inhibit apoptosis through the suppression of caspase activity. It is known that the XIAP protein regulates both caspase-3 and caspase-9 through direct protein-protein interactions. Specifically, the BIR3 domain of XIAP binds to caspase-9 via a ;hotspot' interaction in which the N-terminal residues of caspase-9 bind in a shallow groove on the surface of XIAP. This interaction is regulated via SMAC, the N-terminus of which binds in the same groove, thus displacing caspase-9. The mechanism of suppression of apoptosis by cIAP1 is less clear. The structure of the BIR3 domain of cIAP1 (cIAP1-BIR3) in complex with N-terminal peptides from both SMAC and caspase-9 has been determined. The binding constants of these peptides to cIAP1-BIR3 have also been determined using the surface plasmon resonance technique. The structures show that the peptides interact with cIAP1 in the same way that they interact with XIAP: both peptides bind in a similar shallow groove in the BIR3 surface, anchored at the N-terminus by a charge-stabilized hydrogen bond. The binding data show that the SMAC and caspase-9 peptides bind with comparable affinities (85 and 48 nM, respectively).

Tumor cells extensively utilize the pentose phosphate pathway for the synthesis of ribose. Transketolase is a key enzyme in this pathway and has been suggested as a target for inhibition in the treatment of cancer. In a pharmacodynamic study, nude mice with xenografted HCT-116 tumors were dosed with 1 ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of thiamine, the co-factor of transketolase. Transketolase activity was almost completely suppressed in blood, spleen, and tumor cells, but there was little effect on the activity of the other thiamine-utilizing enzymes alpha-ketoglutarate dehydrogenase or glucose-6-phosphate dehydrogenase. Synthesis and SAR of transketolase inhibitors is described.

Transketolase, a key enzyme in the pentose phosphate pathway, has been suggested as a target for inhibition in the treatment of cancer. Compound 5a ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of the transketolase cofactor thiamine, is a potent transketolase inhibitor but suffers from poor pharmacokinetics due to high clearance and C(max) linked toxicity. An efficient way of improving the pharmacokinetic profile of 5a is to prepare oxidized prodrugs which are slowly reduced in vivo yielding longer, sustained blood levels of the drug. The synthesis of such prodrugs and their evaluation in rodent models is reported.

The determination of accurate binding affinities is critical in drug discovery and development. Several techniques are available for characterizing the binding of small molecules to soluble proteins. The situation is different for integral membrane proteins. Isothermal chemical denaturation has been shown to be a valuable biophysical method to determine, in a direct and label-free fashion, the binding of ligands to soluble proteins. In this study, the application of isothermal chemical denaturation was applied to an integral membrane protein, the A2a G-protein coupled receptor. Binding affinities for a set of 19 small molecule agonists/antagonists of the A2a receptor were determined and found to be in agreement with data from surface plasmon resonance and radioligand binding assays previously reported in the literature. Therefore, isothermal chemical denaturation expands the available toolkit of biophysical techniques to characterize and study ligand binding to integral membrane proteins, specifically G-protein coupled receptors in vitro.

Dengue virus (DENV) is the most significant mosquito-borne viral pathogen in the world and is the cause of dengue fever. The DENV RNA-dependent RNA polymerase (RdRp) is conserved among the four viral serotypes and is an attractive target for antiviral drug development. During initiation of viral RNA synthesis, the polymerase switches from a "closed" to "open" conformation to accommodate the viral RNA template. Inhibitors that lock the "closed" or block the "open" conformation would prevent viral RNA synthesis. Herein, we describe a screening campaign that employed two biochemical assays to identify inhibitors of RdRp initiation and elongation. Using a DENV subgenomic RNA template that promotes RdRp de novo initiation, the first assay measures cytosine nucleotide analogue (Atto-CTP) incorporation. Liberated Atto fluorophore allows for quantification of RdRp activity via fluorescence. The second assay uses the same RNA template but is label free and directly detects RdRp-mediated liberation of pyrophosphates of native ribonucleotides via liquid chromatography-mass spectrometry. The ability of inhibitors to bind and stabilize a "closed" conformation of the DENV RdRp was further assessed in a differential scanning fluorimetry assay. Last, active compounds were evaluated in a renilla luciferase-based DENV replicon cell-based assay to monitor cellular efficacy. All assays described herein are medium to high throughput, are robust and reproducible, and allow identification of inhibitors of the open and closed forms of DENV RNA polymerase.

Malate dehydrogenase specifically oxidizes malate to oxaloacetate. The specificity arises from three arginines in the active site pocket that coordinate the carboxyl groups of the substrate and stabilize the newly forming hydroxyl/keto group during catalysis. Here, the role of Arg-153 in distinguishing substrate specificity is examined by the mutant R153C. The x-ray structure of the NAD binary complex at 2.1 A reveals two sulfate ions bound in the closed form of the active site. The sulfate that occupies the substrate binding site has been translated approximately 2 A toward the opening of the active site cavity. Its new location suggests that the low catalytic turnover observed in the R153C mutant may be due to misalignment of the hydroxyl or ketone group of the substrate with the appropriate catalytic residues. In the NAD.pyruvate ternary complex, the monocarboxylic inhibitor is bound in the open conformation of the active site. The pyruvate is coordinated not by the active site arginines, but through weak hydrogen bonds to the amide backbone. Energy minimized molecular models of unnatural analogues of R153C (Wright, S. K., and Viola, R. E. (2001) J. Biol. Chem. 276, 31151-31155) reveal that the regenerated amino and amido side chains can form favorable hydrogen-bonding interactions with the substrate, although a return to native enzymatic activity is not observed. The low activity of the modified R153C enzymes suggests that precise positioning of the guanidino side chain is essential for optimal orientation of the substrate.

Malate dehydrogenase from Escherichia coli is highly specific for the oxidation of malate to oxaloacetate. The technique of site-specific modulation has been used to alter the substrate binding site of this enzyme. Introduction of a cysteine in place of the active site binding residue arginine 153 results in a mutant enzyme with diminished catalytic activity, but with K(m) values for malate and oxaloacetate that are surprisingly unaffected. Reaction of this introduced cysteine with a series of amino acid analog reagents leads to the incorporation of a range of functional groups at the active site of malate dehydrogenase. The introduction of a positively charged group such as an amine or an amidine at this position results in improved affinity for several inhibitors over that observed with the native enzyme. However, the recovery of catalytic activity is less dramatic, with less than one third of the native activity achieved with the optimal reagents. These modified enzymes do have altered substrate specificity, with alpha-ketoglutarate and hydroxypyruvate no longer functioning as alternative substrates.

Malate dehydrogenase (MDH) from Escherichia coli is highly specific for its keto acid substrate. The placement of the active site-binding groups in MDH effectively discriminates against both the shorter and the longer keto dicarboxylic acids that could potentially serve as alternative substrates. A notable exception to this specificity is the alternative substrate phenylpyruvate. This aromatic keto acid can be reduced by MDH, albeit at a somewhat slower rate and with greatly diminished affinity, despite the presence of several substrate-binding arginyl residues and the absence of a hydrophobic pocket in the active site. The specificity of MDH for phenylpyruvate has now been enhanced, and that for the physiological substrate oxaloacetate has been diminished, through the replacement of one of the binding arginyl residues with several unnatural alkyl and aryl amino acid analogs. This approach, called site-specific modulation, incorporates systematic structural variations at a site of interest. Molecular modeling studies have suggested a structural basis for the affinity of native MDH for phenylpyruvate and a rationale for the improved catalytic activity that is observed with these new, modified phenyllactate dehydrogenases.

Inhibition of the thiamine-utilizing enzyme transketolase (TK) has been linked with diminished tumor cell proliferation. Most thiamine antagonists have a permanent positive charge on the B-ring, and it has been suggested that this charge is required for diphosphorylation by thiamine pyrophosphokinase (TPPK) and binding to TK. We sought to make neutral thiazolium replacements that would be substrates for TPPK, while not necessarily needing thiamine transporters (ThTr1 and ThTr2) for cell penetration. The synthesis, SAR, and structure-based rationale for highly potent non-thiazolium TK antagonists are presented.

The important biosynthetic intermediate chorismate reacts thermally by two competitive pathways, one leading to 4-hydroxybenzoate via elimination of the enolpyruvyl side chain, and the other to prephenate by a facile Claisen rearrangement. Measurements with isotopically labeled chorismate derivatives indicate that both are concerted sigmatropic processes, controlled by the orientation of the enolpyruvyl group. In the elimination reaction of [4-2H]chorismate, roughly 60% of the label was found in pyruvate after 3 h at 60 degrees C. Moreover, a 1.846 +/- 0.057 2H isotope effect for the transferred hydrogen atom and a 1.0374 +/- 0.0005 18O isotope effect for the ether oxygen show that the transition state for this process is highly asymmetric, with hydrogen atom transfer from C4 to C9 significantly less advanced than C-O bond cleavage. In the competing Claisen rearrangement, a very large 18O isotope effect at the bond-breaking position (1.0482 +/- 0.0005) and a smaller 13C isotope effect at the bond-making position (1.0118 +/- 0.0004) were determined. Isotope effects of similar magnitude characterized the transformations catalyzed by evolutionarily unrelated chorismate mutases from Escherichia coli and Bacillus subtilis. The enzymatic reactions, like their solution counterpart, are thus concerted [3,3]-sigmatropic processes in which C-C bond formation lags behind C-O bond cleavage. However, as substantially larger 18O and smaller 13C isotope effects were observed for a mutant enzyme in which chemistry is fully rate determining, the ionic active site may favor a somewhat more polarized transition state than that seen in solution.