Low Affinity Compounds Research Articles

Preparation and Bioevaluation of 18F-Labeled Small-Molecular Radiotracers via Sulfur(VI) Fluoride Exchange Chemistry for Imaging of Programmed Cell Death Protein Ligand 1 Expression in Tumors.

Nowadays, one of the most effective methods of tumor immunotherapy is blocking programmed cell death protein 1/programmed cell death protein ligand 1 (PD-1/PD-L1) immune checkpoints. However, there is still a significant challenge in selecting patients to benefit from immune checkpoint therapies. Positron emission tomography (PET), a noninvasive molecular imaging technique, offers a new approach to accurately detect PD-L1 expression and allows for a better prediction of response to PD-1/PD-L1 target immunotherapy. Here, we designed and synthesized a novel group of aryl fluorosulfate-containing small-molecule compounds (LGSu-1, LGSu-2, LGSu-3, and LGSu-4) based on the phenoxymethyl-biphenyl scaffold. After screening by the time-resolved fluorescence resonance energy transfer (TR-FRET) assay, the most potent compound LGSu-1 (half maximal inhibitory concentration (IC50): 15.53 nM) and the low-affinity compound LGSu-2 (IC50: 189.70 nM) as a control were selected for 18F-radiolabeling by sulfur(VI) fluoride exchange chemistry (SuFEx) to use for PET imaging. [18F]LGSu-1 and [18F]LGSu-2 were prepared by a one-step radiofluorination reaction in over 85% radioconversion and nearly 30% radiochemical yield. In B16-F10 melanoma cell assays, [18F]LGSu-1 (5.00 ± 0.06%AD) showed higher cellular uptake than [18F]LGSu-2 (2.55 ± 0.04%AD), in which cell uptake could be significantly blocked by the nonradioactivity LGSu-1. In vivo experiments, micro-PET imaging of B16-F10 tumor-bearing mice and radiographic autoradiography of tumor sections showed that [18F]LGSu-1 was more effectively accumulated in the tumor due to the higher binding affinity with PD-L1. The above experimental results confirmed the potential of the small-molecule probe LGSu-1 as a targeting PD-L1 imaging tracer in tumor tissues.

Fragment expansion with NUDELs - poised DNA-encoded libraries.

Optimisation of the affinity of lead compounds is a critical challenge in the identification of drug candidates and chemical probes and is a process that takes many years. Fragment-based drug discovery has become established as one of the methods of choice for drug discovery starting with small, low affinity compounds. Due to their low affinity, the evolution of fragments to desirable levels of affinity is often a key challenge. The accepted best method for increasing the potency of fragments is by iterative fragment growing, which can be very time consuming and complex. Here, we introduce a paradigm for fragment hit optimisation using poised DNA-encoded chemical libraries (DELs). The synthesis of a poised DEL, a partially constructed library that retains a reactive handle, allows the coupling of any active fragment for a specific target protein, allowing rapid discovery of potent ligands. This is illustrated for bromodomain-containing protein 4 (BRD4), in which a weakly binding fragment was coupled to a 42-member poised DEL via Suzuki-Miyaura cross coupling resulting in the identification of an inhibitor with 51 nM affinity in a single step, representing an increase in potency of several orders of magnitude from an original fragment. The potency of the compound was shown to arise from the synergistic combination of substructures, which would have been very difficult to discover by any other method and was rationalised by X-ray crystallography. The compound showed attractive lead-like properties suitable for further optimisation and demonstrated BRD4-dependent cellular pharmacology. This work demonstrates the power of poised DELs to rapidly optimise fragments, representing an attractive generic approach to drug discovery.

Correction to “Investigating the Influence of Tracer Kinetics on Competition-Kinetic Association Binding Assays: Identifying the Optimal Conditions for Assessing the Kinetics of Low-Affinity Compounds”

Correction to “Investigating the Influence of Tracer Kinetics on Competition-Kinetic Association Binding Assays: Identifying the Optimal Conditions for Assessing the Kinetics of Low-Affinity Compounds”

Investigating the Influence of Tracer Kinetics on Competition-Kinetic Association Binding Assays: Identifying the Optimal Conditions for Assessing the Kinetics of Low-Affinity Compounds

An increased appreciation of the importance of optimizing drug-binding kinetics has lead to the development of various techniques for measuring the kinetics of unlabeled compounds. One approach is the competition-association kinetic binding method first described in the 1980s. The kinetic characteristics of the tracer employed greatly affects the reliability of estimated kinetic parameters, a barrier to successfully introducing these kinetic assays earlier in the drug discovery process. Using a modeling and Monte Carlo simulation approach, we identify the optimal tracer characteristics for determining the kinetics of the range of unlabeled ligands typically encountered during the different stages of a drug discovery program (i.e., rapidly dissociating, e.g., k off = 10 minute-1 low-affinity "hits" through to slowly dissociating e.g., k off = 0.01 minute-1 high-affinity "candidates"). For more rapidly dissociating ligands (e.g., k off = 10 minute-1), the key to obtaining accurate kinetic parameters was to employ a tracer with a relatively fast off-rate (e.g., k off = 1 minute-1) or, alternatively, to increase the tracer concentration. Reductions in assay start-time ≤1second and read frequency ≤5 seconds significantly improved the reliability of curve fitting. Timing constraints are largely dictated by the method of detection, its inherent sensitivity (e.g., TR-FRET versus radiometric detection), and the ability to inject samples online. Furthermore, we include data from TR-FRET experiments that validate this simulation approach, confirming its practical utility. These insights into the optimal experimental parameters for development of competition-association assays provide a framework for identifying and testing novel tracers necessary for profiling unlabeled competitors, particularly rapidly dissociating low-affinity competitors.

Synthesis, Enzyme Assays and Molecular Docking Studies of Fluorina ted Bioisosteres of Santacruzamate A as Potential HDAC Tracers

Background: Histone deacetylases (HDACs) emerged as important epigenetic regulators of gene expression. Method: In order to identify potential positron emission tomography (PET) tracers for imaging HDACs, we evaluated in vitro and in cellulo activities of some compounds that were reported as potent HDAC2-selective inhibitors. We observed marked differences between reported activity values and the values obtained in our assays for some of the compounds. To understand the structural basis of the activity of some of these inhibitors, we also performed molecular docking studies to understand their interaction patterns and binding modes with HDAC2. Results and Conclusion: We observed the low affinity compounds 4, 6 and 7 did not showed equal number of key ?-? interactions and hydrogen bonding when compared to high affinity compounds, and could be the possible reason for poor inhibition as reflected in in vitro assays. These preliminary experimental and computational results will help to interpret the HDAC affinity values of these key compounds with caution.

Potencies and unblocking kinetic properties of antagonists at recombinant human NMDA receptors in a Xenopus oocytes model.

N-methyl-D-aspartate (NMDA) receptor channels are implicated in a wide range of physiological and pathophysiological processes, and a large number of pharmacological agents have been introduced that target the receptor via diverse mechanisms of action. Amongst others, subunit selectivity (in particular for the NR2B receptor subunit) and rapid unblocking kinetics have been put forward as favourable pharmacological properties of NMDA receptor-targeting drugs. Here, we describe a pharmacological characterization of human recombinant NMDA receptors expressed in Xenopus oocytes in an electrophysiological set-up. Using this approach, we compare inhibitor potencies of several known NMDA receptor ligands as well as unblocking kinetic properties of selected compounds. All compounds tested had similar potencies at receptors containing NR2A or NR2B receptors with the exception of traxoprodil, which was selective for NR2B. The rank order of potency was (+)MK-801 > phencyclidine (PCP) ≈ traxoprodil > memantine≈ ketamine > duloxetine. In line with its proposed rapid dissociation properties, the relatively well-tolerated drug memantine exhibits markedly faster unblocking than ketamine and PCP, similar to the low-affinity compound, duloxetine. Electrophysiological recording in Xenopus oocytes thus allows a relatively convenient comparison of key pharmacological parameters at recombinant human NMDA receptors.

Structure-Based Virtual Screening of MT2 Melatonin Receptor: Influence of Template Choice and Structural Refinement

Developing GPCR homology models for structure-based virtual screening requires the choice of a suitable template and refinement of binding site residues. We explored this systematically for the MT2 melatonin receptor, with the aim to build a receptor homology model that is optimized for the enrichment of active melatoninergic ligands. A set of 12 MT2 melatonin receptor models was built using different GPCR X-ray structural templates and submitted to a virtual screening campaign on a set of compounds composed of 29 known melatonin receptor ligands and 2560 drug-like decoys. To evaluate the effect of including a priori information in receptor models, 12 representative melatonin receptor ligands were placed into the MT2 receptor models in poses consistent with known mutagenesis data and with assessed pharmacophore models. The receptor structures were then adapted to the ligands by induced-fit docking. Most of the 144 ligand-adapted MT2 receptor models showed significant improvements in screening enrichments compared to the unrefined homology models, with some template/refinement combinations giving excellent enrichment factors. The discriminating ability of the models was further tested on the 29 active ligands plus a set of 21 inactive or low-affinity compounds from the same chemical classes. Rotameric states of side chains for some residues, presumed to be involved in the binding process, were correlated with screening effectiveness, suggesting the existence of specific receptor conformations able to recognize active compounds. The top MT2 receptor model was able to identify 24 of 29 active ligands among the first 2% of the screened database. This work provides insights into the use of refined GPCR homology models for virtual screening.

1-Butanol and Gambierol:Low and High Affinity Compounds that Immobilize Charge Movement in Shaker and Kv3.1 Potassium Channels

Voltage-gated K+ (Kv) channels exist as tetramers of α-subunits that contain six transmembrane segments (S1-S6). The S5-S6 segments assemble into the ion permeation pathway that is surrounded by four voltage-sensing domains (VSD). Upon a membrane depolarization the VSD's move independently to the ‘pre-activated state’, and subsequently in a concerted way to the activated state that results in channel opening. It has been reported that 1-alkanols, such as 1-Butanol, inhibit Kv channels by binding to the S4-S5 linker and the bottom part of S6 (S6c), suggesting that they affect the gating machinery. Recently, we demonstrated that the polycyclic-ether toxin gambierol -that most likely binds to S6c outside the permeation pathway- impairs VSD movement in Kv3.1 channels. To determine whether 1-Butanol immobilizes the VSD similarly, we analyzed gating currents of Shaker-W434F channels expressed in HEK-293 cells. Determining WT Shaker's affinity for 1-Butanol based on the fraction of ionic current block yielded an IC50 value of ∼50 mM. Concentrations that yielded full current block reduced charge movement in Shaker-W434F by at least 50%. Based on the loss in gating charge movement an IC50 value of ∼260 mM was obtained which in a model with 4 identical binding sites fits the IC50 value of 50 mM obtained from ionic current inhibition. Kinetic analysis showed that 1-Butanol accelerated the deactivating gating currents similar to 4-AP indicating that the final concerted step could not be passed. However, this final concerted step carries only 5% of the total charge and 1-Butanol reduced charge movement by at least 50%. These data suggest that the mechanism of 1-Butanol is reminiscent to that of gambierol and stabilizes an early/deep closed state. (supported by fellowship CONACyT #176137 to EMM & grant FWO-G0433.12N to DJS)

Distinct States of Methionyl-tRNA Synthetase Indicate Inhibitor Binding by Conformational Selection

To guide development of new drugs targeting methionyl-tRNA synthetase (MetRS) for treatment of human African trypanosomiasis, crystal structure determinations of Trypanosoma brucei MetRS in complex with its substrate methionine and its intermediate product methionyl-adenylate were followed by those of the enzyme in complex with high-affinity aminoquinolone inhibitors via soaking experiments. Drastic changes in conformation of one of the two enzymes in the asymmetric unit allowed these inhibitors to occupy an enlarged methionine pocket anda new so-called auxiliary pocket. Interestingly, a small low-affinity compound caused the same conformational changes, removed the methionine without occupying the methionine pocket, and occupied the previously not existing auxiliary pocket. Analysis of these structures indicates that the binding of the inhibitors is the result of conformational selection, not induced fit.

Chemical Analysis of Complex Organic Mixtures Using Reactive Nanospray Desorption Electrospray Ionization Mass Spectrometry

Reactive nanospray desorption electrospray ionization (nano-DESI) combined with high-resolution mass spectrometry was utilized for the analysis of secondary organic aerosol produced through ozonolysis of limonene (LSOA). Previous studies have shown that LSOA constituents are multifunctional compounds containing at least one aldehyde or ketone groups. In this study, we used the selectivity of the Girard's reagent T (GT) toward carbonyl compounds to examine the utility of reactive nano-DESI for the analysis of complex organic mixtures. In these experiments, 1-100 μM GT solutions were used as the working solvents for reactive nano-DESI analysis. Abundant products from the single addition of GT to LSOA constituents were observed at GT concentrations in excess of 10 μM. We found that LSOA dimeric and trimeric compounds react with GT through a simple addition reaction resulting in formation of the carbinolamine derivative. In contrast, reactions of GT with monomeric species result in the formation of both the carbinolamine and the hydrazone derivatives. In addition, several monomers did not react with GT on the time scale of our experiment. These molecules were characterized by relatively high values of the double bond equivalent and low oxygen content. Furthermore, because addition of a charged GT tag to a neutral molecule eliminates the discrimination against the low proton affinity compounds in the ionization process, reactive nano-DESI analysis enables quantification of individual compounds in the complex mixture. For example, we were able to estimate for the first time the amounts of dimers and trimers in the LSOA mixture. Specifically, we found that the most abundant LSOA dimer was detected at the ~0.5 pg level and the total amount of dimers and trimers in the analyzed sample was ~11 pg. Our results indicate that reactive nano-DESI is a valuable approach for examining the presence of specific functional groups and for the quantification of compounds possessing these groups in complex mixtures.

Influence of bone affinity on the skeletal distribution of fluorescently labeled bisphosphonates in vivo

Bisphosphonates are widely used antiresorptive drugs that bind to calcium. It has become evident that these drugs have differing affinities for bone mineral; however, it is unclear whether such differences affect their distribution on mineral surfaces. In this study, fluorescent conjugates of risedronate, and its lower-affinity analogues deoxy-risedronate and 3-PEHPC, were used to compare the localization of compounds with differing mineral affinities in vivo. Binding to dentine in vitro confirmed differences in mineral binding between compounds, which was influenced predominantly by the characteristics of the parent compound but also by the choice of fluorescent tag. In growing rats, all compounds preferentially bound to forming endocortical as opposed to resorbing periosteal surfaces in cortical bone, 1 day after administration. At resorbing surfaces, lower-affinity compounds showed preferential binding to resorption lacunae, whereas the highest-affinity compound showed more uniform labeling. At forming surfaces, penetration into the mineralizing osteoid was found to inversely correlate with mineral affinity. These differences in distribution at resorbing and forming surfaces were not observed at quiescent surfaces. Lower-affinity compounds also showed a relatively higher degree of labeling of osteocyte lacunar walls and labeled lacunae deeper within cortical bone, indicating increased penetration of the osteocyte canalicular network. Similar differences in mineralizing surface and osteocyte network penetration between high- and low-affinity compounds were evident 7 days after administration, with fluorescent conjugates at forming surfaces buried under a new layer of bone. Fluorescent compounds were incorporated into these areas of newly formed bone, indicating that "recycling" had occurred, albeit at very low levels. Taken together, these findings indicate that the bone mineral affinity of bisphosphonates is likely to influence their distribution within the skeleton.

Biophysical and X-ray Crystallographic Analysis of Mps1 Kinase Inhibitor Complexes,

The dual-specificity protein kinase monopolar spindle 1 (Mps1) is a central component of the mitotic spindle assembly checkpoint (SAC), a sensing mechanism that prevents anaphase until all chromosomes are bioriented on the metaphase plate. Partial depletion of Mps1 protein levels sensitizes transformed, but not untransformed, human cells to therapeutic doses of the anticancer agent Taxol, making it an attractive novel therapeutic cancer target. We have previously determined the X-ray structure of the catalytic domain of human Mps1 in complex with the anthrapyrazolone kinase inhibitor SP600125. In order to validate distinct inhibitors that target this enzyme and improve our understanding of nucleotide binding site architecture, we now report a biophysical and structural evaluation of the Mps1 catalytic domain in the presence of ATP and the aspecific model kinase inhibitor staurosporine. Collective in silico, enzymatic, and fluorescent screens also identified several new lead quinazoline Mps1 inhibitors, including a low-affinity compound termed Compound 4 (Cpd 4), whose interaction with the Mps1 kinase domain was further characterized by X-ray crystallography. A novel biophysical analysis demonstrated that the intrinsic fluorescence of SP600125 changed markedly upon Mps1 binding, allowing spectrophotometric displacement analysis and determination of dissociation constants for ATP-competitive Mps1 inhibitors. By illuminating the structure of the Mps1 ATP-binding site our results provide novel biophysical insights into Mps1-ligand interactions that will be useful for the development of specific Mps1 inhibitors, including those employing a therapeutically validated quinazoline template.

An Evaluation of Explicit Receptor Flexibility in Molecular Docking Using Molecular Dynamics and Torsion Angle Molecular Dynamics

Incorporating receptor flexibility into molecular docking should improve results for flexible proteins. However, the incorporation of explicit all-atom flexibility with molecular dynamics for the entire protein chain may also introduce significant error and "noise" that could decrease docking accuracy and deteriorate the ability of a scoring function to rank native-like poses. We address this apparent paradox by comparing the success of several flexible receptor models in cross-docking and multiple receptor ensemble docking for p38α mitogen-activated protein (MAP) kinase. Explicit all-atom receptor flexibility has been incorporated into a CHARMM-based molecular docking method (CDOCKER) using both molecular dynamics (MD) and torsion angle molecular dynamics (TAMD) for the refinement of predicted protein-ligand binding geometries. These flexible receptor models have been evaluated, and the accuracy and efficiency of TAMD sampling is directly compared to MD sampling. Several flexible receptor models are compared, encompassing flexible side chains, flexible loops, multiple flexible backbone segments, and treatment of the entire chain as flexible. We find that although including side chain and some backbone flexibility is required for improved docking accuracy as expected, docking accuracy also diminishes as additional and unnecessary receptor flexibility is included into the conformational search space. Ensemble docking results demonstrate that including protein flexibility leads to to improved agreement with binding data for 227 active compounds. This comparison also demonstrates that a flexible receptor model enriches high affinity compound identification without significantly increasing the number of false positives from low affinity compounds.

Fragment approaches in structure-based drug discovery

There has been considerable interest recently in what is known as ;fragment-based lead discovery'. The novel feature of the approach is to begin with small low-affinity compounds. The main advantage is that a larger potential chemical diversity can be sampled with fewer compounds, which is particularly important for new target classes. The approach relies on careful design of the fragment library, a method that can detect binding of the fragment to the protein target, determination of the structure of the fragment bound to the target, and the conventional use of structural information to guide compound optimization. In this article the methods are reviewed, and experiences in fragment-based discovery of lead series of compounds against kinases such as PDK1 and ATPases such as Hsp90 are discussed. The examples illustrate some of the key benefits and issues of the approach and also provide anecdotal examples of the patterns seen in selectivity and the binding mode of fragments across different protein targets.

Experimental study of the effect of temperature on ion cluster formation using ion mobility spectrometry

Ion mobility spectrometry offers a robust and effective technique to study ion clusters in ambient conditions. Here, we have experimentally studied the influence of temperature on the positive ion cluster formation of 2-propanol vapor in air, along with parallel measurements for n-butyl acetate vapor in air. For both of these low proton affinity compounds in the ppm concentration range, temperatures below 0 °C tend to favor formation of dimers and trimers. The measurements indicate that approximate estimations for the fractions of these n-mers ( n > 1) in the ion spectra, can be obtained by classical theory for ion induced nucleation. Presence of natural background vapors however slightly blurs the data, especially for the fraction of monomers, so that accurate prediction of the fractions of n-mers in the spectra would require more accurate information on the gas composition. The findings concerning thermal behavior of ions help to understand better ion phenomena also in field conditions.

Discovery of a Highly Active Ligand of Human Pregnane X Receptor: A Case Study from Pharmacophore Modeling and Virtual Screening to “In Vivo” Biological Activity

The human pregnane X receptor (hPXR) is a nuclear receptor that regulates the expression of phase I and II drug-metabolizing enzymes as well as that of drug transporters. In addition, this receptor plays a critical role in cholesterol homeostasis and in protecting tissues from potentially toxic endobiotics. hPXR is activated by a broad spectrum of low-affinity compounds including xenobiotics and endobiotics such as bile acids and their precursors. Crystallographic studies revealed a ligand binding domain (LBD) with a large and conformable binding pocket that is likely to contribute to the ability of hPXR to respond to compounds of varying size and shape. Here, we describe an in silico method that allowed the identification of nine novel hPXR agonists. We further characterize the compound 1-(2-chlorophenyl)-N-[1-(1-phenylethyl)-1H-benzimidazol-5-yl]methanesulfonamide (C2BA-4), a methanesulfonamide that activates PXR specifically and more potently than does the reference compound 4-[2,2-bis(diethoxyphosphoryl)ethenyl]-2,6-ditert-butyl-phenol (SR12813) in our stable cell line expressing a Gal4-PXR and a GAL4 driven luciferase reporter gene. Furthermore treatment of primary human hepatocytes with C2BA-4 results in a marked induction of the mRNA expression of hPXR target genes, such as cytochromes P450 3A4 and 2B6. Finally, C2BA-4 is also able to induce hPXR-mediated in vivo luciferase expression in HGPXR stable bioluminescent cells implanted in mice. The study suggests new directions for the rational design of selective hPXR agonists and antagonists.

The Search for Drug Leads Targeted to the β -Secretase: An Example of the Roles of Computer Assisted Approaches in Drug Discovery

The inhibition of beta-secretase has become a very promising approach to control the onset and progression of Alzheimer's disease due to its involvement in the generation of amyloid plaques. The main goal of the many drug discovery projects targeting this enzyme is the identification of highly specific, non-peptidic compounds with low molecular weight, characteristics that are desirable for drug leads with low toxicity that have to transverse the blood brain barrier. We describe the main approaches used in the discovery of novel inhibitors, including substrate specificity, target structure based design, and high throughput screening (HTS), both in vitro and in silico. We place special emphasis in the receptor based design and in silico HTS, two strategies that make wide use of computer assisted tools. To exemplify the usefulness and versatility of computer tools in this endeavor we use the computer generated 'enzyme's binding site cast' to rationalize qualitatively some salient structural features of known beta-secretase second generation inhibitors, and for guiding the review of many of the ligands whose complexes with the enzyme have been studied by X-ray crystallography. We discuss the use made by other authors of molecular modelling for the understanding of the very special characteristics of ligand binding to beta-secretase and for the design of new inhibitors. Finally, we review the quest for non-peptidic inhibitors that has led to the use of HTS in vitro and in silico. The screening of extensive libraries resulted in a few low affinity compounds that do not fit into the key S1/S1' pockets, indicating that this is not an easy target to block.

APPI-MS: Effects of Mobile Phases and VUV Lamps on the Detection of PAH Compounds

The technique of atmospheric pressure photoionization (APPI) has several advantages over electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), including efficient ionization of nonpolar or low charge affinity compounds, reduced susceptibility to ion suppression, high sensitivity, and large linear dynamic range. These benefits are greatest at low flow rates (i.e., <or=100 microL/min), while at a higher flow, photon absorption and ion-molecule reactions become significant. Under certain circumstances, APPI signal and S/N have been observed to excel at higher flow, which may be due to a nonphotoionzation mechanism. To better understand APPI at higher flow rates, we have selected three lamps (Xe, Kr, and Ar) and four mobile phases typical for reverse-phase, high-pressure liquid chromatography: acetonitrile, methanol, (1:1) acetonitrile:water and (1:1) methanol:water. As test compounds, three polyaromatic hydrocarbons are studied: benzo[a]pyrene, indeno[1,2,3-c, d]pyrene and benz[a]anthracene. We find that solvent photoabsorption cross-section is not the only parameter in explaining relative signal intensity, but that solvent photo-ion chemistry can also play a significant role. Three conclusions from this investigation are: (1) methanol photoionization leads to protonated methanol clusters that can result in chemical ionization of analyte molecule; (2) use of the Ar lamp often results in greater signal and S/N; (3) acetonitrile photoionization is less efficient and resulting clusters are too strongly bound to chemically ionize the analyte efficiently, so that analyte ion formation is dominated by direct photoionization.

The FRB Domain of mTOR: NMR Solution Structure and Inhibitor Design,

The mammalian target of rapamycin (mTOR) is a protein that is intricately involved in signaling pathways controlling cell growth. Rapamycin is a natural product that binds and inhibits mTOR function by interacting with its FKBP-rapamycin-binding (FRB) domain. Here we report on the NMR solution structure of FRB and on further studies aimed at the identification and characterization of novel ligands that target the rapamycin binding pocket. The biological activity of the ligands, and that of rapamycin in the absence of FKBP12, was investigated by assaying the kinase activity of mTOR. While we found that rapamycin binds the FRB domain and inhibits the kinase activity of mTOR even in the absence of FKBP12 (in the low micromolar range), our most potent ligands bind to FRB with similar binding affinity but inhibit the kinase activity of mTOR at much higher concentrations. However, we have also identified one low-affinity compound that is also capable of inhibiting mTOR. Hence, we have identified compounds that can directly mimic rapamycin or can dissociate the FRB binding from the inhibition of the catalytic activity of mTOR. As such, these ligands could be useful in deciphering the complex regulation of mTOR in the cell and in validating the FRB domain as a possible target for the development of novel therapeutic compounds.

Crystal Structures of Proto-oncogene Kinase Pim1: A Target of Aberrant Somatic Hypermutations in Diffuse Large Cell Lymphoma

Pim1, a serine/threonine kinase, is involved in several biological functions including cell survival, proliferation, and differentiation. While pim1 has been shown to be involved in several hematopoietic cancers, it was also recently identified as a target of aberrant somatic hypermutation in diffuse large cell lymphoma (DLCL), the most common form of non-Hodgkin's lymphoma. The crystal structures of Pim1 in apo form and bound with AMPPNP have been solved and several unique features of Pim1 were identified, including the presence of an extra beta-hairpin in the N-terminal lobe and an unusual conformation of the hinge connecting the two lobes of the enzyme. While the apo Pim1 structure is nearly identical with that reported recently, the structure of AMPPNP bound to Pim1 is significantly different. Pim1 is unique among protein kinases due to the presence of a proline residue at position 123 that precludes the formation of the canonical second hydrogen bond between the hinge backbone and the adenine moiety of ATP. One crystal structure reported here shows that changing P123 to methionine, a common residue that offers the backbone hydrogen bond to ATP, does not restore the ATP binding pocket of Pim1 to that of a typical kinase. These unique structural features in Pim1 result in novel binding modes of AMP and a known kinase inhibitor scaffold, as shown by co-crystallography. In addition, the kinase activities of five Pim1 mutants identified in DLCL patients have been determined. In each case, the observed effects on kinase activity are consistent with the predicted consequences of the mutation on the Pim1 structure. Finally, 70 co-crystal structures of low molecular mass, low-affinity compounds with Pim1 have been solved in order to identify novel chemical classes as potential Pim1 inhibitors. Based on the structural information, opportunities for optimization of one specific example are discussed.