Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity.

The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.

Fragment-based design of 3-aminopyridine-derived amides as potent inhibitors of human nicotinamide phosphoribosyltransferase (NAMPT).

The fragment-based identification of two novel and potent biochemical inhibitors of the nicotinamide phosphoribosyltransferase (NAMPT) enzyme is described. These compounds (51 and 63) incorporate an amide moiety derived from 3-aminopyridine, and are thus structurally distinct from other known anti-NAMPT agents. Each exhibits potent inhibition of NAMPT biochemical activity (IC50=19 and 15 nM, respectively) as well as robust antiproliferative properties in A2780 cell culture experiments (IC50=121 and 99 nM, respectively). However, additional biological studies indicate that only inhibitor 51 exerts its A2780 cell culture effects via a NAMPT-mediated mechanism. The crystal structures of both 51 and 63 in complex with NAMPT are also independently described.

Fragment-based identification and optimization of a class of potent pyrrolo[2,1-f][1,2,4]triazine MAP4K4 inhibitors.

MAP4K4 has been shown to regulate key cellular processes that are tied to disease pathogenesis. In an effort to generate small molecule MAP4K4 inhibitors, a fragment-based screen was carried out and a pyrrolotriazine fragment with excellent ligand efficiency was identified. Further modification of this fragment guided by X-ray crystal structures and molecular modeling led to the discovery of a series of promising compounds with good structural diversity and physicochemical properties. These compounds exhibited single digit nanomolar potency and compounds 35 and 44 achieved good in vivo exposure.

Identification of substituted 3-hydroxy-2-mercaptocyclohex-2-enones as potent inhibitors of human lactate dehydrogenase.

A novel class of 3-hydroxy-2-mercaptocyclohex-2-enone-containing inhibitors of human lactate dehydrogenase (LDH) was identified through a high-throughput screening approach. Biochemical and surface plasmon resonance experiments performed with a screening hit (LDHA IC50=1.7 µM) indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of this screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.18 µM). Two crystal structures of optimized compounds bound to human LDHA were obtained and explained many of the observed structure-activity relationships. In addition, an optimized inhibitor exhibited good pharmacokinetic properties after oral administration to rats (F=45%).

The transferrin receptor modulates Hfe-dependent regulation of hepcidin expression.

Hemochromatosis is caused by mutations in HFE, a protein that competes with transferrin (TF) for binding to transferrin receptor 1 (TFR1). We developed mutant mouse strains to gain insight into the role of the Hfe/Tfr1 complex in regulating iron homeostasis. We introduced mutations into a ubiquitously expressed Tfr1 transgene or the endogenous Tfr1 locus to promote or prevent the Hfe/Tfr1 interaction. Under conditions favoring a constitutive Hfe/Tfr1 interaction, mice developed iron overload attributable to inappropriately low expression of the hormone hepcidin. In contrast, mice carrying a mutation that interferes with the Hfe/Tfr1 interaction developed iron deficiency associated with inappropriately high hepcidin expression. High-level expression of a liver-specific Hfe transgene in Hfe-/- mice was also associated with increased hepcidin production and iron deficiency. Together, these models suggest that Hfe induces hepcidin expression when it is not in complex with Tfr1.

Specific Btk inhibition suppresses B cell- and myeloid cell-mediated arthritis.

Bruton's tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes FcγRIII-induced TNFα, IL-1ß and IL-6 production. Accordingly, in myeloid- and FcγR-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.

Identification of 2-amino-5-aryl-pyrazines as inhibitors of human lactate dehydrogenase.

A 2-amino-5-aryl-pyrazine was identified as an inhibitor of human lactate dehydrogenase A (LDHA) via a biochemical screening campaign. Biochemical and biophysical experiments demonstrated that the compound specifically interacted with human LDHA. Structural variation of the screening hit resulted in improvements in LDHA biochemical inhibition and pharmacokinetic properties. A crystal structure of an improved compound bound to human LDHA was also obtained and it explained many of the observed structure-activity relationships.

Identification of substituted 2-thio-6-oxo-1,6-dihydropyrimidines as inhibitors of human lactate dehydrogenase.

A novel 2-thio-6-oxo-1,6-dihydropyrimidine-containing inhibitor of human lactate dehydrogenase (LDH) was identified by high-throughput screening (IC50=8.1 µM). Biochemical, surface plasmon resonance, and saturation transfer difference NMR experiments indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of the screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.48 µM). A crystal structure of an optimized compound bound to human LDHA was obtained and explained many of the observed structure-activity relationships.

Docking for fragment inhibitors of AmpC beta-lactamase.

Fragment screens for new ligands have had wide success, notwithstanding their constraint to libraries of 1,000-10,000 molecules. Larger libraries would be addressable were molecular docking reliable for fragment screens, but this has not been widely accepted. To investigate docking's ability to prioritize fragments, a library of >137,000 such molecules were docked against the structure of beta-lactamase. Forty-eight fragments highly ranked by docking were acquired and tested; 23 had K(i) values ranging from 0.7 to 9.2 mM. X-ray crystal structures of the enzyme-bound complexes were determined for 8 of the fragments. For 4, the correspondence between the predicted and experimental structures was high (RMSD between 1.2 and 1.4 A), whereas for another 2, the fidelity was lower but retained most key interactions (RMSD 2.4-2.6 A). Two of the 8 fragments adopted very different poses in the active site owing to enzyme conformational changes. The 48% hit rate of the fragment docking compares very favorably with "lead-like" docking and high-throughput screening against the same enzyme. To understand this, we investigated the occurrence of the fragment scaffolds among larger, lead-like molecules. Approximately 1% of commercially available fragments contain these inhibitors whereas only 10(-7)% of lead-like molecules do. This suggests that many more chemotypes and combinations of chemotypes are present among fragments than are available among lead-like molecules, contributing to the higher hit rates. The ability of docking to prioritize these fragments suggests that the technique can be used to exploit the better chemotype coverage that exists at the fragment level.

Surface plasmon resonance based assay for the detection and characterization of promiscuous inhibitors.

Promiscuous binders achieve enzyme inhibition using a nonspecific aggregation-type binding mechanism to proteins. These compounds are a source of false-positive hits in biochemical inhibition assays and should be removed from screening hit lists because they are not good candidates to initiate medicinal chemistry programs. We introduce a robust approach to identify these molecules early in the lead generation process using real time surface plasmon resonance based biosensors to observe the behavior of the binding interactions between promiscuous compounds and proteins. Furthermore, the time resolution of the assay reveals a number of distinct mechanisms that promiscuous compounds employ to inhibit enzyme function and indicate that the type of mechanism can vary depending on the protein target. A classification scheme for these compounds is presented that can be used to rapidly characterize the hits from high-throughput screens and eliminate compounds with a nonspecific mechanism of inhibition.

Thermodynamic characterization of pyrazole and azaindole derivatives binding to p38 mitogen-activated protein kinase using Biacore T100 technology and van't Hoff analysis.

Biacore T100 technology was used in conjunction with a van't Hoff analysis to characterize the thermodynamic binding parameters of 85 small-molecule inhibitors of adenosine triphosphate (ATP) binding to p38 mitogen-activated protein (MAP) kinase. The compounds were selected from a large panel of azaindole and pyrazole derivatives for which IC(50) data exist. We showed a strong relationship between the K(D) and IC(50) of a compound, but only a modest relationship between k(off) and IC(50) was detected and an apparent relationship between a compound's k(on) and its IC(50) could not be discerned. Similarly, a correlation between a compound's IC(50) and its thermodynamic parameters DeltaH degrees and DeltaS degrees could not be established. The lack of a predominant kinetic or thermodynamic signature associated with the inhibitory potential of these compounds demonstrates that there exists, even within a single well-defined system, a library of kinetic routes or, alternatively, a library of initial and final enthalpic and entropic states from which to effect inhibition. As a complement to these studies, selected double mutant thermodynamic cycles were performed to probe the energetic coupling, if any, between common sites of fluorination in both the azaindole and pyrazole classes and two different substituents. Although both cycles indicated negligible coupling free energies, both revealed significant coupling enthalpies, an observation made in other similarly dissected systems. The possible significance and caveats associated with these findings along with the advantages of using Biacore technology to derive thermodynamic parameters in drug discovery efforts are discussed.

Fragment-based discovery of hepatitis C virus NS5b RNA polymerase inhibitors.

Non-nucleoside inhibitors of HCV NS5b RNA polymerase were discovered by a fragment-based lead discovery approach, beginning with crystallographic fragment screening. The NS5b binding affinity and biochemical activity of fragment hits and inhibitors was determined by surface plasmon resonance (Biacore) and an enzyme inhibition assay, respectively. Crystallographic fragment screening hits with approximately 1-10mM binding affinity (K(D)) were iteratively optimized to give leads with approximately 200nM biochemical activity and low microM cellular activity in a Replicon assay.

Discovery of triazolinone non-nucleoside inhibitors of HIV reverse transcriptase.

Novel non-nucleoside inhibitors of HIV-RT that contain pyridazinone isosteres were prepared, and a series of triazolinones were found to be potent inhibitors of HIV replication. These compounds were active against several NNRTI-resistant virus strains. Pharmacokinetic studies indicated that inhibitor 7e has good bioavailability in rats. Several fragments of inhibitor 7c were prepared, and the binding of these compounds to HIV-RT was analyzed by surface plasmon resonance spectroscopy.

Mechanism for multiple ligand recognition by the human transferrin receptor.

Transferrin receptor 1 (TfR) plays a critical role in cellular iron import for most higher organisms. Cell surface TfR binds to circulating iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes, where low pH promotes iron to dissociate from transferrin (Tf) in a TfR-assisted process. The iron-free form of Tf (apo-Tf) remains bound to TfR and is recycled to the cell surface, where the complex dissociates upon exposure to the slightly basic pH of the blood. Fe-Tf competes for binding to TfR with HFE, the protein mutated in the iron-overload disease hereditary hemochromatosis. We used a quantitative surface plasmon resonance assay to determine the binding affinities of an extensive set of site-directed TfR mutants to HFE and Fe-Tf at pH 7.4 and to apo-Tf at pH 6.3. These results confirm the previous finding that Fe-Tf and HFE compete for the receptor by binding to an overlapping site on the TfR helical domain. Spatially distant mutations in the TfR protease-like domain affect binding of Fe-Tf, but not iron-loaded Tf C-lobe, apo-Tf, or HFE, and mutations at the edge of the TfR helical domain affect binding of apo-Tf, but not Fe-Tf or HFE. The binding data presented here reveal the binding footprints on TfR for Fe-Tf and apo-Tf. These data support a model in which the Tf C-lobe contacts the TfR helical domain and the Tf N-lobe contacts the base of the TfR protease-like domain. The differential effects of some TfR mutations on binding to Fe-Tf and apo-Tf suggest differences in the contact points between TfR and the two forms of Tf that could be caused by pH-dependent conformational changes in Tf, TfR, or both. From these data, we propose a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf.

The molecular mechanism for receptor-stimulated iron release from the plasma iron transport protein transferrin.

Human transferrin receptor 1 (TfR) binds iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes where iron is released in a TfR-facilitated process. Consistent with our hypothesis that TfR binding stimulates iron release from Fe-Tf at acidic pH by stabilizing the apo-Tf conformation, a TfR mutant (W641A/F760A-TfR) that binds Fe-Tf, but not apo-Tf, cannot stimulate iron release from Fe-Tf, and less iron is released from Fe-Tf inside cells expressing W641A/F760A-TfR than cells expressing wild-type TfR (wtTfR). Electron paramagnetic resonance spectroscopy shows that binding at acidic pH to wtTfR, but not W641A/F760A-TfR, changes the Tf iron binding site > or =30 A from the TfR W641/F760 patch. Mutation of Tf histidine residues predicted to interact with the W641/F760 patch eliminates TfR-dependent acceleration of iron release. Identification of TfR and Tf residues critical for TfR-facilitated iron release, yet distant from a Tf iron binding site, demonstrates that TfR transmits long-range conformational changes and stabilizes the conformation of apo-Tf to accelerate iron release from Fe-Tf.

Atom-Atom-Path similarity and Sphere Exclusion clustering: tools for prioritizing fragment hits.

BACKGROUND: After performing a fragment based screen the resulting hits need to be prioritized for follow-up structure elucidation and chemistry. This paper describes a new similarity metric, Atom-Atom-Path (AAP) similarity that is used in conjunction with the Directed Sphere Exclusion (DISE) clustering method to effectively organize and prioritize the fragment hits. The AAP similarity rewards common substructures and recognizes minimal structure differences. The DISE method is order-dependent and can be used to enrich fragments with properties of interest in the first clusters. RESULTS: The merit of the software is demonstrated by its application to the MAP4K4 fragment screening hits using ligand efficiency (LE) as quality measure. The first clusters contain the hits with the highest LE. The clustering results can be easily visualized in a LE-over-clusters scatterplot with points colored by the members' similarity to the corresponding cluster seed. The scatterplot enables the extraction of preliminary SAR. CONCLUSIONS: The detailed structure differentiation of the AAP similarity metric is ideal for fragment-sized molecules. The order-dependent nature of the DISE clustering method results in clusters ordered by a property of interest to the teams. The combination of both allows for efficient prioritization of fragment hit for follow-ups. Graphical abstractAAP similarity computation and DISE clustering visualization.

Development of a Model Protein Interaction Pair as a Benchmarking Tool for the Quantitative Analysis of 2-Site Protein-Protein Interactions.

A significant challenge in the molecular interaction field is to accurately determine the stoichiometry and stepwise binding affinity constants for macromolecules having >1 binding site. The mission of the Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource Facilities (ABRF) is to show how biophysical technologies are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies [such as biosensor, microcalorimetry, or analytic ultracentrifugation (AUC)]. In the present work, the MIRG has developed a robust model protein interaction pair consisting of a bivalent variant of the Bacillus amyloliquefaciens extracellular RNase barnase and a variant of its natural monovalent intracellular inhibitor protein barstar. It is demonstrated that this system can serve as a benchmarking tool for the quantitative analysis of 2-site protein-protein interactions. The protein interaction pair enables determination of precise binding constants for the barstar protein binding to 2 distinct sites on the bivalent barnase binding partner (termed binase), where the 2 binding sites were engineered to possess affinities that differed by 2 orders of magnitude. Multiple MIRG laboratories characterized the interaction using isothermal titration calorimetry (ITC), AUC, and surface plasmon resonance (SPR) methods to evaluate the feasibility of the system as a benchmarking model. Although general agreement was seen for the binding constants measured using solution-based ITC and AUC approaches, weaker affinity was seen for surface-based method SPR, with protein immobilization likely affecting affinity. An analysis of the results from multiple MIRG laboratories suggests that the bivalent barnase-barstar system is a suitable model for benchmarking new approaches for the quantitative characterization of complex biomolecular interactions.

Internal motions prime cIAP1 for rapid activation.

Cellular inhibitor of apoptosis 1 (cIAP1) is a ubiquitin ligase with critical roles in the control of programmed cell death and NF-κB signaling. Under normal conditions, the protein exists as an autoinhibited monomer, but proapoptotic signals lead to its dimerization, activation and proteasomal degradation. This view of cIAP1 as a binary switch has been informed by static structural studies that cannot access the protein's dynamics. Here, we use NMR spectroscopy to study micro- and millisecond motions of specific domain interfaces in human cIAP1 and use time-resolved small-angle X-ray scattering to observe the global conformational changes necessary for activation. Although motions within each interface of the 'closed' monomer are insufficient to activate cIAP1, they enable associations with catalytic partners and activation factors. We propose that these internal motions facilitate rapid peptide-induced opening and dimerization of cIAP1, which undergoes a dramatic spring-loaded structural transition.

Tailoring small molecules for an allosteric site on procaspase-6.

Although they represent attractive therapeutic targets, caspases have so far proven recalcitrant to the development of drugs targeting the active site. Allosteric modulation of caspase activity is an alternate strategy that potentially avoids the need for anionic and electrophilic functionality present in most active-site inhibitors. Caspase-6 has been implicated in neurodegenerative disease, including Huntington's and Alzheimer's diseases. Herein we describe a fragment-based lead discovery effort focused on caspase-6 in its active and zymogen forms. Fragments were identified for procaspase-6 using surface plasmon resonance methods and subsequently shown by X-ray crystallography to bind a putative allosteric site at the dimer interface. A fragment-merging strategy was employed to produce nanomolar-affinity ligands that contact residues in the L2 loop at the dimer interface, significantly stabilizing procaspase-6. Because rearrangement of the L2 loop is required for caspase-6 activation, our results suggest a strategy for the allosteric control of caspase activation with drug-like small molecules.

Structure of the BRAF-MEK complex reveals a kinase activity independent role for BRAF in MAPK signaling.

Numerous oncogenic mutations occur within the BRAF kinase domain (BRAF(KD)). Here we show that stable BRAF-MEK1 complexes are enriched in BRAF(WT) and KRAS mutant (MT) cells but not in BRAF(MT) cells. The crystal structure of the BRAF(KD) in a complex with MEK1 reveals a face-to-face dimer sensitive to MEK1 phosphorylation but insensitive to BRAF dimerization. Structure-guided studies reveal that oncogenic BRAF mutations function by bypassing the requirement for BRAF dimerization for activity or weakening the interaction with MEK1. Finally, we show that conformation-specific BRAF inhibitors can sequester a dormant BRAF-MEK1 complex resulting in pathway inhibition. Taken together, these findings reveal a regulatory role for BRAF in the MAPK pathway independent of its kinase activity but dependent on interaction with MEK.