Design, stereoselective synthesis, and biological evaluation of novel tri-cyclic compounds as inhibitor of apoptosis proteins (IAP) antagonists.

We recently reported the discovery of octahydropyrrolo[1,2-a]pyrazine A as a lead compound for an inhibitor of apoptosis proteins (IAP) antagonist. To develop IAP antagonists with favorable PK profiles, we designed novel tri-cyclic compounds, octahydro-1H-cyclopropa[4,5]pyrrolo[1,2-a]pyrazines 1 and 2 based on co-crystal structural analysis of A with cellular IAP-1 (cIAP-1). The additional cyclopropane moiety was used to block the predicted metabolic site of compound A without detriment to the binding affinity for cIAP. Compounds 1 and 2 were stereoselectively synthesized via intermediates 4a and 5b', which were obtained by Simmons-Smith cyclopropanation of ethylester 3a and silyl ether 3b'. Compounds 1 and 2 showed strong growth inhibition in MDA-MB-231 breast cancer cells and improved metabolic stability in comparison to A. Compound 2 exhibited significant in vivo PD effects to increase tumor necrosis factor-alpha mRNA in a dose dependent manner.

Conserved structural chemistry for incision activity in structurally non-homologous apurinic/apyrimidinic endonuclease APE1 and endonuclease IV DNA repair enzymes.

Non-coding apurinic/apyrimidinic (AP) sites in DNA form spontaneously and as DNA base excision repair intermediates are the most common toxic and mutagenic in vivo DNA lesion. For repair, AP sites must be processed by 5' AP endonucleases in initial stages of base repair. Human APE1 and bacterial Nfo represent the two conserved 5' AP endonuclease families in the biosphere; they both recognize AP sites and incise the phosphodiester backbone 5' to the lesion, yet they lack similar structures and metal ion requirements. Here, we determined and analyzed crystal structures of a 2.4 Å resolution APE1-DNA product complex with Mg(2+) and a 0.92 Å Nfo with three metal ions. Structural and biochemical comparisons of these two evolutionarily distinct enzymes characterize key APE1 catalytic residues that are potentially functionally similar to Nfo active site components, as further tested and supported by computational analyses. We observe a magnesium-water cluster in the APE1 active site, with only Glu-96 forming the direct protein coordination to the Mg(2+). Despite differences in structure and metal requirements of APE1 and Nfo, comparison of their active site structures surprisingly reveals strong geometric conservation of the catalytic reaction, with APE1 catalytic side chains positioned analogously to Nfo metal positions, suggesting surprising functional equivalence between Nfo metal ions and APE1 residues. The finding that APE1 residues are positioned to substitute for Nfo metal ions is supported by the impact of mutations on activity. Collectively, the results illuminate the activities of residues, metal ions, and active site features for abasic site endonucleases.

Structure-activity relationships and key structural feature of pyridyloxybenzene-acylsulfonamides as new, potent, and selective peroxisome proliferator-activated receptor (PPAR) γ Agonists.

In our search for a novel class of non-TZD, non-carboxylic acid peroxisome proliferator-activated receptor (PPAR) γ agonists, we explored alternative lipophilic templates to replace benzylpyrazole core of the previously reported agonist 1. Introduction of a pentylsulfonamide group into arylpropionic acids derived from previous in-house PPARγ ligands succeeded in the identification of 2-pyridyloxybenzene-acylsulfonamide 2 as a lead compound. Docking studies of compound 2 suggested that a substituent para to the central benzene ring should be incorporated to effectively fill the Y-shaped cavity of the PPARγ ligand-binding domain (LBD). This strategy led to significant improvement of PPARγ activity. Further optimization to balance in vitro activity and metabolic stability allowed the discovery of the potent, selective and orally efficacious PPARγ agonist 8f. Structure-activity relationship study as well as detailed analysis of the binding mode of 8f to the PPARγ-LBD revealed the essential structural features of this series of ligands.

A new class of non-thiazolidinedione, non-carboxylic-acid-based highly selective peroxisome proliferator-activated receptor (PPAR) γ agonists: design and synthesis of benzylpyrazole acylsulfonamides.

Herein, we describe the design, synthesis, and structure-activity relationships of novel benzylpyrazole acylsulfonamides as non-thiazolidinedione (TZD), non-carboxylic-acid-based peroxisome proliferator-activated receptor (PPAR) γ agonists. Docking model analysis of in-house weak agonist 2 bound to the reported PPARγ ligand binding domain suggested that modification of the carboxylic acid of 2 would help strengthen the interaction of 2 with the TZD pocket and afford non-carboxylic-acid-based agonists. In this study, we used an acylsulfonamide group as the ring-opening analog of TZD as an isosteric replacement of carboxylic acid moiety of 2; further, preliminary modification of the terminal alkyl chain on the sulfonyl group gave the lead compound 3c. Subsequent optimization of the resulting compound gave the potent agonists 25c, 30b, and 30c with high metabolic stability and significant antidiabetic activity. Further, we have described the difference in binding mode of the carboxylic-acid-based agonist 1 and acylsulfonamide 3d.

Design, synthesis, and structure-activity relationships of spirolactones bearing 2-ureidobenzothiophene as acetyl-CoA carboxylases inhibitors.

The co-crystal structure of the human acetyl-coenzyme A 2 (ACC2) carboxyl transferase domain and the reported compound CP-640186 (1b) suggested that two carbonyl groups are essential for potent ACC2 inhibition. By focusing on enhancing the interactions between the two carbonyl groups and the amino acid residues Gly(2162) and Glu(2230), we used ligand- and structure-based drug design to discover spirolactones bearing a 2-ureidobenzothiophene moiety.

Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists.

Chemokine receptors are critical regulators of cell migration in the context of immune surveillance, inflammation, and development. The G protein-coupled chemokine receptor CXCR4 is specifically implicated in cancer metastasis and HIV-1 infection. Here we report five independent crystal structures of CXCR4 bound to an antagonist small molecule IT1t and a cyclic peptide CVX15 at 2.5 to 3.2 angstrom resolution. All structures reveal a consistent homodimer with an interface including helices V and VI that may be involved in regulating signaling. The location and shape of the ligand-binding sites differ from other G protein-coupled receptors and are closer to the extracellular surface. These structures provide new clues about the interactions between CXCR4 and its natural ligand CXCL12, and with the HIV-1 glycoprotein gp120.

Structure-based design and synthesis of pyrrole derivatives as MEK inhibitors.

A novel series of pyrrole inhibitors of MEK kinase has been developed using structure-based drug design. Optimization of the series led to the identification of potent inhibitors with good pharmaceutical properties.

2-{3-[4-(Alkylsulfinyl)phenyl]-1-benzofuran-5-yl}-5-methyl-1,3,4-oxadiazole derivatives as novel inhibitors of glycogen synthase kinase-3beta with good brain permeability.

Glycogen synthase kinase 3beta (GSK-3beta) inhibition is expected to be a promising therapeutic approach for treating Alzheimer's disease. Previously we reported a series of 1,3,4-oxadiazole derivatives as potent and highly selective GSK-3beta inhibitors, however, the representative compounds 1a,b showed poor pharmacokinetic profiles. Efforts were made to address this issue by reducing molecular weight and lipophilicity, leading to the identification of oxadiazole derivatives containing a sulfinyl group, (S)-9b and (S)-9c. These compounds exhibited not only highly selective and potent inhibitory activity against GSK-3beta but also showed good pharmacokinetic profiles including favorable BBB penetration. In addition, (S)-9b and (S)-9c given orally to mice significantly inhibited cold water stress-induced tau hyperphosphorylation in mouse brain.

Design, synthesis and structure-activity relationships of 1,3,4-oxadiazole derivatives as novel inhibitors of glycogen synthase kinase-3beta.

Glycogen synthase kinase-3beta (GSK-3beta) is implicated in abnormal hyperphosphorylation of tau protein and its inhibitors are expected to be a promising therapeutic agents for the treatment of Alzheimer's disease. Here we report design, synthesis and structure-activity relationships of a novel series of oxadiazole derivatives as GSK-3beta inhibitors. Among these inhibitors, compound 20x showed highly selective and potent GSK-3beta inhibitory activity in vitro and its binding mode was determined by obtaining the X-ray co-crystal structure of 20x and GSK-3beta.

Structural insights into the conformational selectivity of STI-571 and related kinase inhibitors.

STI-571 (Gleevec) is a highly successful cancer drug due to its activity as an inhibitor of the Abelson cytoplasmic tyrosine kinase (Abl), which is constitutively active in a majority of patients with chronic myelogenous leukemia. STI-571 also inhibits two type III receptor tyrosine kinases, c-Kit and platelet-derived growth factor receptor, and functions by targeting inactive conformations of these kinases. This review focuses on recent developments in X-ray co-crystal structure analyses of STI-571 bound to Abl and the c-Kit receptor tyrosine kinase domain, and also three other relevant kinase inhibitor co-crystal structures. The similar structural features of these inactive kinases suggest they will be useful for the successful drug discovery and development of specific and targeted gene-based cancer drugs.

Switching on kinases: oncogenic activation of BRAF and the PDGFR family.

The cytoplasmic serine/threonine kinase BRAF and receptor tyrosine kinases of the platelet-derived growth factor receptor (PDGFR) family are frequently activated in cancer by mutations of an equivalent amino acid. Structural studies have provided important insights into why these very different kinases share similar oncogenic hot spots and why the PDGFR juxtamembrane region is also a frequent oncogenic target. This research has implications for other kinases that are mutated in human tumours and for the treatment of cancer using kinase inhibitors.

Structural basis for the autoinhibition and STI-571 inhibition of c-Kit tyrosine kinase.

The activity of the c-Kit receptor protein-tyrosine kinase is tightly regulated in normal cells, whereas deregulated c-Kit kinase activity is implicated in the pathogenesis of human cancers. The c-Kit juxtamembrane region is known to have an autoinhibitory function; however the precise mechanism by which c-Kit is maintained in an autoinhibited state is not known. We report the 1.9-A resolution crystal structure of native c-Kit kinase in an autoinhibited conformation and compare it with active c-Kit kinase. Autoinhibited c-Kit is stabilized by the juxtamembrane domain, which inserts into the kinase-active site and disrupts formation of the activated structure. A 1.6-A crystal structure of c-Kit in complex with STI-571 (Imatinib or Gleevec) demonstrates that inhibitor binding disrupts this natural mechanism for maintaining c-Kit in an autoinhibited state. Together, these results provide a structural basis for understanding c-Kit kinase autoinhibition and will facilitate the structure-guided design of specific inhibitors that target the activated and autoinhibited conformations of c-Kit kinase.

Crystal structures of active fully assembled substrate- and product-bound complexes of UDP-N-acetylmuramic acid:L-alanine ligase (MurC) from Haemophilus influenzae.

UDP-N-acetylmuramic acid:L-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg(2+) and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-L-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn(2+) have been determined to 1.85- and 1.7-A resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the gamma-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates.

Structure of a c-kit product complex reveals the basis for kinase transactivation.

The c-Kit proto-oncogene is a receptor protein-tyrosine kinase associated with several highly malignant human cancers. Upon binding its ligand, stem cell factor (SCF), c-Kit forms an active dimer that autophosphorylates itself and activates a signaling cascade that induces cell growth. Disease-causing human mutations that activate SCF-independent constitutive expression of c-Kit are found in acute myelogenous leukemia, human mast cell disease, and gastrointestinal stromal tumors. We report on the phosphorylation state and crystal structure of a c-Kit product complex. The c-Kit structure is in a fully active form, with ordered kinase activation and phosphate-binding loops. These results provide key insights into the molecular basis for c-Kit kinase transactivation to assist in the design of new competitive inhibitors targeting activated mutant forms of c-Kit that are resistant to current chemotherapy regimes.

Structure and activity of a thermostable thymine-DNA glycosylase: evidence for base twisting to remove mismatched normal DNA bases.

The repair of T:G mismatches in DNA is key for maintaining bacterial restriction/modification systems and gene silencing in higher eukaryotes. T:G mismatch repair can be initiated by a specific mismatch glycosylase (MIG) that is homologous to the helix-hairpin-helix (HhH) DNA repair enzymes. Here, we present a 2.0 A resolution crystal structure and complementary mutagenesis results for this thermophilic HhH MIG enzyme. The results suggest that MIG distorts the target thymine nucleotide by twisting the thymine base approximately 90 degrees away from its normal anti position within DNA. We propose that functionally significant differences exist in DNA repair enzyme extrahelical nucleotide binding and catalysis that are characteristic of whether the target base is damaged or is a normal base within a mispair. These results explain why pure HhH DNA glycosylases and combined glycosylase/AP lyases cannot be interconverted by simply altering their functional group chemistry, and how broad-specificity DNA glycosylase enzymes may weaken the glycosylic linkage to allow a variety of damaged DNA bases to be excised.