Novel reversible methionine aminopeptidase-2 (MetAP-2) inhibitors based on purine and related bicyclic templates.

The natural product fumagillin 1 and derivatives like TNP-470 2 or beloranib 3 bind to methionine aminopeptidase 2 (MetAP-2) irreversibly. This enzyme is critical for protein maturation and plays a key role in angiogenesis. In this paper we describe the synthesis, MetAP-2 binding affinity and structural analysis of reversible MetAP-2 inhibitors. Optimization of enzymatic activity of screening hit 10 (IC50: 1µM) led to the most potent compound 27 (IC50: 0.038µM), with a concomitant improvement in LLE from 2.1 to 4.2. Structural analysis of these MetAP-2 inhibitors revealed an unprecedented conformation of the His339 side-chain imidazole ring being co-planar sandwiched between the imidazole of His331 and the aryl-ether moiety, which is bound to the purine scaffold. Systematic alteration and reduction of H-bonding capability of this metal binding moiety induced an unexpected 180° flip for the triazolo[1,5-a]pyrimdine bicyclic template.

Discovery of novel 7-azaindoles as PDK1 inhibitors.

A combined screening strategy using HTS together with focused kinase library and virtual screening led to the identification of diverse chemical series as PDK1 inhibitors. We focused our medicinal chemistry efforts on 7-azaindoles with low micromolar IC50s (e.g., 16: IC50=1.1µM) in the biochemical PDK1 assay. Our structure-guided optimization efforts considered also PDK1 X-ray structures with weaker binding fragments and resulted in 7-azaindoles with significantly improved biochemical PDK1 potency in the two-digit nanomolar range. However, the most potent analogues only showed moderate activities in a cellular mechanistic assay (42: IC50=2.3µM) together with either low microsomal stability or low permeability. The described structure-activity relationship together with PDK1 X-ray structures and early ADME data provided the basis for our subsequent hit-to-lead program.

Lersivirine, a nonnucleoside reverse transcriptase inhibitor with activity against drug-resistant human immunodeficiency virus type 1.

The nonnucleoside reverse transcriptase inhibitors (NNRTIs) are key components of highly active antiretroviral therapy (HAART) for the treatment of human immunodeficiency virus type 1 (HIV-1). A major problem with the first approved NNRTIs was the emergence of mutations in the HIV-1 reverse transcriptase (RT), in particular K103N and Y181C, which led to resistance to the entire class. We adopted an iterative strategy to synthesize and test small molecule inhibitors from a chemical series of pyrazoles against wild-type (wt) RT and the most prevalent NNRTI-resistant mutants. The emerging candidate, lersivirine (UK-453,061), binds the RT enzyme in a novel way (resulting in a unique resistance profile), inhibits over 60% of viruses bearing key RT mutations, with 50% effective concentrations (EC(50)s) within 10-fold of those for wt viruses, and has excellent selectivity against a range of human targets. Altogether lersivirine is a highly potent and selective NNRTI, with excellent efficacy against NNRTI-resistant viruses.

The discovery and optimization of hexahydro-2H-pyrano[3,2-c]quinolines (HHPQs) as potent and selective inhibitors of the mitotic kinesin-5.

Here we describe the discovery and optimization of hexahydro-2H-pyrano[3,2-c]quinolines (HHPQs) as potent and selective inhibitors of the mitotic kinesin-5 originally found during a high-throughput screening (HTS) campaign sampling our in-house compound collection. The compounds optimized subsequently and characterized herein were potently inhibiting the ATPase activity of Kinesin-5 and also exhibited consistent cellular activity, in that cells arrested in mitosis and apoptosis induction could be observed. X-ray crystallographic data demonstrated that these inhibitors bind in an allosteric pocket of Kinesin-5 distant from the nucleotide and microtubule binding sites. The selected clinical candidate EMD 534085 caused strong growth inhibition in human tumor xenograft models using Colo 205 colon carcinoma cells at doses below 30mg/kg administered twice weekly without showing severe toxicity as determined by loss of body weight.

Novel indazole non-nucleoside reverse transcriptase inhibitors using molecular hybridization based on crystallographic overlays.

A major problem associated with non-nucleoside reverse transcriptase inhibitors (NNRTIs) for the treatment of HIV is their lack of resilience to mutations in the reverse transcriptase (RT) enzyme. Using structural overlays of the known inhibitors efavirenz and capravirine complexed in RT as a starting point, and structure-based drug design techniques, we have created a novel series of indazole NNRTIs that possess excellent metabolic stability and mutant resilience.

Matuzumab binding to EGFR prevents the conformational rearrangement required for dimerization.

An increasing number of therapeutic antibodies targeting tumors that express the epidermal growth factor receptor (EGFR) are in clinical use or late stages of clinical development. Here we investigate the molecular basis for inhibition of EGFR activation by the therapeutic antibody matuzumab (EMD72000). We describe the X-ray crystal structure of the Fab fragment of matuzumab (Fab72000) in complex with isolated domain III from the extracellular region of EGFR. Fab72000 interacts with an epitope on EGFR that is distinct from the ligand-binding region on domain III and from the cetuximab/Erbitux epitope. Matuzumab blocks ligand-induced receptor activation indirectly by sterically preventing the domain rearrangement and local conformational changes that must occur for high-affinity ligand binding and receptor dimerization.

Role of the N-terminal extension of the (betaalpha)8-barrel enzyme indole-3-glycerol phosphate synthase for its fold, stability, and catalytic activity.

Indole-3-glycerol phosphate synthase (IGPS) catalyzes the fifth step in the biosynthesis of tryptophan. It belongs to the large and versatile family of (betaalpha)(8)-barrel enzymes but has an unusual N-terminal extension of about 40 residues. Limited proteolysis with trypsin of IGPS from both Sulfolobus solfataricus (sIGPS) and Thermotoga maritima (tIGPS) removes about 25 N-terminal residues and one of the two extra helices contained therein. To assess the role of the extension, the N-terminally truncated variants sIGPSDelta(1-26) and tIGPSDelta(1-25) were produced recombinantly in Escherichia coli, purified, and characterized in comparison to the wild-type enzymes. Both sIGPSDelta(1-26) and tIGPSDelta(1-25) have unchanged oligomerization states and turnover numbers. In contrast, their Michaelis constants for the substrate 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate are increased, and their resistance toward unfolding induced by heat and guanidinium chloride is decreased. sIGPSDelta(1-26) was crystallized, and its X-ray structure was solved at 2.8 A resolution. The comparison with the known structure of sIGPS reveals small differences that account for its reduced substrate affinity and protein stability. The structure of the core of sIGPSDelta(1-26) is, however, unchanged compared to sIGPS, explaining its retained catalytic activity and consistent with the idea that it evolved from the same ancestor as the phosphoribosyl anthranilate isomerase and the alpha-subunit of tryptophan synthase. These (betaalpha)(8)-barrel enzymes catalyze the reactions preceding and following IGPS in tryptophan biosynthesis but lack an N-terminal extension.

The crystal structure of indoleglycerol-phosphate synthase from Thermotoga maritima. Kinetic stabilization by salt bridges.

The crystal structure of the thermostable indoleglycerol-phosphate synthase from Thermotoga maritima (tIGPS) was determined at 2.5 A resolution. It was compared with the structures of the thermostable sIGPS from Sulfolobus solfataricus and of the thermolabile eIGPS from Escherichia coli. The main chains of the three (beta alpha)(8)-barrel proteins superimpose closely, and the packing of side chains in the beta-barrel cores, as well as the architecture of surface loops, is very similar. Both thermostable proteins have, however, 17 strong salt bridges, compared with only 10 in eIGPS. The number of additional salt bridges in tIGPS and sIGPS correlates well with their reduced rate of irreversible thermal inactivation at 90 degrees C. Only 3 of 17 salt bridges in tIGPS and sIGPS are topologically conserved. The major difference between the two proteins is the preference for interhelical salt bridges in sIGPS and intrahelical ones in tIGPS. The different implementation of salt bridges in the closely related proteins suggests that the stabilizing effect of salt bridges depends rather on the sum of their individual contributions than on their location. This observation is consistent with a protein unfolding mechanism where the simultaneous breakdown of all salt bridges is the rate-determining step.