Alan Hruza

Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation.

Crystal structures of human endothelial nitric oxide synthase (eNOS) and human inducible NOS (iNOS) catalytic domains were solved in complex with the arginine substrate and an inhibitor S-ethylisothiourea (SEITU), respectively. The small molecules bind in a narrow cleft within the larger active-site cavity containing heme and tetrahydrobiopterin. Both are hydrogen-bonded to a conserved glutamate (eNOS E361, iNOS E377). The active-site residues of iNOS and eNOS are nearly identical. Nevertheless, structural comparisons provide a basis for design of isozyme-selective inhibitors. The high-resolution, refined structures of eNOS (2.4 Å resolution) and iNOS (2.25 Å resolution) reveal an unexpected structural zinc situated at the intermolecular interface and coordinated by four cysteines, two from each monomer.

Zinc mediated dimer of human interferon-α2b revealed by X-ray crystallography

BACKGROUND The human alpha-interferon (huIFN-alpha) family displays broad spectrum antiviral, antiproliferative and immunomodulatory activities on a variety of cell types. The diverse biological activities of the IFN-alphas are conveyed to cells through specific interactions with cell-surface receptors. Despite considerable effort, no crystal structure of a member of this family has yet been reported, because the quality of the protein crystals have been unsuitable for crystallographic studies. Until now, structural models of the IFN-alphas have been based on the structure of murine IFN-beta (muIFN-beta). These models are likely to be inaccurate, as the amino acid sequence of muIFN-beta differs significantly from the IFN-alphas at proposed receptor-binding sites. Structural information on a huIFN-alpha subtype would provide an improved basis for modeling the structures of the entire IFN-alpha family. RESULTS The crystal structure of recombinant human interferon-alpha 2b (huIFN-alpha 2b) has been determined at 2.9 A resolution. HuIFN-alpha 2b exists in the crystal as a noncovalent dimer, which associates in a novel manner. Unlike other structurally characterized cytokines, extensive interactions in the dimer interface are mediated by a zinc ion (Zn2+). The overall fold of huIFN-alpha 2b is most similar to the structure of muIFN-beta. Unique to huIFN-alpha 2b is a 3(10) helix in the AB loop which is held to the core of the molecule by a disulfide bond. CONCLUSIONS The structure of huIFN-alpha 2b provides an accurate model for analysis of the > 15 related type 1 interferon molecules. HuIFN-alpha 2b displays considerable structural similarity with muIFN-beta, interleukin-10 and interferon-gamma, which also bind related class 2 cytokine receptors. From these structural comparisons and numerous studies on the effects of mutations on biological activity, we have identified protein surfaces that appear to be important in receptor activation. This study also reveals the potential biological importance of the huIFN-alpha 2b dimer.

Discovery of Dinaciclib (SCH 727965): A Potent and Selective Inhibitor of Cyclin-Dependent Kinases

Inhibition of cyclin-dependent kinases (CDKs) has emerged as an attractive strategy for the development of novel oncology therapeutics. Herein is described the utilization of an in vivo screening approach with integrated efficacy and tolerability parameters to identify candidate CDK inhibitors with a suitable balance of activity and tolerability. This approach has resulted in the identification of SCH 727965, a potent and selective CDK inhibitor that is currently undergoing clinical evaluation.

Discovery of a Potent, Injectable Inhibitor of Aurora Kinases Based on the Imidazo-[1,2-a]-Pyrazine Core

The imidazo-[1,2-a]-pyrazine (1) is a dual inhibitor of Aurora kinases A and B with modest cell potency (IC50 = 250 nM) and low solubility (5 μM). Lead optimization guided by the binding mode led to the acyclic amino alcohol 12k (SCH 1473759), which is a picomolar inhibitor of Aurora kinases (TdF K d Aur A = 0.02 nM and Aur B = 0.03 nM) with improved cell potency (phos-HH3 inhibition IC50 = 25 nM) and intrinsic aqueous solubility (11.4 mM). It also demonstrated efficacy and target engagement in human tumor xenograft mouse models.

Discovery of Novel, Dual Mechanism ERK Inhibitors by Affinity Selection Screening of an Inactive Kinase

An affinity-based mass spectrometry screening technology was used to identify novel binders to both nonphosphorylated and phosphorylated ERK2. Screening of inactive ERK2 identified a pyrrolidine analogue 1 that bound to both nonphosphorylated and phosphorylated ERK2 and inhibited ERK2 kinase activity. Chemical optimization identified compound 4 as a novel, potent, and highly selective ERK1,2 inhibitor which not only demonstrated inhibition of phosphorylation of ERK substrate p90RSK but also demonstrated inhibition of ERK1,2 phosphorylation on the activation loop. X-ray cocrystallography revealed that upon binding of compound 4 to ERK2, Tyr34 undergoes a rotation (flip) along with a shift in the poly-Gly rich loop to create a new binding pocket into which 4 can bind. This new binding mode represents a novel mechanism by which high affinity ATP-competitive compounds may achieve excellent kinase selectivity.

Binding affinities and geometries of various metal ligands in peptide deformylase inhibitors

Removal of the N-terminal formyl group from newly synthesized proteins by the enzyme peptide deformylase (PDF) is essential for normal growth of bacteria but not higher organisms. Recently, PDF has been explored as a target for novel antibiotics. Screening a collection of natural products for antimicrobial activity identified actinonin and two matlystatin analogs as potent PDF inhibitors. A number of synthetic analogs of these natural products were prepared and their inhibitory potency determined. Previous work has shown that PDF is an iron metalloproteinase also containing a catalytic glutamic acid residue. Ligation of the ferrous cation is an essential feature of potent inhibitors. The structures of actinonin, a matlystatin analog and a synthetic inhibitor complexed with PDF were determined by crystallography. A quantum mechanics/molecular mechanics (QM/MM) method was used to reproduce the geometry of known complexes, to predict the protonation state in the active site and to predict the geometry of additional complexes. The requirement for protonation of the active site glutamate anion is an important factor in understanding the potency of inhibitors with acidic iron-ligating groups such as hydroxamate and carboxylate. Even though potent inhibitors of PDF have been discovered, their bacteriostatic mechanism of action and the rapid development of resistance in vitro may limit their potential as antibacterial drugs.

Discovery of a 3-(4-Pyrimidinyl) Indazole (MLi-2), an Orally Available and Selective Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitor that Reduces Brain Kinase Activity

Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein which contains a kinase domain and GTPase domain among other regions. Individuals possessing gain of function mutations in the kinase domain such as the most prevalent G2019S mutation have been associated with an increased risk for the development of Parkinsons disease (PD). Given this genetic validation for inhibition of LRRK2 kinase activity as a potential means of affecting disease progression, our team set out to develop LRRK2 inhibitors to test this hypothesis. A high throughput screen of our compound collection afforded a number of promising indazole leads which were truncated in order to identify a minimum pharmacophore. Further optimization of these indazoles led to the development of MLi-2 (1): a potent, highly selective, orally available, brain-penetrant inhibitor of LRRK2.

Isolation and structure elucidation of two novel deformylase inhibitors produced by Streptomyces sp.

Abstract Sch 382582 ( 1 ) and Sch 382583 ( 2 ), two novel pseudopeptides, were isolated from fermentation broth of Streptomyces sp. as bacteria peptide deformylase inhibitors. Structure elucidation of 1 and 2 was accomplished by extensive 2D NMR spectroscopic studies including NOESY, HMQC-TOCSY and HMBC experiments, and the relative stereochemistry was determined by X-ray crystallography. Both compounds displayed potent inhibitory activity against E. coli deformylase.

Discovery of 1-(1H-Pyrazolo[4,3-c]pyridin-6-yl)urea Inhibitors of Extracellular Signal-Regulated Kinase (ERK) for the Treatment of Cancers

The ERK/MAPK pathway plays a central role in the regulation of critical cellular processes and is activated in more than 30% of human cancers. Specific BRAF and MEK inhibitors have shown clinical efficacy in patients for the treatment of BRAF-mutant melanoma. However, the majority of responses are transient, and resistance is often associated with pathway reactivation of the ERK signal pathway. Acquired resistance to these agents has led to greater interest in ERK, a downstream target of the MAPK pathway. De novo design efforts of a novel scaffold derived from SCH772984 by employing hydrogen bond interactions specific for ERK in the binding pocket identified 1-(1H-pyrazolo[4,3-c]pyridin-6-yl)ureas as a viable lead series. Sequential SAR studies led to the identification of highly potent and selective ERK inhibitors with low molecular weight and high LE. Compound 21 exhibited potent target engagement and strong tumor regression in the BRAF(V600E) xenograft model.

Synthesis, properties, and applications of diazotrifluropropanoyl-containing photoactive analogs of farnesyl diphosphate containing modified linkages for enhanced stability.

Photoactive analogs of farnesyl diphosphate (FPP) are useful probes in studies of enzymes that employ this molecule as a substrate. Here, we describe the preparation and properties of two new FPP analogs that contain diazotrifluoropropanoyl photophores linked to geranyl diphosphate via amide or ester linkages. The amide‐linked analog (3) was synthesized in 32P‐labeled form from geraniol in seven steps. Experiments with Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) showed that 3 is an alternative substrate for the enzyme. Photolysis experiments with [32P]3 demonstrate that this compound labels the β‐subunits of both farnesyltransferase and geranylgeranyltransferase (types 1 and 2). However, the amide‐linked probe 3 undergoes a rearrangement to a photochemically unreactive isomeric triazolone upon long term storage making it inconvenient to use. To address this stability issue, the ester‐linked analog 4 was prepared in six steps from geraniol. Computational analysis and X‐ray crystallographic studies suggest that 4 binds to protein farnesyl transferase (PFTase) in a similar fashion as FPP. Compound 4 is also an alternative substrate for PFTase, and a 32P‐labeled form selectively photocrosslinks the β‐subunit of ScPFTase as well as E. coli farnesyldiphosphate synthase and a germacrene‐producing sesquiterpene synthase from Nostoc sp. strain PCC7120 (a cyanobacterial source). Finally, nearly exclusive labeling of ScPFTase in crude E. coli extract was observed, suggesting that [32P]4 manifests significant selectivity and should hence be useful for identifying novel FPP‐utilizing enzymes in crude protein preparations.