Discovery of a highly selective chemical inhibitor of matrix metalloproteinase-9 (MMP-9) that allosterically inhibits zymogen activation.

Aberrant activation of matrix metalloproteinases (MMPs) is a common feature of pathological cascades observed in diverse disorders, such as cancer, fibrosis, immune dysregulation, and neurodegenerative diseases. MMP-9, in particular, is highly dynamically regulated in several pathological processes. Development of MMP inhibitors has therefore been an attractive strategy for therapeutic intervention. However, a long history of failed clinical trials has demonstrated that broad-spectrum MMP inhibitors have limited clinical utility, which has spurred the development of inhibitors selective for individual MMPs. Attaining selectivity has been technically challenging because of sequence and structural conservation across the various MMPs. Here, through a biochemical and structural screening paradigm, we have identified JNJ0966, a highly selective compound that inhibited activation of MMP-9 zymogen and subsequent generation of catalytically active enzyme. JNJ0966 had no effect on MMP-1, MMP-2, MMP-3, MMP-9, or MMP-14 catalytic activity and did not inhibit activation of the highly related MMP-2 zymogen. The molecular basis for this activity was characterized as an interaction of JNJ0966 with a structural pocket in proximity to the MMP-9 zymogen cleavage site near Arg-106, which is distinct from the catalytic domain. JNJ0966 was efficacious in reducing disease severity in a mouse experimental autoimmune encephalomyelitis model, demonstrating the viability of this therapeutic approach. This discovery reveals an unprecedented pharmacological approach to MMP inhibition, providing an opportunity to improve selectivity of future clinical drug candidates. Targeting zymogen activation in this manner may also allow for pharmaceutical exploration of other enzymes previously viewed as intractable drug targets.

Screening and characterization of human monoglyceride lipase active site inhibitors using orthogonal binding and functional assays.

Endocannabinoids such as 2-arachidonylglycerol (2-AG) are ligands for cannabinoid receptors that contribute to the transmission and modulation of pain signals. The antinociceptive effect of exogenous 2-AG suggests that inhibition of monoglyceride lipase (MGLL), the enzyme responsible for degrading 2-AG and arresting signaling, may be a target for pain modulation. Here we describe the characterization of MGLL ligands following a high-throughput screening campaign. Ligands were discovered using ThermoFluor, a label-free affinity-based screening tool that measures ligand binding via modulation of protein thermal stability. A kinetic fluorescent assay using the substrate 4-methylcoumarin butyrate was used to counterscreen confirmed HTS positives. A comparison of results from binding and inhibition assays allowed elucidation of compound mechanism of action. We demonstrate the limit of each technology and the benefits of using orthogonal assay techniques in profiling compounds.

Optimization of a pyrazole hit from FBDD into a novel series of indazoles as ketohexokinase inhibitors.

A series of indazoles have been discovered as KHK inhibitors from a pyrazole hit identified through fragment-based drug discovery (FBDD). The optimization process guided by both X-ray crystallography and solution activity resulted in lead-like compounds with good pharmaceutical properties.

How to avoid rediscovering the known.

For anyone who has participated in a screening exercise in a pharmaceutical or biotech setting with the aim to discover hits against protein targets, it is evident that a single screening exercise does not always offer hits of sufficient quality to be progressed into a lead compound. Often, more than one screen is needed. The premise in conducting a new screening exercise is to find "better" hits that are chemically and pharmacologically more attractive. As we move into challenging, new target classes, the need of new methods to find tractable hits is ever more urgent and the availability of differentiated backup compounds is crucial to sustain a clinical program. The obvious alternate routes to conduct the new screen include an improved compound library, a larger compound library, and different or more sensitive detection methods. As many of us have experienced firsthand, repeating a screen without drastically changing the chemical nature of the compound library or information content of the readout likely will offer only variations of the original hits. This chapter describes the strategies to adopt in fragment-based lead discovery to avoid rediscovering the known.

Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 Å resolution.

A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.

Electron density guided fragment-based lead discovery of ketohexokinase inhibitors.

A fragment-based drug design paradigm has been successfully applied in the discovery of lead series of ketohexokinase inhibitors. The paradigm consists of three iterations of design, synthesis, and X-ray crystallographic screening to progress low molecular weight fragments to leadlike compounds. Applying electron density of fragments within the protein binding site as defined by X-ray crystallography, one can generate target specific leads without the use of affinity data. Our approach contrasts with most fragment-based drug design methodology where solution activity is a main design guide. Herein we describe the discovery of submicromolar ketohexokinase inhibitors with promising druglike properties.

SAR of tertiary carbinamine derived BACE1 inhibitors: role of aspartate ligand amine pKa in enzyme inhibition.

The optimization of tertiary carbinamine derived inhibitors of BACE1 from its discovery as an unstable lead to low nanomolar cell active compounds is described. Five-membered heterocycles are reported as stable and potency enhancing linkers. In the course of this work, we have discovered a clear trend where the activity of inhibitors at a given assay pH is dependent on pK(a) of the amino group that interacts directly with the catalytic aspartates. The potency of compounds as inhibitors of Alphabeta production in a cell culture assay correlated much better with BACE1 enzyme potency measured at pH 7.5 than at pH 4.5.

Kinesin spindle protein (KSP) inhibitors. 9. Discovery of (2S)-4-(2,5-difluorophenyl)-n-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide (MK-0731) for the treatment of taxane-refractory cancer.

Inhibition of kinesin spindle protein (KSP) is a novel mechanism for treatment of cancer with the potential to overcome limitations associated with currently employed cytotoxic agents. Herein, we describe a C2-hydroxymethyl dihydropyrrole KSP inhibitor ( 11) that circumvents hERG channel binding and poor in vivo potency, issues that limited earlier compounds from our program. However, introduction of the C2-hydroxymethyl group caused 11 to be a substrate for cellular efflux by P-glycoprotein (Pgp). Utilizing knowledge garnered from previous KSP inhibitors, we found that beta-fluorination modulated the p K a of the piperidine nitrogen and reduced Pgp efflux, but the resulting compound ( 14) generated a toxic metabolite in vivo. Incorporation of fluorine in a strategic, metabolically benign position by synthesis of an N-methyl-3-fluoro-4-(aminomethyl)piperidine urea led to compound 30 that has an optimal in vitro and metabolic profile. Compound 30 (MK-0731) was recently studied in a phase I clinical trial in patients with taxane-refractory solid tumors.

Structure-based design of novel groups for use in the P1 position of thrombin inhibitor scaffolds. Part 2: N-acetamidoimidazoles.

Guided by X-ray crystallography of thrombin-inhibitor complexes and molecular modeling, alkylation of the N1 nitrogen of the imidazole P1 ligand of the pyridinoneacetamide thrombin inhibitor 1 with various acetamide moieties furnished inhibitors with significantly improved thrombin potency, trypsin selectivity, functional in vitro anticoagulant potency and in vivo antithrombotic efficacy. In the pyrazinoneacetamide series, oral bioavailability was also improved.

Structural basis for elastolytic substrate specificity in rodent alpha-chymases.

Divergence of substrate specificity within the context of a common structural framework represents an important mechanism by which new enzyme activity naturally evolves. We present enzymological and x-ray structural data for hamster chymase-2 (HAM2) that provides a detailed explanation for the unusual hydrolytic specificity of this rodent alpha-chymase. In enzymatic characterization, hamster chymase-1 (HAM1) showed typical chymase proteolytic activity. In contrast, HAM2 exhibited atypical substrate specificity, cleaving on the carboxyl side of the P1 substrate residues Ala and Val, characteristic of elastolytic rather than chymotryptic specificity. The 2.5-A resolution crystal structure of HAM2 complexed to the peptidyl inhibitor MeOSuc-Ala-Ala-Pro-Ala-chloromethylketone revealed a narrow and shallow S1 substrate binding pocket that accommodated only a small hydrophobic residue (e.g. Ala or Val). The different substrate specificities of HAM2 and HAM1 are explained by changes in four S1 substrate site residues (positions 189, 190, 216, and 226). Of these, Asn(189), Val(190), and Val(216) form an easily identifiable triplet in all known rodent alpha-chymases that can be used to predict elastolytic specificity for novel chymase-like sequences. Phylogenetic comparison defines guinea pig and rabbit chymases as the closest orthologs to rodent alpha-chymases.

Kinesin spindle protein (KSP) inhibitors. Part 6: Design and synthesis of 3,5-diaryl-4,5-dihydropyrazole amides as potent inhibitors of the mitotic kinesin KSP.

3,5-diaryl-4,5-dihydropyrazoles were discovered to be potent KSP inhibitors with excellent in vivo potency. These enzyme inhibitors possess desirable physical properties that can be readily modified by incorporation of a weakly basic amine. Careful adjustment of amine basicity was essential for preserving cellular potency in a multidrug resistant cell line while maintaining good aqueous solubility.

Optimization of a pyrazoloquinolinone class of Chk1 kinase inhibitors.

The development of 2,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-ones as inhibitors of Chk1 kinase is described. Introduction of a fused ring at the C7/C8 positions of the pyrazoloquinolinone provided an increase in potency while guidance from overlapping inhibitor bound Chk1 X-ray crystal structures contributed to the discovery of a potent and solubilizing propyl amine moiety in compound 52 (Chk1 IC(50)=3.1 nM).

Kinesin spindle protein (KSP) inhibitors. Part 7: Design and synthesis of 3,3-disubstituted dihydropyrazolobenzoxazines as potent inhibitors of the mitotic kinesin KSP.

Observations from two structurally related series of KSP inhibitors led to the proposal and discovery of dihydropyrazolobenzoxazines that possess ideal properties for cancer drug development. The synthesis and characterization of this class of inhibitors along with relevant pharmacokinetic and in vivo data are presented. The synthesis is highlighted by a key [3+2] cycloaddition to form the pyrazolobenzoxazine core followed by diastereospecific installation of a quaternary center.

Kinesin spindle protein (KSP) inhibitors. Part 8: Design and synthesis of 1,4-diaryl-4,5-dihydropyrazoles as potent inhibitors of the mitotic kinesin KSP.

Inspired by previous efforts in the pyrazolobenzoxazine class of KSP inhibitors, the design and synthesis of 1,4-diaryl-4,5-dihydropyrazole inhibitors of KSP are described. Crystallographic evidence of binding mode and in vivo potency data is also highlighted.

Synthesis and evaluation of substituted benzoisoquinolinones as potent inhibitors of Chk1 kinase.

From HTS lead 1, a novel benzoisoquinolinone class of ATP-competitive Chk1 inhibitors was devised and synthesized via a photochemical route. Using X-ray crystallography as a guide, potency was rapidly enhanced through the installation of a tethered basic amine designed to interact with an acidic residue (Glu91) in the enzyme pocket. Further SAR was explored at the solvent front and near to the H1 pocket and resulted in the discovery of low MW, sub-nanomolar inhibitors of Chk1.

Design, synthesis, and SAR of macrocyclic tertiary carbinamine BACE-1 inhibitors.

This Letter describes the design and synthesis of tertiary carbinamine macrocyclic inhibitors of the beta-secretase (BACE-1) enzyme. These macrocyclic inhibitors, some of which incorporate novel P2 substituents, display a 2- to 100-fold increase in potency relative to the previously described acyclic analogs while affording greater stability.

Discovery of oxadiazoyl tertiary carbinamine inhibitors of beta-secretase (BACE-1).

We describe the discovery and optimization of tertiary carbinamine derived inhibitors of the enzyme beta-secretase (BACE-1). These novel non-transition-state-derived ligands incorporate a single primary amine to interact with the catalytic aspartates of the target enzyme. Optimization of this series provided inhibitors with intrinsic and functional potency comparable to evolved transition state isostere derived inhibitors of BACE-1.

Development of 6-substituted indolylquinolinones as potent Chek1 kinase inhibitors.

Through a comparison of X-ray co-crystallographic data for 1 and 2 in the Chek1 active site, it was hypothesized that the affinity of the indolylquinolinone series (2) for Chek1 kinase would be improved via C6 substitution into the hydrophobic region I (HI) pocket. An efficient route to 6-bromo-3-indolyl-quinolinone (9) was developed, and this series was rapidly optimized for potency by modification at C6. A general trend was observed among these low nanomolar Chek1 inhibitors that compounds with multiple basic amines, or elevated polar surface area (PSA) exhibited poor cell potency. Minimization of these parameters (basic amines, PSA) resulted in Chek1 inhibitors with improved cell potency, and preliminary pharmacokinetic data are presented for several of these compounds.

3-(Indol-2-yl)indazoles as Chek1 kinase inhibitors: Optimization of potency and selectivity via substitution at C6.

The development of 3-(indol-2-yl)indazoles as inhibitors of Chek1 kinase is described. Introduction of amides and heteroaryl groups at the C6 position of the indazole ring system provided sufficient Chek1 potency and selectivity over Cdk7 to permit escape from DNA damage-induced arrest in a cellular assay. Enzyme potency against Chek1 was optimized by the incorporation of a hydroxymethyl triazole moiety in compound 21 (Chek1 IC(50)=0.30nM) that was shown by X-ray crystallography to displace one of three highly conserved water molecules in the HI region of the ATP-binding cleft.