ABSTRACT
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.
Subject(s)
Enzyme Precursors/antagonists & inhibitors , Enzyme Precursors/chemistry , Matrix Metalloproteinase 9/chemistry , Matrix Metalloproteinase Inhibitors/chemistry , Allosteric Regulation , Animals , COS Cells , Catalytic Domain , Chlorocebus aethiops , Enzyme Precursors/genetics , Enzyme Precursors/metabolism , Humans , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Protein DomainsABSTRACT
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.
Subject(s)
Catalytic Domain/drug effects , Enzyme Assays/methods , Enzyme Inhibitors/pharmacology , Monoacylglycerol Lipases/antagonists & inhibitors , Arachidonic Acids/chemistry , Endocannabinoids , Enzyme Inhibitors/chemistry , Glycerides/chemistry , High-Throughput Screening Assays , Humans , Hydrolysis , Inhibitory Concentration 50 , Kinetics , Monoacylglycerol Lipases/chemistry , Monoacylglycerol Lipases/metabolism , Protein Binding , Solubility , Substrate SpecificityABSTRACT
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.
Subject(s)
Drug Discovery , Fructokinases/antagonists & inhibitors , Indazoles/pharmacology , Pyrazoles/chemistry , Crystallography, X-Ray , Dose-Response Relationship, Drug , Indazoles/chemical synthesis , Indazoles/chemistry , Models, Molecular , Molecular Structure , Stereoisomerism , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Drug Discovery/methods , Drug Evaluation, Preclinical/methods , Small Molecule Libraries , Pharmaceutical Preparations/metabolism , Pharmacokinetics , Protein BindingABSTRACT
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.
Subject(s)
Monoacylglycerol Lipases/antagonists & inhibitors , Monoacylglycerol Lipases/chemistry , Protein Structure, Secondary , Protein Structure, Tertiary , Arachidonic Acids/chemistry , Arachidonic Acids/metabolism , Cannabinoid Receptor Modulators/chemistry , Cannabinoid Receptor Modulators/metabolism , Catalytic Domain , Crystallography, X-Ray , Endocannabinoids , Glycerides/chemistry , Glycerides/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Molecular Structure , Monoacylglycerol Lipases/genetics , Monoacylglycerol Lipases/metabolism , Mutagenesis, Site-Directed , Protein Binding , Static ElectricityABSTRACT
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.
Subject(s)
Fructokinases/antagonists & inhibitors , Indazoles/chemical synthesis , Models, Molecular , Piperidines/chemical synthesis , Animals , Caco-2 Cells , Cell Membrane Permeability , Crystallography, X-Ray , Electrons , Humans , In Vitro Techniques , Indazoles/chemistry , Indazoles/pharmacokinetics , Male , Microsomes, Liver/metabolism , Molecular Structure , Piperidines/chemistry , Piperidines/pharmacokinetics , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Kinesins/antagonists & inhibitors , Neoplasms/enzymology , Piperidines/pharmacology , Pyrroles/pharmacology , Taxoids/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Antineoplastic Agents/therapeutic use , Enzyme Inhibitors/therapeutic use , Humans , Neoplasms/drug therapy , Taxoids/therapeutic useABSTRACT
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.
Subject(s)
Anticoagulants/pharmacology , Antithrombins/pharmacology , Drug Design , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Thrombin/antagonists & inhibitors , Administration, Oral , Animals , Anticoagulants/chemical synthesis , Anticoagulants/chemistry , Anticoagulants/pharmacokinetics , Antithrombins/chemical synthesis , Antithrombins/chemistry , Antithrombins/pharmacokinetics , Biological Availability , Chlorides , Crystallography, X-Ray , Dogs , Ferric Compounds/pharmacology , Imidazoles/chemistry , Imidazoles/pharmacokinetics , Macaca mulatta , Models, Molecular , Molecular Structure , Partial Thromboplastin Time , Rats , Structure-Activity Relationship , Thrombin/chemistry , Thrombin/metabolism , Trypsin/metabolismABSTRACT
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.
Subject(s)
Drug Design , Hydrogen/chemistry , Kinesins/antagonists & inhibitors , Kinesins/metabolism , Mitosis/drug effects , Pyrazoles/chemistry , Pyrazoles/pharmacology , Binding Sites , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Structure , Pyrazoles/chemical synthesis , Structure-Activity RelationshipABSTRACT
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).
Subject(s)
Protein Kinase Inhibitors/pharmacology , Protein Kinases/drug effects , Quinolones/pharmacology , Checkpoint Kinase 1 , Crystallography, X-Ray , Models, Molecular , Protein Kinase Inhibitors/chemistry , Quinolones/chemistry , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Benzoxazines/chemistry , Benzoxazines/pharmacology , Drug Design , Kinesins/antagonists & inhibitors , Kinesins/metabolism , Mitosis/drug effects , Pyrazoles/chemistry , Animals , Benzoxazines/chemical synthesis , Benzoxazines/pharmacokinetics , Cell Line , Dogs , Humans , Hydrogen/chemistry , Molecular Structure , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Indazoles/chemistry , Indazoles/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Kinases/chemistry , Protein Kinases/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Checkpoint Kinase 1 , Crystallography, X-Ray , Humans , Indazoles/metabolism , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/metabolism , Structure-Activity RelationshipABSTRACT
Molecular modeling in combination with X-ray crystallographic information was employed to identify a region of the kinesin spindle protein (KSP) binding site not fully utilized by our first generation inhibitors. We discovered that by appending a propylamine substituent at the C5 carbon of a dihydropyrazole core, we could effectively fill this unoccupied region of space and engage in a hydrogen-bonding interaction with the enzyme backbone. This change led to a second generation compound with increased potency, a 400-fold enhancement in aqueous solubility at pH 4, and improved dog pharmacokinetics relative to the first generation compound.
Subject(s)
Drug Design , Kinesins/antagonists & inhibitors , Pyrazoles/chemistry , Pyrazoles/pharmacology , Alkylation , Allosteric Site , Amination , Animals , Crystallography, X-Ray , Dogs , Hydroxylation , Kinesins/chemistry , Kinesins/metabolism , Mitosis , Models, Molecular , Molecular Structure , Pyrazoles/chemical synthesis , Pyrazoles/pharmacokinetics , Solubility , Structure-Activity Relationship , WaterABSTRACT
2,4-Diaryl-2,5-dihydropyrroles have been discovered to be novel, potent and water-soluble inhibitors of KSP, an emerging therapeutic target for the treatment of cancer. A potential concern for these basic KSP inhibitors (1 and 2) was hERG binding that can be minimized by incorporation of a potency-enhancing C2 phenol combined with neutral N1 side chains. Aqueous solubility was restored to these, and other, non-basic inhibitors, through a phosphate prodrug strategy.
Subject(s)
Ether-A-Go-Go Potassium Channels/metabolism , Kinesins/antagonists & inhibitors , Prodrugs , Pyrroles/chemical synthesis , Pyrroles/pharmacology , Animals , Area Under Curve , Dogs , Protein Binding , Pyrroles/metabolism , Pyrroles/pharmacokinetics , Rats , Solubility , Spindle Apparatus/chemistry , WaterABSTRACT
The evolution of 2,4-diaryl-2,5-dihydropyrroles as inhibitors of KSP is described. Introduction of basic amide and urea moieties to the dihydropyrrole nucleus enhanced potency and aqueous solubility, simultaneously, and provided compounds that caused mitotic arrest of A2780 human ovarian carcinoma cells with EC(50)s<10nM. Ancillary hERG activity was evaluated for this series of inhibitors.
Subject(s)
Kinesins/antagonists & inhibitors , Pyrroles/chemistry , Pyrroles/pharmacology , Cell Line, Tumor , Female , Humans , Models, Molecular , Ovarian Neoplasms/pathology , Pyrroles/chemical synthesis , Spindle Apparatus/chemistryABSTRACT
A series of monopyrrolinone-based HIV-1 protease inhibitors possessing rationally designed P2' side chains have been synthesized and evaluated for activity against wild-type HIV-1 protease. The most potent inhibitor displays subnanomolar potency in vitro for the wild-type HIV-1 protease. Additionally, the monopyrrolinone inhibitors retain potency in cellular assays against clinically significant mutant forms of the virus. X-ray structures of these inhibitors bound in the wild-type enzyme reveal important insights into the observed biological activity.
Subject(s)
HIV Protease Inhibitors , HIV Protease/drug effects , Pyrrolidinones , Animals , Cattle , Crystallography, X-Ray , Drug Design , HIV Protease Inhibitors/chemical synthesis , HIV Protease Inhibitors/chemistry , HIV Protease Inhibitors/pharmacology , Humans , In Vitro Techniques , Microbial Sensitivity Tests , Models, Molecular , Molecular Conformation , Mutation , Pyrrolidinones/chemical synthesis , Pyrrolidinones/chemistry , Pyrrolidinones/pharmacology , Structure-Activity RelationshipABSTRACT
Thrombin-inhibitor X-ray crystal structures, in combination with the installation of binding elements optimized within the pyrazinone series of thrombin inhibitors, were utilized to transform a weak triazolopyrimidine lead into a series of potent oxazolopyridines. A modification intended to attenuate plasma protein binding (i.e., conversion of the P3 pyridine to a piperidine) conferred significant factor Xa activity to this series. Ultimately, these dual thrombin/factor Xa inhibitors demonstrated excellent in vitro and in vivo anticoagulant efficacy.
Subject(s)
Antithrombins/chemistry , Antithrombins/pharmacology , Factor Xa Inhibitors , Pyridines/chemistry , Pyridines/pharmacology , Crystallography, X-Ray , Models, Molecular , Molecular StructureABSTRACT
The type 1 insulin-like growth factor receptor (IGF-1R) is often overexpressed on tumor cells and is believed to play an important role in anchorage-independent proliferation. Additionally, cell culture studies have indicated that IGF-1R confers increased resistance to apoptosis caused by radiation or chemotherapeutic agents. Thus, inhibitors of the intracellular kinase domain of this receptor may have utility for the clinical treatment of cancer. As part of an effort to develop clinically useful inhibitors of IGF-1R kinase, a novel class of pyrrole-5-carboxaldehyde compounds was investigated. The compounds exhibited selectivity against the closely related insulin receptor kinase intrinsically and in cell-based assays. The inhibitors formed a reversible, covalent adduct at the kinase active site, and treatment of such adducts with sodium borohydride irreversibly inactivated the enzyme. Analysis of a tryptic digest of a covalently modified IGF-1R kinase fragment revealed that the active site Lys1003 had been reductively alkylated with the aldehyde inhibitor. Reductive alkylation of the insulin receptor kinase with one of these inhibitors led to a similarly inactivated enzyme which was examined by X-ray crystallography. The crystal structure confirmed the modification of the active site lysine side chain and revealed details of the key interactions between the inhibitor and enzyme.
Subject(s)
Aldehydes/chemistry , Protein Kinase Inhibitors/chemistry , Pyrroles/chemistry , Receptor, IGF Type 1/antagonists & inhibitors , Receptor, Insulin/antagonists & inhibitors , Receptor, Insulin/chemistry , Aldehydes/metabolism , Amino Acid Sequence , Binding Sites , Borohydrides/chemistry , Cell Line , Crystallography, X-Ray , Enzyme Activation , Humans , Molecular Sequence Data , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Phosphorylation , Protein Kinase Inhibitors/metabolism , Protein Structure, Tertiary , Pyrroles/metabolism , Receptor, Insulin/metabolism , Schiff Bases/chemistry , Structure-Activity RelationshipABSTRACT
As many processes in the preclinical drug discovery process become highly parallel, the need to also produce a large number of different proteins in parallel has become acute, such as for protein crystallization and activity screening. In turn, the requisite DNA constructions to produce these proteins must now be done at a rate that requires automated cloning procedures, each with an intrinsic low failure probability per sample. The high-throughput cloning solutions presented here achieve production of 192 different expression plasmids at a success rate of greater than 95% of the targeted open reading frames. Time for completion of the set by one person is reduced to approximately 11 working days, starting with polymerase chain reactions for a number of source clones and ending with purified expression plasmids. Achievement of this throughput utilizes the following: (1) the Beckman Coulter (Fullerton, CA) Biomek FX liquid handler for most manipulations, (2) Gateway cloning technology (Invitrogen Corp., Carlsbad, CA), and (3) computer programs designed for parallel processing of all sample information, including primer design and the resulting DNA and protein sequence assembly. Exemplary data are presented for discovery of a form of the Rho-kinase that crystallizes (ROCK2).