ABSTRACT
HIV/AIDS continues to be a menace to public health. Several drugs currently on the market have successfully improved the ability to manage the viral burden in infected patients. However, new drugs are needed to combat the rapid emergence of mutated forms of the virus that are resistant to existing therapies. Currently, approved drugs target three of the four major enzyme activities encoded by the virus that are critical to the HIV life cycle. Although a number of inhibitors of HIV RNase H activity have been reported, few inhibit by directly engaging the RNase H active site. Here, we describe structures of naphthyridinone-containing inhibitors bound to the RNase H active site. This class of compounds binds to the active site via two metal ions that are coordinated by catalytic site residues, D443, E478, D498, and D549. The directionality of the naphthyridinone pharmacophore is restricted by the ordering of D549 and H539 in the RNase H domain. In addition, one of the naphthyridinone-based compounds was found to bind at a second site close to the polymerase active site and non-nucleoside/nucleotide inhibitor sites in a metal-independent manner. Further characterization, using fluorescence-based thermal denaturation and a crystal structure of the isolated RNase H domain reveals that this compound can also bind the RNase H site and retains the metal-dependent binding mode of this class of molecules. These structures provide a means for structurally guided design of novel RNase H inhibitors.
Subject(s)
Enzyme Inhibitors/metabolism , HIV Reverse Transcriptase/antagonists & inhibitors , HIV Reverse Transcriptase/chemistry , HIV-1/drug effects , Naphthyridines/metabolism , Ribonuclease H, Human Immunodeficiency Virus/antagonists & inhibitors , Ribonuclease H, Human Immunodeficiency Virus/chemistry , Binding Sites , Catalytic Domain , Cations/metabolism , Crystallography, X-Ray , HIV , HIV Reverse Transcriptase/metabolism , HIV-1/chemistry , Humans , Metals/metabolism , Models, Molecular , Protein Binding , Protein Structure, Tertiary , Ribonuclease H, Human Immunodeficiency Virus/metabolismABSTRACT
A high throughput screening campaign was designed to identify allosteric inhibitors of Chk1 kinase by testing compounds at high concentration. Activity was then observed at K(m) for ATP and at near-physiological concentrations of ATP. This strategy led to the discovery of a non-ATP competitive thioquinazolinone series which was optimized for potency and stability. An X-ray crystal structure for the complex of our best inhibitor bound to Chk1 was solved, indicating that it binds to an allosteric site approximately 13A from the ATP binding site. Preliminary data is presented for several of these compounds.
Subject(s)
Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Kinases/drug effects , Quinazolines/chemical synthesis , Quinazolines/pharmacology , Binding Sites , Checkpoint Kinase 1 , Combinatorial Chemistry Techniques , Crystallography, X-Ray , Humans , Molecular Conformation , Molecular Structure , Protein Kinase Inhibitors/chemistry , Protein Kinases/chemistry , Protein Kinases/metabolism , Quinazolines/chemistryABSTRACT
The 'NMDA hypofunction hypothesis of schizophrenia' can be tested in a number of ways. DAO is the enzyme primarily responsible for the metabolism of d-serine, a co-agonist for the NMDA receptor. We identified novel DAO inhibitors, in particular, acid 1, which demonstrated moderate potency for DAO in vitro and ex vivo, and raised plasma d-serine levels after dosing ip to rats. In parallel, analogues were prepared to survey the SARs of 1.
Subject(s)
Carboxylic Acids/chemical synthesis , Carboxylic Acids/pharmacology , D-Amino-Acid Oxidase/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Pyrroles/chemical synthesis , Pyrroles/pharmacology , Animals , Carboxylic Acids/chemistry , Combinatorial Chemistry Techniques , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Molecular Conformation , Molecular Structure , Pyrroles/chemistry , Rats , Schizophrenia/drug therapy , Serine/analysis , Serine/bloodABSTRACT
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.
Subject(s)
Enzyme Inhibitors/pharmacology , Indoles/chemistry , Protein Kinases/drug effects , Quinolones/chemistry , Animals , Binding Sites , Checkpoint Kinase 1 , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Humans , Hydrophobic and Hydrophilic Interactions , Protein Kinases/metabolism , Structure-Activity RelationshipABSTRACT
A small molecule nonpeptide inhibitor of beta-secretase has been developed, and its binding has been defined through crystallographic determination of the enzyme-inhibitor complex. The molecule is shown to bind to the catalytic aspartate residues in an unprecedented manner in the field of aspartyl protease inhibition. Additionally, the complex reveals a heretofore unknown S(3) subpocket that is created by the inhibitor. This structure has served an important role in the design of newer beta-secretase inhibitors.
Subject(s)
Acetamides/chemistry , Aspartic Acid Endopeptidases/chemistry , Benzamides/chemistry , Benzenesulfonates/chemistry , Protease Inhibitors/chemistry , Amyloid Precursor Protein Secretases , Binding Sites , Combinatorial Chemistry Techniques , Crystallography, X-Ray , Endopeptidases , Hydrogen Bonding , Models, Molecular , Molecular Structure , Stereoisomerism , Structure-Activity RelationshipABSTRACT
HIV-1 protease is one of several key enzymes required for the replication and maturation of HIV-1 virus. An almost two-decade research effort by academic and pharmaceutical institutions resulted in the successful commercialization of seven drugs that are potent inhibitors of HIV-1 protease activity and which, if used correctly, are highly effective in managing viral load. However, identification of clinical viral isolates that are resistant to these drugs indicates that this is a significant problem and that new classes of inhibitors are continually needed. Screening of microbial extracts followed by bioassay-guided isolation led to the discovery of a natural hinnuliquinone, a C(2)-symmetric bis-indolyl quinone natural product that inhibited the wild-type and a clinically resistant (A44) strain of HIV-1 protease with K(i) values of 0.97 and 1.25microM, respectively. Crystallographic analysis of the inhibitor-bound HIV-1 protease helped explain the importance of the C(2)-symmetry of hinnuliquinone for activity. Details of the isolation, biological activity, and crystallographic analysis of the inhibitor-bound protease are herein described.
Subject(s)
Benzoquinones/chemistry , Benzoquinones/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , HIV Protease Inhibitors/chemistry , HIV Protease Inhibitors/metabolism , Indoles/chemistry , Indoles/metabolism , Quinones , Catalytic Domain , Dimerization , HIV Infections/drug therapy , HIV Protease/metabolism , HIV Protease Inhibitors/therapeutic use , HIV-1/enzymology , Humans , Models, Molecular , Molecular Conformation , Molecular Structure , Quercus/microbiology , Quinones/chemistry , Quinones/metabolismABSTRACT
Expression and purification of human beta-secretase (BACE1) in bacteria have been plagued with issues concerning solubility, inhomogeneous N-terminus, and lack of enzymic activity. Several forms of the mature human BACE1 have been expressed in Escherichia coli with different N-terminal extensions and without the C-terminus transmembrane domain. Although each of the proteins expresses in inclusion bodies, a generalized protocol has been developed to solubilize, refold, and purify these BACE1 variants. The resultant proteins are homogeneous and monodispersed in solution. Each possesses a unique N-terminus. Activity assays using the peptide substrate 7-methoxycoumarin-4-yl-SEVNLDAEFK-2,4-dinitrophenyl-RR, corresponding to the beta-secretase cleavage sequence in the amyloid precursor protein with the Swedish mutations of N(670)L(671) substituting for the residues K(670)M(671), reveal a kcat and KM of 9.3 min(-1) and 55 microM, respectively.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , Carrier Proteins/metabolism , Protein Folding , Amino Acid Sequence , Amyloid Precursor Protein Secretases , Amyloid beta-Protein Precursor , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/isolation & purification , Crystallization , Endopeptidases , Enzyme Activation , Escherichia coli/genetics , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Protease Nexins , Receptors, Cell Surface , Substrate SpecificityABSTRACT
We report here the first inhibitor-bound structure of a mitotic motor protein. The 1.9 A resolution structure of the motor domain of KSP, bound with the small molecule monastrol and Mg2+ x ADP, reveals that monastrol confers inhibition by "induced-fitting" onto the protein some 12 A away from the catalytic center of the enzyme, resulting in the creation of a previously non-existing binding pocket. The structure provides new insights into the biochemical and mechanical mechanisms of the mitotic motor domain. Inhibition of KSP provides a novel mechanism to arrest mitotic spindle formation, a target of several approved and investigative anti-cancer agents. The structural information gleaned from this novel pocket offers a new angle for the design of anti-mitotic agents.
