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Narcolepsy type 1 (NT1) is a chronic neurological disorder that impairs the brain’s ability to control sleep-wake cycles. Current therapies are limited to the management of symptoms with modest effectiveness and substantial adverse effects. Agonists of the orexin receptor 2 (OX2R) have shown promise as novel therapeutics that directly target the pathophysiology of the disease. However, identification of drug-like OX2R agonists has proven difficult. Here we report cryo-electron microscopy structures of active-state OX2R bound to an endogenous peptide agonist and a small-molecule agonist. The extended carboxy-terminal segment of the peptide reaches into the core of OX2R to stabilize an active conformation, while the small-molecule agonist binds deep inside the orthosteric pocket, making similar key interactions. Comparison with antagonist-bound OX2R suggests a molecular mechanism that rationalizes both receptor activation and inhibition. Our results enable structure-based discovery of therapeutic orexin agonists for the treatment of NT1 and other hypersomnia disorders., Agonists of the orexin receptor 2 (OX2R) show promise in the treatment of narcolepsy. Cryo-EM structures of active-state OX2R bound to an endogenous peptide agonist and a small-molecule agonist suggest a molecular mechanism that rationalizes both receptor activation and inhibition.

3-Hydroxy-4-pyridinones and 5-hydroxy-4-pyrimidinones were identified as inhibitors of catechol-O-methyltransferase (COMT) in a high-throughput screen. These heterocyclic catechol mimics exhibit potent inhibition of the enzyme and an improved toxicity profile versus the marketed nitrocatechol inhibitors tolcapone and entacapone. Optimization of the series was aided by X-ray cocrystal structures of the novel inhibitors in complex with COMT and cofactors SAM and Mg(2+). The crystal structures suggest a mechanism of inhibition for these heterocyclic inhibitors distinct from previously disclosed COMT inhibitors.

O-GlcNAc hydrolase (OGA) catalyzes removal of βα-linked N-acetyl-D-glucosamine from serine and threonine residues. We report crystal structures of Homo sapiens OGA catalytic domain in apo and inhibited states, revealing a flexible dimer that displays three unique conformations and is characterized by subdomain α-helix swapping. These results identify new structural features of the substrate-binding groove adjacent to the catalytic site and open new opportunities for structural, mechanistic and drug discovery activities.

Reduced dopamine neurotransmission in the prefrontal cortex has been implicated as causal for the negative symptoms and cognitive deficit associated with schizophrenia; thus, a compound which selectively enhances dopamine neurotransmission in the prefrontal cortex may have therapeutic potential. Inhibition of catechol-O-methyltransferase (COMT, EC 2.1.1.6) offers a unique advantage, since this enzyme is the primary mechanism for the elimination of dopamine in cortical areas. Since membrane bound COMT (MB-COMT) is the predominant isoform in human brain, a high throughput screen (HTS) to identify novel MB-COMT specific inhibitors was completed. Subsequent optimization led to the identification of novel, non-nitrocatechol COMT inhibitors, some of which interact specifically with MB-COMT. Compounds were characterized for in vitro efficacy versus human and rat MB and soluble (S)-COMT. Select compounds were administered to male Wistar rats, and ex vivo COMT activity, compound levels in plasma and cerebrospinal fluid (CSF), and CSF dopamine metabolite levels were determined as measures of preclinical efficacy. Finally, novel non-nitrocatechol COMT inhibitors displayed less potent uncoupling of the mitochondrial membrane potential (MMP) compared to tolcapone as well as nonhepatotoxic entacapone, thus mitigating the risk of hepatotoxicity.

Prolylcarboxypeptidase (PrCP) is a lysosomal serine carboxypeptidase that cleaves a variety of C-terminal amino acids adjacent to proline and has been implicated in diseases such as hypertension and obesity. Here, the robust production, purification and crystallization of glycosylated human PrCP from stably transformed CHO cells is described. Purified PrCP yielded crystals belonging to space group R32, with unit-cell parameters a = b = 181.14, c = 240.13 A, that diffracted to better than 2.8 A resolution.

Prostasin (also called channel activating protease-1 (CAP1)) is an extracellular serine protease implicated in the modulation of fluid and electrolyte regulation via proteolysis of the epithelial sodium channel. Several disease states, particularly hypertension, can be affected by modulation of epithelial sodium channel activity. Thus, understanding the biochemical function of prostasin and developing specific agents to inhibit its activity could have a significant impact on a widespread disease. We report the expression of the prostasin proenzyme in Escherichia coli as insoluble inclusion bodies, refolding and activating via proteolytic removal of the N-terminal propeptide. The refolded and activated enzyme was shown to be pure and monomeric, with kinetic characteristics very similar to prostasin expressed from eukaryotic systems. Active prostasin was crystallized, and the structure was determined to 1.45Å resolution. These apoprotein crystals were soaked with nafamostat, allowing the structure of the inhibited acyl-enzyme intermediate structure to be determined to 2.0Å resolution. Comparison of the inhibited and apoprotein forms of prostasin suggest a mechanism of regulation through stabilization of a loop which interferes with substrate recognition.

The crystal structure of the wild-type unactivated kinase domain (IGFRK-0P) of insulin-like growth factor-1 receptor has been reported previously at 2.7 A resolution [Munshi et al. (2002), J. Biol. Chem. 277, 38797-38802]. In order to obtain a high-resolution structure, a number of variants of IGFRK-0P were prepared and screened for crystallization. A double mutant with E1067A and E1069A substitutions within the kinase-insert region resulted in crystals that diffracted to 1.5 A resolution. Overall, the structure of the mutant IGFRK-0P is similar to that of the wild-type IGFRK-0P structure, with the exception of the previously disordered kinase-insert region in the wild type having become fixed. In addition, amino-acid residues 947-952 at the N-terminus are well defined in the mutant structure. The monomeric protein structure is folded into two lobes connected by a hinge region, with the catalytic center situated at the interface of the two lobes. Two molecules of IGFRK-0P in the asymmetric unit are associated as a dimer and two different types of dimers with their ATP-binding clefts either facing towards or away from each other are observed. The current refined model consists of a dimer and 635 water molecules.

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2'-substituted nucleosides as inhibitors of HCV replication. The 5'-triphosphates of 2'-C-methyladenosine and 2'-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2'-O-methylcytidine inhibit HCV RNA replication. The 5'-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2'-C-methyladenosine triphosphate than 2'-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2'-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2'-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication.

The x-ray structure of the unactivated kinase domain of insulin-like growth factor-1 receptor (IGFRK-0P) is reported here at 2.7 A resolution. IGFRK-0P is composed of two lobes connected by a hinge region. The N-terminal lobe of the kinase is a twisted β-sheet flanked by a single helix, and the C-terminal lobe comprises eight α-helices and four short β-strands. The ATP binding pocket and the catalytic center reside at the interface of the two lobes. Despite the overall similarity to other receptor tyrosine kinases, three notable conformational modifications are observed: 1) this kinase adopts a more closed structure, with its two lobes rotated further toward each other; 2) the conformation of the proximal end of the activation loop (residues 1121–1129) is different; 3) the orientation of the nucleotide-binding loop is altered. Collectively, these alterations lead to a different ATP-binding pocket that might impact on inhibitor designs for IGFRK-0P. Two molecules of IGFRK-0P are seen in the asymmetric unit; they are associated as a dimer with their ATP binding clefts facing each other. The ordered N terminus of one monomer approaches the active site of the other, suggesting that the juxtamembrane region of one molecule could come into close proximity to the active site of the other.

L-735,524 is a potent, orally bioavailable inhibitor of human immunodeficiency virus (HIV) protease currently in a Phase II clinical trial. We report here the three-dimensional structure of L-735,524 complexed to HIV-2 protease at 1.9-A resolution, as well as the structure of the native HIV-2 protease at 2.5-A resolution. The structure of HIV-2 protease is found to be essentially identical to that of HIV-1 protease. In the crystal lattice of the HIV-2 protease complexed with L-735,524, the inhibitor is chelated to the active site of the homodimeric enzyme in one orientation. This feature allows an unambiguous assignment of protein-ligand interactions from the electron density map. Both Fourier and difference Fourier maps reveal clearly the closure of the flap domains of the protease upon L-735,524 binding. Specific interactions between the enzyme and the inhibitor include the hydroxy group of the hydroxyaminopentane amide moiety of L-735,524 ligating to the carboxyl groups of the essential Asp-25 and Asp-25' enzymic residues and the amide oxygens of the inhibitor hydrogen bonding to the backbone amide nitrogen of Ile-50 and Ile-50' via an intervening water molecule. A second bridging water molecule is found between the amide nitrogen N2 of L-735,524 and the carboxyl oxygen of Asp-29'. Although other hydrogen bonds also add to binding, an equally significant contribution to affinity arises from hydrophobic interactions between the protease and the inhibitor throughout the pseudo-symmetric S1/S1', S2/S2', and S3/S3' regions of the enzyme. Except for its pyridine ring, all lipophilic moieties (t-butyl, indanyl, benzyl, and piperidyl) of L-735,524 are rigidly defined in the active site.

Assembly of viral capsids for replication of herpes simplex virus requires the proteolytic processing of the assembly protein ICP35. The protease responsible for this process is encoded within the 635-amino acid open reading frame of the UL26 gene of the virus. A simple purification scheme is given in this report for the native, mature form of the protease expressed in Escherichia coli. The scheme allows the preparation of milligram quantities of purified enzyme for elucidation of kinetic mechanism as well as for structural studies. Utilizing a 13-residue peptide substrate representing the natural cleavage site that releases the protease, kcat and Km values of the purified native enzyme are 2.0 min-1 and 0.88 mM, respectively. Thus, peptide cleavage is less efficient than reported for other viral proteases. The possibility exists that viral or cellular factors are involved in vivo for activation of the protease for herpes capsid maturation.

The kinetics and equilibrium properties were investigated for the interconversion between the active dimer of human immunodeficiency virus 1 (HIV-1) protease and its inactive monomeric subunits. The equilibrium dissociation constant (Kd) of the dimeric protease as well as the monomer association rate were obtained by monitoring the fluorescence change of an active-site-directed fluorescent probe (L-737244) upon its binding to the protease. The Kd of the HIV-1 protease is strongly pH dependent. At pH 5.5 where the enzyme is most active catalytically, the extrapolated values of Kd are 0.75 and 3.4 nM at 30 and 37 degrees C, respectively. The rate constant for HIV-1 monomer association, approximately 4 x 10(5) M-1 s-1, is within the range commonly observed for protein-protein interactions. Dimer dissociation was further scrutinized in the presence of an inactive, point mutant form of the enzyme. As a result of subunit exchange between the native and mutant enzymes and the formation of an inactive heterodimer, there was a time-dependent decrease in the activity of the native protease. Enzyme activity could be reinstated with the addition of an active-site-directed inhibitor (L-365862) which selectively binds active dimers. The rate of dimer dissociation was found to also decrease with pH. At pH 5.5 and 30 degrees C, the half-life for subunit dissociation is about 0.5 h. The slow dissociation, coupled with the high stability for dimer association, attests to the importance of allowing sufficient time for dimer-monomer equilibration in kinetic assays in order to avoid reaching erroneous conclusions in studies of dimer dissociation.

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).

The quaternary state of the human cytomegalovirus (hCMV) protease has been analyzed in relation to its catalysis of peptide hydrolysis. Based on results obtained from steady state kinetics, size exclusion chromatography, and velocity sedimentation, the hCMV protease exists in a monomer-dimer equilibrium. Dimerization of the protease is enhanced by the presence of glycerol and high concentrations of enzyme. Isolation of monomeric and dimeric species eluted from a size exclusion column, followed by immediate assay, identifies the dimer as the active species. Activity measurements conducted with a range of enzyme concentrations are also consistent with a kinetic model in which only the dimeric hCMV protease is active. Using this model, the dissociation constant of the protease is 6.6 microM in 10% glycerol and 0.55 microM in 20% glycerol at 30 degrees C and pH 7.5.

The catalytic efficiency of the mature HSV-1 protease has been examined as a function of solvent composition. With the peptide substrate HTYLQASEKFKMWG-amide, the specificity constant (kcat/Km) at pH 7.5 for cleavage is 5.2 M-1 s-1. This value increases to 38 M-1 s-1 when 25% glycerol is present in the reaction mixture. It was found that glycerol activation is but one case of the general phenomenon of HSV-1 protease activation by kosmotropes, or water structure-forming cosolvents. For example, an 860-fold increase in the protease activity (kcat/Km = 4500 M-1 s-1) occurs in the presence of 0.8 M sodium citrate. Similarly, the presence of 0.8 M sodium phosphate activates the catalytic efficiency by 420-fold (kcat/Km = 2200 M-1 s-1). The extent of HSV-1 protease activation by various anions correlates with the Hofmeister series. Both the susceptibility to proteolysis by trypsin and the protein fluorescence spectra of the HSV-1 protease change in the presence of activating solvents, suggesting a conformational change accompanying activation.

Analysis of mutational effects in the human immunodeficiency virus type-1 (HIV-1) provirus has revealed that as few as four amino acid side-chain substitutions in the HIV-1 protease (M46I/L63P/V82T/I84V) suffice to yield viral variants cross-resistant to a panel of protease inhibitors either in or being considered for clinical trials (Condra, J. H., Schleif, W. A., Blahy, O. M., Gadryelski, L. J., Graham, D. J., Quintero, J. C., Rhodes, A., Robbins, H. L., Roth, E., Shivaprakash, M., Titus, D., Yang, T., Teppler, H., Squires, K. E., Deutsch, P. J., and Emini, E. A. (1995) Nature 374, 569-571). As an initial effort toward elucidation of the molecular mechanism of drug resistance in AIDS therapies, the three-dimensional structure of the HIV-1 protease mutant containing the four substitutions has been determined to 2.4-A resolution with an R factor of 17.1%. The structure of its complex with MK639, a protease inhibitor of the hydroxyaminopentane amide class of peptidomimetics currently in Phase III clinical trials, has been resolved at 2.0 A with an R factor of 17.0%. These structures are compared with those of the wild-type enzyme and its complex with MK639 (Chen, Z., Li, Y., Chen, E., Hall, D. L., Darke, P. L., Culberson, C., Shafer, J., and Kuo, L. C. (1994) J. Biol. Chem. 269, 26344-26348). There is no gross structural alteration of the protease due to the site-specific mutations. The C alpha tracings of the two native structures are identical with a root-mean-square deviation of 0.5 A, and the four substituted side chains are clearly revealed in the electron density map. In the MK639-bound form, the V82T substitution introduces an unfavorable hydrophilic moiety for binding in the active site and the I84V substitution creates a cavity (unoccupied by water) that should lead to a decrease in van der Waals contacts with the inhibitor. These changes are consistent with the observed 70-fold increase in the Ki value (approximately 2.5 kcal/mol) for MK639 as a result of the mutations in the HIV-1 protease. The role of the M46I and L63P substitutions in drug resistance is not obvious from the crystallographic data, but they induce conformational perturbations (0.9-1.1 A) in the flap domain of the native enzyme and may affect the stability and/or activity of the enzyme unrelated directly to binding.