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The development and optimization of a series of quinolinylpurines as potent and selective PI3Kδ kinase inhibitors with excellent physicochemical properties are described. This medicinal chemistry effort led to the identification of 1 (AMG319), a compound with an IC50 of 16 nM in a human whole blood assay (HWB), excellent selectivity over a large panel of protein kinases, and a high level of in vivo efficacy as measured by two rodent disease models of inflammation.

We have previously shown that the aminooxazoline xanthene scaffold can generate potent and orally efficacious BACE1 inhibitors although certain of these compounds exhibited potential hERG liabilities. In this article, we describe 4-aza substitution on the xanthene core as a means to increase BACE1 potency while reducing hERG binding affinity. Further optimization of the P3 and P2' side chains resulted in the identification of 42 (AMG-8718), a compound with a balanced profile of BACE1 potency, hERG binding affinity, and Pgp recognition. This compound produced robust and sustained reductions of CSF and brain Aβ levels in a rat pharmacodynamic model and exhibited significantly reduced potential for QTc elongation in a cardiovascular safety model.

Small molecule activators of glucokinase have shown robust efficacy in both preclinical models and humans. However, overactivation of glucokinase (GK) can cause excessive glucose turnover, leading to hypoglycemia. To circumvent this adverse side effect, we chose to modulate GK activity by targeting the endogenous inhibitor of GK, glucokinase regulatory protein (GKRP). Disrupting the GK-GKRP complex results in an increase in the amount of unbound cytosolic GK without altering the inherent kinetics of the enzyme. Herein we report the identification of compounds that efficiently disrupt the GK-GKRP interaction via a previously unknown binding pocket. Using a structure-based approach, the potency of the initial hit was improved to provide 25 (AMG-1694). When dosed in ZDF rats, 25 showed both a robust pharmacodynamic effect as well as a statistically significant reduction in glucose. Additionally, hypoglycemia was not observed in either the hyperglycemic or normal rats.

β-Secretase inhibitors are potentially disease-modifying treatments for Alzheimer's disease. Previous efforts in our laboratory have resulted in hydroxyethylamine-derived inhibitors such as 1 with low nanomolar potency against β-site amyloid precursor protein cleaving enzyme (BACE). When dosed intravenously, compound 1 was also shown to significantly reduce Aβ40 levels in plasma, brain, and cerebral spinal fluid. Herein, we report further optimizations that led to the discovery of inhibitor 16 as a novel, potent, and orally efficacious BACE inhibitor.

A series of potent hydroxyethyl amine (HEA) derived inhibitors of β-site APP cleaving enzyme (BACE1) was optimized to address suboptimal pharmacokinetics and poor CNS partitioning. This work identified a series of benzodioxolane analogues that possessed improved metabolic stability and increased oral bioavailability. Subsequent efforts focused on improving CNS exposure by limiting susceptibility to Pgp-mediated efflux and identified an inhibitor which demonstrated robust and sustained reduction of CNS β-amyloid (Aβ) in Sprague-Dawley rats following oral administration.

We have previously shown that hydroxyethylamines can be potent inhibitors of the BACE1 enzyme and that the generation of BACE1 inhibitors with CYP 3A4 inhibitory activities in this scaffold affords compounds (e.g., 1) with sufficient bioavailability and pharmacokinetic profiles to reduce central amyloid-β peptide (Aβ) levels in wild-type rats following oral dosing. In this article, we describe further modifications of the P1-phenyl ring of the hydroxyethylamine series to afford potent, dual BACE1/CYP 3A4 inhibitors which demonstrate improved penetration into the CNS. Several of these compounds caused robust reduction of Aβ levels in rat CSF and brain following oral dosing, and compound 37 exhibited an improved cardiovascular safety profile relative to 1.

Using fragment-based screening of a focused fragment library, 2-aminoquinoline 1 was identified as an initial hit for BACE1. Further SAR development was supported by X-ray structures of BACE1 cocrystallized with various ligands and molecular modeling studies to expedite the discovery of potent compounds. These strategies enabled us to integrate the C-3 side chain on 2-aminoquinoline 1 extending deep into the P2' binding pocket of BACE1 and enhancing the ligand's potency. We were able to improve the BACE1 potency to subnanomolar range, over 10(6)-fold more potent than the initial hit (900 μM). Further elaboration of the physical properties of the lead compounds to those more consistent with good blood-brain barrier permeability led to inhibitors with greatly improved cellular activity and permeability. Compound 59 showed an IC(50) value of 11 nM on BACE1 and cellular activity of 80 nM. This compound was advanced into rat pharmacokinetic and pharmacodynamic studies and demonstrated significant reduction of Aβ levels in cerebrospinal fluid (CSF).

A telomerase assay has been developed for high-throughput screening in 96-well microtiter plates. A crude cell lysate which adds telomere repeats to a biotinylated DNA primer is the source of telomerase. The telomerase-extended primer is hybridized to a digoxigenin-labeled telomere antisense DNA probe. The hybrid is further processed by enzyme-linked immunosorbent assay (ELISA) as follows. The biotinylated hybrid is captured on streptavidin-coated microtiter plates. The immobilized hybrid is probed with alkaline phosphatase-antidigoxigenin and detected via chemiluminescent readout. The limit of detection of a chemically synthesized tetra-telomere repeat was about 10 attomoles. Apparent telomerase activity was detected in lysates of 293T cells. The signal to background for the assay (ratio of signal for the complete assay mixture divided by the signal for the assay mixture without primer) was around 10. An automated system that performed unattended runs of up to 17 96-well microtiter plates in 8h was constructed.

BACE1 inhibition to prevent Aβ peptide formation is considered to be a potential route to a disease-modifying treatment for Alzheimer’s disease. Previous efforts in our laboratory using a combined structure- and property-based approach have resulted in the identification of aminooxazoline xanthenes as potent BACE1 inhibitors. Herein, we report further optimization leading to the discovery of inhibitor 15 as an orally available and highly efficacious BACE1 inhibitor that robustly reduces CSF and brain Aβ levels in both rats and nonhuman primates. In addition, compound 15 exhibited low activity on the hERG ion channel and was well tolerated in an integrated cardiovascular safety model.

The optimization of a series of aminooxazoline xanthene inhibitors of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is described. An early lead compound showed robust Aβ lowering activity in a rat pharmacodynamic model, but advancement was precluded by a low therapeutic window to QTc prolongation in cardiovascular models consistent with in vitro activity on the hERG ion channel. While the introduction of polar groups was effective in reducing hERG binding affinity, this came at the expense of higher than desired Pgp-mediated efflux. A balance of low Pgp efflux and hERG activity was achieved by lowering the polar surface area of the P3 substituent while retaining polarity in the P2' side chain. The introduction of a fluorine in position 4 of the xanthene ring improved BACE1 potency (5-10-fold). The combination of these optimized fragments resulted in identification of compound 40, which showed robust Aβ reduction in a rat pharmacodynamic model (78% Aβ reduction in CSF at 10 mg/kg po) and also showed acceptable cardiovascular safety in vivo.

The proteome of normal male urine from a commercial pooled source has been examined using direct liquid chromatography-tandem mass spectrometry (LC-MS/MS). The entire urinary protein mixture was denatured, reduced and enzymatically digested prior to LC-MS/MS analysis using a hybrid-quadrupole time-of-flight mass spectrometer (Q-TOF) to perform data-dependent ion selection and fragmentation. To fragment as many peptides as possible, the mixture was analyzed four separate times, with the mass spectrometer selecting ions for fragmentation from a subset of the entire mass range for each run. This approach requires only an autosampler on the HPLC for automation ( i.e, unattended operation). Across these four analyses, 1.450 peptide MS/MS spectra were matched to 751 sequences to identify 124 gene products (proteins and translations of expressed sequence tags). Interestingly, the experimental time for these analyses was less than that required to run a single two-dimensional gel.

In an attempt to identify peptides that may be involved in the obese phenotype observed in CpEfat/CpEfat mice (deficient in Carboxypeptidase E, CpE) samples from fourteen neuroendocrine tissues in wild-type and CpEfat/CpEfat mice were obtained. Peptides were purified from these tissues and potential CpE substrate peptides were enriched using an anhydrotrypsin column that captures peptides with basic C-termini. Bound peptides were subjected to tryptic digestion and followed by liquid chromatography-mass spectrometry analysis. The relative levels of CpEfat/CpEfat versus wild-type peptides were determined by comparison of the ion intensities. Peptide ions elevated in the CpEfat/CpEfat samples were identified by targeted liquid chromatography-tandem mass spectrometry. From those ions, 27 peptides derived from known neuropeptides (including CpE substrates) were identified, together with another 25 peptides from proteins not known to be components of the neuropeptide processing pathway. The known CpE substrates identified included the recently discovered proSAAS, granin-like neuroendocrine peptide precursor that inhibits prohormone processing. The approach demonstrated the feasibility of using an affinity-based method for identifying differences in specific classes of peptides between normal and mutant mice.

We report here the backbone 1HN, 15N, 13Cα, 13CO, and 1Hα NMR assignmentsfor the catalytic domain of human fibroblast collagenase (HFC). Three independentassignment pathways (matching 1H, 13Cα, and 13CO resonances) were used to establishsequential connections. The connections using 13Cα resonances were obtained fromHNCOCA and HNCA experiments; 13CO connections were obtained from HNCO andHNCACO experiments. The sequential proton assignment pathway was established from a 3D(1H/15N) NOESY-HSQC experiment. Amino acid typing was accomplished using 13C and15N chemical shifts, specific labeling of 15N-Leu, and spin pattern recognition from DQF-COSY. The secondary structure was determined by analyzing the 3D (1H/15N) NOESY-HSQC. A preliminary NMR structure calculation of HFC was found to be in agreement withrecent X-ray structures of human fibroblast collagenase and human neutrophil collagenase aswell as similar to recent NMR structures of a highly homologous protein, stromelysin. Allthree helices were located; a five-stranded β-sheet (four parallel strands, one antiparallelstrand) was also determined. β-Sheet regions were identified by cross-stranddαN and dNN connections and by strong intraresidue dαN correlations, and were corroborated byobserving slow amide proton exchange. Chemical shift changes in a selectively 15N-labeledsample suggest that substantial structural changes occur in the active site cleft on the bindingof an inhibitor.

In the nuclei of hepatocytes, glucokinase regulatory protein (GKRP) modulates the activity of glucokinase (GK), a key regulator of glucose homeostasis. Currently, direct activators of GK (GKAs) are in development for the treatment of type 2 diabetes. However, this approach is generally associated with a risk of hypoglycemia. To mitigate such risk, we target the GKRP regulation, which indirectly restores GK activity. Here we describe a screening strategy to look specifically for GKRP modulators, in addition to traditional GKAs. Two high-throughput screening campaigns were performed with our compound libraries using a luminescence assay format, one with GK alone and the other with a GK/GKRP complex in the presence of sorbitol-6-phosphate (S6P). By a subtraction method in the hit triage process of these campaigns, we discovered two close analogs that bind GKRP specifically with sub-µM potency to a site distinct from where fructose-1-phosphate binds. These small molecules are first-in-class allosteric modulators of the GK/GKRP interaction and are fully active even in the presence of S6P. Activation of GK by this particular mechanism, without altering the enzymatic profile, represents a novel pharmacologic modality of intervention in the GK/GKRP pathway.

We describe a systematic study of how macrocyclization in the P₁-P₃ region of hydroxyethylamine-based inhibitors of β-site amyloid precursor protein (APP)-cleaving enzyme (BACE1) modulates in vitro activity. This study reveals that in a number of instances macrocyclization of bis-terminal dienes leads to improved potency toward BACE1 and selectivity against cathepsin D (CatD), as well as greater amyloid β-peptide (Aβ)-lowering activity in HEK293T cells stably expressing APPSW. However, for several closely related analogs the benefits of macrocyclization are attenuated by the effects of other structural features in different regions of the molecules. X-ray crystal structures of three of these novel macrocyclic inhibitors bound to BACE1 revealed their binding conformations and interactions with the enzyme.

Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic β-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

In our natural products screening programme for the discovery of competitive inhibitors of the matrix metalloproteinases, stromelysin and collagenase, we have isolated an active compound, betulinic acid (1), from the plant Doliocarpus verruculosus. Betulinic acid inhibits stromelysin and collagenase with K i values of 2.2 and 1.3 μM, respectively. The analogous C-28 alcohol, betulin, was a less potent inhibitor of these proteases. We therefore postulate that the moiety of 1-carboxyl-3-(2-propenyl)-cyclopentane in betulinic acid may mimic the hydroxamate-containing residue of actinonin (3), a known stromelysin and collagenase inhibitor. Because a hydroxamate can serve as a bidentate ligand for the active site zinc, we synthesized the hydroxamate of 3-acetoxy-betulinic acid (2), hoping to observe an increase in potency relative to 1. However, the K i values against stromelysin and collagenase were essentially equal to those of 1. These data suggest that the rigid cyclopentyl ring is probably restricting the tight binding as seen in the flexible peptidal hydroxamates, or the acid moiety is not interacting directly with the active site zinc of the metalloproteinases.

Pharmacokinetic parameters for a series of C-terminally modified hydroxamate dipeptides were evaluated by in vitro and in vivo models. The presence of a tertiary base at the C-terminus significantly reduced biliary excretion and increased plasma half-life. Moreover, introduction of a thioether functionality produced a more favorable pharmacokinetic profile compared to the corresponding oxo- and aza-analogs.

Structure-based drug design (SBDD) and traditional SAR have guided the development of potent and selective hydroxamate inhibitors which contain heteroatom-based modifications of the P1′ group. These inhibitors may help delineate the in vivo roles of specific MMPs in normal and disease states.

Gelatinase B is potently inhibited by peptide hydroxamates, including molecules that have a R 1 ′ group which is larger than the side chains of the natural amino acids.

Human fibroblast collagenase belongs to a family of matrix metalloproteinases which have been implicated in a number of connective tissue disorders ranging from rheumatoid arthritis to tumor invasion. To examine the active site of this enzyme by biophysical studies, a 19 kDa recombinant truncated mature collagenase (mCL-t) was prepared. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry have been utilized for the characterization of mCL-t. The molecular weights measured by these techniques identified the presence of two closely related protein components separated by approximately 100 Da. Edman sequence analysis demonstrated that the two protein components differ from each other by an amino terminal valine, consistent with the mass spectrometric data. In addition, the molecular weight of mCL-t determined by mass spectrometry did not agree with that calculated from the reported sequence. To identify the origin of this discrepancy, the DNA sequence of the mCL-t clone was examined. Several differences were noted between the DNA sequence of mCL-t and the published collagenase gene sequence. When these differences were taken into account, the measured molecular weights were found to be in good agreement with that calculated for the modified sequence. In separate experiments, both ESI and MALDI mass spectrometry have been used to determine molecular weights of mCL-t samples enriched with stable isotopes 15N and (15N + 13C). The measured molecular weights demonstrated a 97% (15N) and 99% (15N + 13C) incorporation of labeled isotopes in the two samples.

A structure- and property-based drug design approach was employed to identify aminooxazoline xanthenes as potent and selective human β-secretase inhibitors. These compounds exhibited good isolated enzyme, cell potency, and selectivity against the structurally related aspartyl protease cathepsin D. Our efforts resulted in the identification of a potent, orally bioavailable CNS penetrant compound that exhibited in vivo efficacy. A single oral dose of compound 11a resulted in a significant reduction of CNS Aβ40 in naive rats.

Phosphoinositide 3-kinase α (PI3Kα) is a lipid kinase that plays a key regulatory role in several cellular processes. The mutation or amplification of this kinase in humans has been implicated in the growth of multiple tumor types. Consequently, PI3Kα has become a target of intense research for drug discovery. Our studies began with the identification of benzothiazole compound 1 from a high throughput screen. Extensive SAR studies led to the discovery of sulfonamide 45 as an early lead, based on its in vitro cellular potency. Subsequent modifications of the central pyrimidine ring dramatically improved enzyme and cellular potency and led to the identification of chloropyridine 70. Further arylsulfonamide SAR studies optimized in vitro clearance and led to the identification of 82 as a potent dual inhibitor of PI3K and mTOR. This molecule exhibited potent enzyme and cell activity, low clearance, and high oral bioavailability. In addition, compound 82 demonstrated tumor growth inhibition in U-87 MG, A549, and HCT116 tumor xenograft models.

AKT/PKB is a phosphoinositide-dependent serine/threonine protein kinase that plays a critical role in the signal transduction of receptors. It also serves as an oncogene in the tumorigenesis of cancer cells when aberrantly activated by genetic lesions of the PTEN tumor suppressor, phosphatidylinositol 3-kinase, and receptor tyrosine kinase overexpression. Here we have characterized and compared kinetic mechanisms of the three AKT isoforms. Initial velocity studies revealed that all AKT isozymes follow the sequential kinetic mechanism by which an enzyme-substrate ternary complex forms before the product release. The empirically derived kinetic parameters are apparently different among the isoforms. AKT2 showed the highest Km value for ATP, and AKT3 showed the highest kcat value. The patterns of product inhibition of AKT1, AKT2, and AKT3 by ADP were all consistent with an ordered substrate addition mechanism with ATP binding to the enzymes prior to the peptide substrate. Further analysis of steady state kinetics of AKT1 in the presence of dead-end inhibitors supported the finding and suggested that the AKT family of kinases catalyzes reactions via an Ordered Bi Bi sequential mechanism with ATP binding to the enzyme prior to peptide substrate and ADP being released after the phosphopeptide product. These results suggest that ATP is an initiating factor for the catalysis of AKT enzymes and may play a role in the regulation AKT enzyme activity in cells.

Affinity chromatography, employing the extracellular domain of the Sea receptor, was used to enrich Sea-binding proteins from chicken serum. One isolated protein bound both a Sea-immunoglobulin fusion protein and an antisera raised against murine macrophage stimulating protein. Amino-terminal sequencing of the dual-reactive protein yielded sequences which were identical to the predicted alpha and beta subunits of chicken macrophage stimulating protein. The partially purified chicken macrophage stimulating protein caused autophosphorylation of the Sea receptor. Previous work showed that recombinant expression of fully activatible human or mouse macrophage stimulating protein required a specific Cys to Ala substitution (Wahl, R. C., Costigan, V. J., Batac, J. P., Chen, K., Cam, L., Courchesne, P. L., Patterson, S. D. Zhang, K., and Pacifici, R. E. (1997) J. Biol. Chem. 272, 1-4). Therefore, we expressed both the wild type and the specific Cys to Ala form of chicken macrophage stimulating protein as recombinant proteins. After proteolytic activation, only conditioned media from COS cells transfected with the C665A chicken macrophage stimulating protein, but not from wild type chicken macrophage-stimulating protein, or control vector, was detected by the Sea-immunoglobulin fusion protein in Western blotting experiments. Conditioned media containing the C665A chicken macrophage-stimulating protein readily caused Sea phosphorylation, while conditioned media containing the wild type chicken macrophage-stimulating protein was only effective at inducing receptor phosphorylation at high concentrations. In addition to receptor phosphorylation, the C665A chicken macrophage-stimulating protein induced phosphorylation of Shc, Erk1, and Erk 2. We conclude that macrophage-stimulating protein is a ligand of the Sea receptor protein-tyrosine kinase.

In this study, structure-based drug design of matrix metalloproteinase inhibitors [human fibroblast collagenase (HFC), human fibroblast stromelysin (HFS), and human neutrophil collagenase (HNC)] was utilized in the development of potent hydroxamates which contain novel, heteroatom-based modifications of the P 1 ' group. A series containing a P 1 ' butyramide group resulted in a nanomolar potent and selective HNC inhibitor as well as a dual HFS/HNC inhibitor. Benzylic ethers with a four- or five-carbon methylene linker in the P 1 ' position also produced nanomolar potent HFS/HNC inhibition and micromolar potent HFC inhibition as expected. Surprisingly, the phenolic ethers of the same overall length as the benzylic ethers showed nanomolar potencies against HFC, as well as HFS and HNC. The potency profile of the phenolic ethers was optimized by structure-activity relationships of the phenolic group and the C-terminal amide. These inhibitors may help elucidate the in vivo roles of matrix metalloproteinases in normal and disease states.

Molecular analogs of amino acids can be incorporated into proteins. The amino acid analog selenomethionine (SeMet) has been shown to be efficiently incorporated into the proteins of growing Escherichia coli. SeMet-containing proteins are known to produce sufficiently strong anomalous scatter permitting the solution of the selenomethionyl crystal structure by multiwavelength anomalous diffraction (MAD) techniques. The recombinant protein chosen for these studies is mature, truncated neutrophil collagenase (rmNC-t). The rmNC-t protein is a monomer of 163 amino acid residues featuring one active site and two Met residues. We developed a T7 polymerase expression system allowing incorporation of SeMet into rmNC-t protein produced in E. coli. Substitution of Met with SeMet was accomplished by culturing E. coli DL41(DE3), a SeMet-tolerant strain with metA lesion, in a defined medium containing SeMet as the sole source of Met. The SeMet-labeled rmNC-t was isolated from inclusion bodies by solubilizing in urea, purified by anion column chromatography, and then refolded in the presence of Ca2+ and Zn2+. Analysis of SeMet-labeled rmNC-t demonstrated that Met replacement was 100%. Enzymatic characterization revealed no obvious differences in activity or inhibitor binding between rmNC-t and the SeMet-labeled product. We have produced pure, active SeMet-labeled rmNC-t in sufficient quantities for macromolecular crystallography studies.

Human neutrophil collagenase (HNC) is a member of a family of matrix metalloproteinases (MMP). HNC is capable of cleaving all three alpha-chains of types I, II and III collagens. In rheumatoid and osteo-arthritis, MMP members have been implicated in the pathology associated with these diseases due to the accelerated breakdown of the extracellular matrix of articular cartilage. A cDNA coding for the HNC catalytic domain (lacking both the propeptide and C-terminal fragments) was sub-cloned into the pETlla prokaryotic expression vector. The cloned fragment encodes a protein that extends from amino acids (aa) Met100 through Gly262 of the full-length proenzyme, which as a result, would not require proteolytic or chemical activation. The HNC construct was expressed in Escherichia coli and recombinant mature, truncated neutrophil collagenase (re-mNC-t) was produced at high levels (approx. 30% of total bacterial protein). The re-mNC-t protein was extracted from inclusion bodies by solubilization in 6 M urea, followed by ion-exchange chromatography. The protein was refolded to an active conformation in the presence of Ca2+ and Zn2+. A final purification step on size-exclusion chromatography yielded 30 mg per liter of active re-mNC-t with minor autodegradative products. Alternatively, hydroxamate affinity chromatography was used to obtain pure, non-degraded re-mNC-t (20-25 mg per liter). The catalytic activity of re-mNC-t was abolished by known MMP inhibitors and the Ki measurement against actinonin was similar to that of HNC prepared from human blood.

The X-ray crystal structure of a 19 kDa active fragment of human fibroblast collagenase has been determined by the multiple isomorphous replacement method and refined at 1.56 A resolution to an R-factor of 17.4%. The current structure includes a bound hydroxamate inhibitor, 88 waters and three metal atoms (two zincs and a calcium). The overall topology of the enzyme, comprised of a five stranded β-sheet and three α-helices, is similar to the thermolysin-like metalloproteinases. There are some important differences between the collagenase and thermolysin families of enzymes. The active site zinc ligands are all histidines (His-218, His-222, and His-228). The presence of a second zinc ion in a structural role is a unique feature of the matrix metalloproteinases. The binding properties of the active site cleft are more dependent on the main chain conformation of the enzyme (and substrate) compared with thermolysin. A mechanism of action for peptide cleavage similar to that of thermolysin is proposed for fibroblast collagenase. © 1994 Wiley-Liss, Inc.

The crystal structure of the catalytic domain of human neutrophil collagenase complexed with a peptide transition state analogue has been determined to a resolution of 2.1 A. The structure of the neutrophil enzyme, when compared with the three dimensional structure of the corresponding human f ibroblast collagenase, shows differences in the first, S1′, of the three enzyme specificity subsites on the carboxy–terminal side of the substrate scissile bond. The S1′ pocket in the neutrophil collagenase is significantly larger than the equivalent site in the f ibroblast enzyme, suggesting that the former enzyme has a broader range of possible substrates. Such differences also suggest approaches for the design of selective matrix metalloproteinase inhibitors.

Publisher Summary Collagens and proteoglycans are the major organic molecules of cartilage and bone that are the primary endogenous substrate for the collagenases and an important substrate for stromelysin respectively. The collagenases and stromelysin are extracellular, calcium dependent, zinc endoproteinases whose substrates include the macromolecular components of the extracellular matrix. Of the approximately 10 human matrix metalloproteinases (MMPs) that have been tentatively identified to date, six have been isolated and have had their enzymatic activity studied: these are fibroblast collagenase (HFC or MMP-1), stromelysin (HFS or MMP-3), neutrophil collagenase (HNC or MMP-8), 72 kDa gelatinase (HFG or MMP-2), 92 kDdneutrophil gelatinase (HNG or MMP-9), and uterine metalloproteinase (PUMP-1 or MMP-7). This chapter discusses the biochemistry and inhibition of three of these enzymes: hybrid fiber-coaxia (HFC), HNC, and HFS. Substantial progress has occurred in the cloning, isolation, and purification of HFC and HFS in quantities sufficient for mechanistic and structural studies. Peptide-based assays have been developed that are much more accurate and reliable than assays based on the hydrolysis of biological macromolecules. Although potent, peptide-based inhibitors of HFC have been designed, studies of the activity in vivo have appeared. It is only when inhibitors with in vivo activity are obtained then the role of the MMPs in disorders, such as arthritis and peridontal disease, can be established.

Reconstitution of the apoprotein of the molybdoenzyme nitrate reductase in extracts of the Neurospora crassa mutant nit-1 with molybdenum cofactor released by denaturation of purified molybdoenzymes is efficient in the absence of exogenous MoO42− under defined conditions. Evidence is presented that this molybdate-independent reconstitution is due to transfer of intact Mo cofactor, a complex of Mo and molybdopterin (MPT), the organic constituent of the cofactor. This complex can be separated from denatured protein by gel filtration, and from excess MoO42− by reverse-phase HPLC. Sulfite oxidase, native xanthine dehydrogenase, and cyanolyzed xanthine dehydrogenase are equipotent Mo cofactor donors. Other well-studied inactive forms of xanthine dehydrogenase are also shown to be good cofactor sources. Using xanthine dehydrogenase specifically radiolabeled in the cyanolyzable sulfur, it is shown that this terminal ligand of Mo is rapidly removed from Mo cofactor under the conditions used for reconstitution.