Peptidomimetic inhibitors of the human cytomegalovirus protease

The development of peptidomimetic inhibitors of the human cytomegalovirus (HCMV) protease showing sub-micromolar potency in an enzymatic assay is described. Selective substitution of the amino acid residues of these inhibitors led to the identification of tripeptide inhibitors showing improvements in inhibitor potency of 27-fold relative to inhibitor 39 based upon the natural tetrapeptide sequence. Small side chains at P1 were well tolerated by this enzyme, a fact consistent with previous observations. The S2 binding pocket of HCMV protease was very permissive, tolerating lipophilic and basic residues. The substitutions tried at P3 indicated that a small increase in inhibitor potency could be realized by the substitution of a tert-leucine residue for valine. Substitutions of the N-terminal capping group did not significantly affect inhibitor potency. Pentafluoroethyl ketones, alpha,alpha-difluoro-beta-keto amides, phosphonates and alpha-keto amides were all effective substitutions for the activated carbonyl component and gave inhibitors which were selective for HCMV protease. A slight increase in potency was observed by lengthening the P1' residue of the alpha-keto amide series of inhibitors. This position also tolerated a variety of groups making this a potential site for future modifications which could modulate the physicochemical properties of these molecules.     

The herpesvirus protease: mechanistic studies and discovery of inhibitors of the human cytomegalovirus protease

This paper is a review of the literature on the possible association between osteoporosis and oral bone loss, with an emphasis on radiological studies. Such an association was first suggested in 1960. Subsequent histomorphometric and microradiographic studies showed that after the age of 50 there was a marked increase in the cortical porosity of the mandible, with this increase being greater in the alveolar bone than the mandibular body; and that with this increase in porosity, there was a concomitant decrease in bone mass, which appeared to be more pronounced in females than in males, with the loss in bone mineral content estimated to be 1.5% per year in females and 0.9% in males. These studies also demonstrated a considerable amount of variation in the amounts of cortical and trabecular bone within and among individuals. Subsequent clinical studies reported associations between the bone densities of jaws and (1) metacarpals, (2) forearm bones, (3) vertebrae and (4) femurs. These studies indicated that women had lower mandibular bone mineral content (BMC) than men and that age-related loss of bone was more pronounced in women after the age of 50 years than in men of the same age, as was the case for the rest of the body. It was suggested that systemic factors responsible for osteoporotic bone loss may combine with local factors (periodontal diseases) to increase rates of periodontal alveolar bone loss. Although not all studies found associations between osteoporosis and oral bone loss, the conclusion of this review is that such an association exists; yet additional longitudinal investigations are needed to confirm this, and before the implications of this association could be fully utilized in clinical dentistry, inexpensive methods must be developed for sensitive and specific measures of oral bone loss.     

Structure of the human cytomegalovirus protease catalytic domain reveals a novel serine protease fold and catalytic triad

Proteolytic processing of capsid assembly protein precursors by herpesvirus proteases is essential for virion maturation. A 2.5 A crystal structure of the human cytomegalovirus protease catalytic domain has been determined by X-ray diffraction. The structure defines a new class of serine protease with respect to global-fold topology and has a catalytic triad consisting of Ser-132, His-63, and His-157 in contrast with the Ser-His-Asp triads found in other serine proteases. However, catalytic machinery for activating the serine nucleophile and stabilizing a tetrahedral transition state is oriented similarly to that for members of the trypsin-like and subtilisin-like serine protease families. Formation of the active dimer is mediated primarily by burying a helix of one protomer into a deep cleft in the protein surface of the other.     

Coevolutionary analysis of resistance-evading peptidomimetic inhibitors of HIV-1 protease

We have developed a coevolutionary method for the computational design of HIV-1 protease inhibitors selected for their ability to retain efficacy in the face of protease mutation. For HIV-1 protease, typical drug design techniques are shown to be ineffective for the design of resistance-evading inhibitors: An inhibitor that is a direct analogue of one of the natural substrates will be susceptible to resistance mutation, as will inhibitors designed to fill the active site of the wild-type or a mutant enzyme. Two design principles are demonstrated: (i) For enzymes with broad substrate specificity, such as HIV-1 protease, resistance-evading inhibitors are best designed against the immutable properties of the active site-the properties that must be conserved in any mutant protease to retain the ability to bind and cleave all of the native substrates. (ii) Robust resistance-evading inhibitors can be designed by optimizing activity simultaneously against a large set of mutant enzymes, incorporating as much of the mutational space as possible.     

Inhibition of human cytomegalovirus protease N(o) with monocyclic beta-lactams

Monocyclic beta-lactams have been identified as potent and selective inhibitors of the human cytomegalovirus protease (HCMV) N(o). Two series of these inhibitors are described, a peptidyl series of compounds and non-peptidic molecules featuring lower molecular weights. The SAR work that lead to the discovery of these inhibitors, together with their synthesis is also disclosed.     

NMR and topochemical studies of peptidomimetic HIV-I protease inhibitors containing a cis-epoxide amide isostere

NMR and topochemical studies of irreversible HIV-1 protease inhibitors containing a cis-epoxide as amide isostere have been carried out to identify conformational preference of the inhibitors in solution. The inhibitors prefer to adopt extended conformations similar to the beta-strand in solution.     

Unique fold and active site in cytomegalovirus protease

Human herpesviruses are responsible for a variety of diseases. They are divided into three subfamilies: alpha includes herpes simplex viruses (HSV-1 and HSV-2) and varicella-zoster virus (VZV); beta includes cytomegalovirus (CMV) and human herpesvirus-6 (HHV-6); and gamma includes Epstein-Barr virus (EBV). Each virus encodes a serine protease that is essential for its replication and is a potential target for therapeutic intervention. Human CMV is a ubiquitous opportunistic pathogen that can result in life-threatening infections in congenitally infected infants, immunocompromised individuals and immunosuppressed cancer or transplant patients. Here we report the crystal structure of human CMV protease at 2.5 angstroms resolution. The structure reveals a fold that has not been reported for any other serine protease, and an active site consisting of a novel catalytic triad in which the third member is a histidine instead of an aspartic acid, or possibly a catalytic tetrad consisting of a serine, two histidines and an aspartic acid. An unusual dimer interface that is important to the protease activity has also been identified.     

Benzothiopyran-4-one based reversible inhibitors of the human cytomegalovirus (HCMV) protease

A novel class of CMV protease inhibitors based on a benzothiopyran-S,S-dioxide nucleus has been discovered. Enzyme kinetic data supports a reversible mode of inhibition for a representative member of this class, 2-(3-pyridyl-N-oxide)benzothiopyran-4-one-S,S-dioxide, 1. Experiments in the presence and absence of the disulfide reducing agent DTT suggest that the inhibition by 1 is not due to oxidative inactivation of the enzyme. Also presented are results of some SAR studies of the benzothiopyranone ring system.     

Quanolirones I and II, two new human cytomegalovirus protease inhibitors produced by Streptomyces sp. WC76535

The aim of the study was to assess whether changes in the interposition of body compartments affect the results of body composition measurements by dual-energy x-ray absorptiometry (DEXA) in the fan-beam mode. Thirty healthy subjects underwent two sequential measurements: the first was performed in the supine position as described by the manufacturer, and the second in the prone position. Estimates of body composition were compared between the two measurements. Mean body weight did not differ between measurements ([mean+/-SD] supine vprone, 68.561+/-12.461 v 68.589+/-12.469 kg). Mean bone mineral content (BMC) was lower in the prone position versus the supine position. When the head was excluded, this difference reached statistical significance (supine v prone, 1,738+/-361 v 1,688+/-360 g, P=.0001). The mean fat tissue mass (FTM) was lower and lean tissue mass (LTM) higher in the prone measurements. When the head was excluded, the mean FTM difference between the two measurements became greater (FTM supine v prone, 25.129+/-10.445 v 24.030+/-10.388 kg, P=.0001; LTM supine v prone, 37.309+/-9.357 v 38.246+/-9.150 kg, P=.0001). It is concluded that the positioning of the patient on the examination table affects DEXA body composition measurements by the fan-beam mode. This could imply a lack of accuracy of the method, which may be due to subtle changes in regional tissue depth and fat distribution caused by patient repositioning.     

The 26S-proteasome: regulation and substrate recognition

The authors report their experience of biliary ileus, namely 8 cases out of 533 intestinal occlusions of the small bowel operated from 1982-1994. The patients included 5 women and 3 men with a F/M ratio = 1.7. Mean age was 79 years. Past medical history involving the biliary tract was only recorded in 3 cases. At the time of hospitalization occlusive symptoms had been present for between 2 and 7 days: pain in 7 patients, abdomen closed to gas and feces in 6, and vomiting in 7. Direct abdominal X-ray was only performed in 3 cases. A diagnosis of intestinal occlusion was made in 6 cases and biliary ileus in 2. During surgery it became clear that the occlusion was primarily localized at the level of the terminal ileum. Cholecysto-duodenal fistula was present in 5 cases. Enterolithotomy alone was performed in 5 cases. In 4 patients postoperative progress was normal leading to recovery. Two patients died: one immediately after surgery and the other on day 12. In conclusion, the authors confirm the increasingly frequent presence of cholecysto-duodenal fistula and the increasing age of patients, with a prevalence of females. They also emphasize the importance of an early diagnosis, supported by direct abdominal radiography so that surgery can be performed as rapidly as possible. Lastly, the need to perform cholecystectomy at a subsequent date is underlined.     

Investigation of the covalent modification of the catalytic triad of human cytomegalovirus protease by pseudo-reversible beta-lactam inhibitors and a peptide chloromethylketone

An investigation into the interaction between human cytomegalovirus (HCMV) protease and several beta-lactams, with characterization of the resulting acylenzymes using mass spectrometry, is reported. The time dependence of the inhibitors is highlighted by making comparisons of values obtained for inhibition and acylation. Analysis of inactivated HCMV protease revealed a beta-lactam: protease stoichiometry of 1. Subsequent enzymatic digestion with trypsin, peptide mapping using liquid chromatography coupled with electrospray ionization mass spectrometry and sequencing by nanoelectrospray tandem mass spectrometry (NanoES-MS/MS) allowed the identification of the site of covalent modification and confirmed Ser 132 as the active site hydroxyl nucleophile. Further, treatment of the protease with a peptide chloromethylketone and sequence analysis using NanoES-MS/MS of the alkylated enzyme confirmed His 63 as the active site imidazole nucleophile.     

Synthesis of oligopeptide and peptidomimetic libraries

The elaboration of peptide libraries prepared by either chemical or biological methods in a format useful for discovery of peptides with specific biological activities was first introduced in the 1980s. A virtual explosion of activity in this area has occurred recently, and the basic approaches have been applied to a wide variety of chemistries and for all manner of biological, chemical and physical targets. Recent advances include new synthetic methodologies, new analytical methods, new design methods and new assay procedures.     

General protease assay method coupling solid-phase substrate extraction and capillary electrophoresis

Capillary electrophoresis with laser-induced fluorescence detection was used to develop a universal, highly specific protease assay. In this method, a peptide, biotinylated at the N-terminus, is labeled with fluorescein at a lysine residue near the C-terminus. Impurities are removed from the fluorescence labeling mixture by solid-phase extraction of the substrate on immobilized streptavidin, followed by extensive washing. The purified fluorescent substrate is dissociated from the streptavidin and incubated with the protease. The peptide sequence between the biotin and fluorescent label contains the cleavage sequence of the protease of interest. After cleavage, the fluorescent product does not contain a biotin group. A second solid-phase extraction is used to remove unreacted substrate to dramatically lower the background signal. The product is detected by capillary electrophoresis, which provides powerful discrimination against products generated by nonspecific proteases. With chymotrypsin as a test protease, product was detected with as little as 10 pg/mL (4.6 x 10(-13) M) chymotrypsin, or 5 amol of enzyme in the 10-microL sample volume.     

Molecular recognition of cyclic urea HIV-1 protease inhibitors

As long as the threat of human immunodeficiency virus (HIV) protease drug resistance still exists, there will be a need for more potent antiretroviral agents. We have therefore determined the crystal structures of HIV-1 protease in complex with six cyclic urea inhibitors: XK216, XK263, DMP323, DMP450, XV638, and SD146, in an attempt to identify 1) the key interactions responsible for their high potency and 2) new interactions that might improve their therapeutic benefit. The structures reveal that the preorganized, C2 symmetric scaffolds of the inhibitors are anchored in the active site of the protease by six hydrogen bonds and that their P1 and P2 substituents participate in extensive van der Waals interactions and hydrogen bonds. Because all of our inhibitors possess benzyl groups at P1 and P1', their relative binding affinities are modulated by the extent of their P2 interactions, e.g. XK216, the least potent inhibitor (Ki (inhibition constant) = 4.70 nM), possesses the smallest P2 and the lowest number of P2-S2 interactions; whereas SD146, the most potent inhibitor (Ki = 0.02 nM), contains a benzimidazolylbenzamide at P2 and participates in fourteen hydrogen bonds and approximately 200 van der Waals interactions. This analysis identifies the strongest interactions between the protease and the inhibitors, suggests ways to improve potency by building into the S2 subsite, and reveals how conformational changes and unique features of the viral protease increase the binding affinity of HIV protease inhibitors.     

The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition

The structure of a truncated form of the gamma-subunit of phosphorylase kinase (PHKgammat) has been solved in a ternary complex with a non-hydrolysable ATP analogue (adenylyl imidodiphosphate, AMPPNP) and a heptapeptide substrate related in sequence to both the natural substrate and to the optimal peptide substrate. Kinetic characterization of the phosphotransfer reaction confirms the peptide to be a good substrate, and the structure allows identification of key features responsible for its high affinity. Unexpectedly, the substrate peptide forms a short anti-parallel beta-sheet with the kinase activation segment, the region which in other kinases plays an important role in regulation of enzyme activity. This anchoring of the main chain of the substrate peptide at a fixed distance from the gamma-phosphate of ATP explains the selectivity of PHK for serine/threonine over tyrosine as a substrate. The catalytic core of PHK exists as a dimer in crystals of the ternary complex, and the relevance of this phenomenon to its in vivo recognition of dimeric glycogen phosphorylase b is considered.     

Mechanism of substrate recognition by the ribotoxin, alpha-sarcin

The substrate recognition and catalytic mechanisms of alpha-sarcin were explored with kinetic method by using synthetic 25-mer RNA mimicking the alpha-sarcin/ricin loop in 23S rRNA of E. coli ribosomes. The oligomer containing deoxy-G at the site of alpha-sarcin (G14) was a potent competitive inhibitor. The RNA having deoxy-G8 however, increases the Kcat value by about five times but without significant alteration on Km. Surprisingly, the deletion of G8 makes the oligomer become a strong noncompetitive inhibitor of the enzyme. These results suggested that there are at least two sites in the RNA substrate which are recognized by alpha-sarcin, one is the G8 bulge or at around its neighbor and the other is the GAGA in the sarcin/ricin loop of the rRNA.     

Conservation of substrate recognition mechanisms by tRNA splicing endonucleases

Accuracy in transfer RNA (tRNA) splicing is essential for the formation of functional tRNAs, and hence for gene expression, in both Eukaryotes and Archaea. The specificity for recognition of the tRNA precursor (pre-tRNA) resides in the endonuclease, which removes the intron by making two independent endonucleolytic cleavages. Although the eukaryal and archaeal enzymes appear to use different features of pre-tRNAs to determine the sites of cleavage, analysis of hybrid pre-tRNA substrates containing eukaryal and archaeal sequences, described here, reveals that the eukaryal enzyme retains the ability to use the archaeal recognition signals. This result indicates that there may be a common ancestral mechanism for recognition of pre-tRNA by proteins.     

Strain-specific neutralization of human cytomegalovirus isolates by human sera

Induction of an effective antibody response against human cytomegalovirus (HCMV) is an important defense mechanism since it is potentially capable of neutralizing infectious viruses. We have analyzed the extent of HCMV strain-specific neutralization capacity in human sera. Nine recent HCMV isolates and their corresponding sera were investigated in cross-neutralization assays. We observed differences, independent of the overall neutralization capacity, in the 50% neutralization titers of the sera against individual strains, differences that ranged from 8-fold to more than 60-fold. For one isolate, complete resistance to neutralization by two human sera was observed. The neutralization capacity of human sera was not influenced by the presence of various concentrations (up to 100-fold excess) of noninfectious envelope glycoproteins, an inherent contamination of virus preparations from recent HCMV isolates. This indicated that the decisive parameter for neutralization is the titer of the neutralizing antibodies and that neutralization is largely independent of the concentration of virus. Analysis with transplant patients revealed that during primary infection strain-specific and strain-common antibodies are produced asynchronously. Thus, our data demonstrate that the induction of strain-specific neutralizing antibodies is a common event during infection with HCMV and that it might have important implications for the course of the infection and the development of anti-HCMV vaccines.     

Design and synthesis of new potent C2-symmetric HIV-1 protease inhibitors. Use of L-mannaric acid as a peptidomimetic scaffold

A study on the use of derivatized carbohydrates as C2-symmetric HIV-1 protease inhibitors has been undertaken. L-Mannaric acid (6) was bis-O-benzylated at C-2 and C-5 and subsequently coupled with amino acids and amines to give C2-symmetric products based on C-terminal duplication. Potent HIV protease inhibitors, 28 Ki = 0.4 nM and 43 Ki = 0.2 nM, have been discovered, and two synthetic methodologies have been developed, one whereby these inhibitors can be prepared in just three chemical steps from commercially available materials. A remarkable increase in potency going from IC50 = 5000 nM (23) to IC50 = 15 nM (28) was observed upon exchanging -COOMe for -CONHMe in the inhibitor, resulting in the net addition of one hydrogen bond interaction between each of the two -NH- groups and the HIV protease backbone (Gly 48/148). The X-ray crystal structures of 43 and of 48 have been determined (Figures 5 and 6), revealing the binding mode of these inhibitors which will aid further design.