Resistance to HIV protease inhibitors: a comparison of enzyme inhibition and antiviral potency.

Resistance of HIV-1 to protease inhibitors has been associated with changes at residues Val82 and Ile84 of HIV-1 protease (HIV PR). Using both an enzyme assay with a peptide substrate and a cell-based infectivity assay, we examined the correlation between the inhibition constants for enzyme activity (Ki values) and viral replication (IC90 values) for 5 active site mutants and 19 protease inhibitors. Four of the five mutations studied (V82F, V82A, I84V, and V82F/I84V) had been identified as conferring resistance during in vitro selection using a protease inhibitor. The mutant protease genes were expressed in Escherichia coli for preparation of enzyme, and inserted into the HXB2 strain of HIV for test of antiviral activity. The inhibitors included saquinavir, indinavir, nelfinavir, 141W94, ritonavir (all in clinical use), and 14 cyclic ureas with a constant core structure and varying P2, P2' and P3, P3' groups. The single mutations V82F and I84V caused changes with various inhibitors ranging from 0.3- to 86-fold in Ki and from 0.1- to 11-fold in IC90. Much larger changes compared to wild type were observed for the double mutation V82F/I84V both for Ki (10-2000-fold) and for IC90 (0.7-377-fold). However, there were low correlations (r2 = 0.017-0.53) between the mutant/wild-type ratio of Ki values (enzyme resistance) and the mutant/wild-type ratio of viral IC90 values (antiviral resistance) for each of the HIV proteases and the viruses containing the identical enzyme. Assessing enzyme resistance by "vitality values", which adjust the Ki values with the catalytic efficiencies (kcat/Km), caused no significant improvement in the correlation with antiviral resistance. Therefore, our data suggest that measurements of enzyme inhibition with mutant proteases may be poorly predictive of the antiviral effect in resistant viruses even when mutations are restricted to the protease gene.

Nonpeptide cyclic cyanoguanidines as HIV-1 protease inhibitors: synthesis, structure-activity relationships, and X-ray crystal structure studies.

Comparison of the high-resolution X-ray structures of the native HIV-1 protease and its complexes with the inhibitors suggested that the enzyme flaps are flexible. The movement at the tip of the flaps could be as large as 7 A. On the basis of this observation, cyclic cyanoguanidines have been designed, synthesized, and evaluated as HIV-1 protease (PR) inhibitors. Cyclic cyanoguanidines were found to be very potent inhibitors of HIV-1 protease. The choice of cyclic cyanoguanidines over cyclic guanidines was based on the reduced basicity of the former. X-ray structure studies of the HIV PR complex with cyclic cyanoguanidine demonstrated that in analogy to cyclic urea, cyclic cyanoguanidines also displace the unique structural water molecule. The structure-activity relationship of the cyclic cyanoguanidines is compared with that of the corresponding cyclic urea analogues. The differences in binding constants of the two series of compounds have been rationalized using high-resolution X-ray structure information.

Improved cyclic urea inhibitors of the HIV-1 protease: synthesis, potency, resistance profile, human pharmacokinetics and X-ray crystal structure of DMP 450.

BACKGROUND: Effective HIV protease inhibitors must combine potency towards wild-type and mutant variants of HIV with oral bioavailability such that drug levels in relevant tissues continuously exceed that required for inhibition of virus replication. Computer-aided design led to the discovery of cyclic urea inhibitors of the HIV protease. We set out to improve the physical properties and oral bioavailability of these compounds. RESULTS: We have synthesized DMP 450 (bis-methanesulfonic acid salt), a water-soluble cyclic urea compound and a potent inhibitor of HIV replication in cell culture that also inhibits variants of HIV with single amino acid substitutions in the protease. DMP 450 is highly selective for HIV protease, consistent with displacement of the retrovirus-specific structural water molecule. Single doses of 10 mg kg-1 DMP 450 result in plasma levels in man in excess of that required to inhibit wild-type and several mutant HIVs. A plasmid-based, in vivo assay model suggests that maintenance of plasma levels of DMP 450 near the antiviral IC90 suppresses HIV protease activity in the animal. We did identify mutants that are resistant to DMP 450, however; multiple mutations within the protease gene caused a significant reduction in the antiviral response. CONCLUSIONS: DMP 450 is a significant advance within the cyclic urea class of HIV protease inhibitors due to its exceptional oral bioavailability. The data presented here suggest that an optimal cyclic urea will provide clinical benefit in treating AIDS if it combines favorable pharmacokinetics with potent activity against not only single mutants of HIV, but also multiply-mutant variants.

Potency and selectivity of inhibition of human immunodeficiency virus protease by a small nonpeptide cyclic urea, DMP 323.

DMP 323 is a potent inhibitor of the protease of human immunodeficiency virus (HIV), with antiviral activity against both HIV type 1 and HIV type 2. This compound is representative of a class of small, novel, nonpeptide cyclic urea inhibitors of HIV protease that were designed on the basis of three-dimensional structural information and three-dimensional database searching. We report here studies of the kinetics of DMP 323 inhibition of the cleavage of peptide and HIV-1 gag polyprotein substrates. DMP 323 acts as a rapidly binding, competitive inhibitor of HIV protease. DMP 323 is as potent against both peptide and viral polyprotein substrates as A-80987, Q8024, and Ro-31-8959, which are among the most potent inhibitors of HIV protease described in the literature to date. Incubation with human plasma or serum did not decrease the effective potency of DMP 323 for HIV protease, suggesting that plasma protein binding is of a low affinity relative to that of HIV protease. DMP 323 was also assessed for its ability to inhibit the mammalian proteases renin, pepsin, cathepsin D, cathepsin G, and chymotrypsin. No inhibition of greater than 12% was observed for any of these enzymes at concentrations of DMP 323 that were 350 to 40,000 times higher than that required to inhibit the viral protease 50%.

Rational design of potent, bioavailable, nonpeptide cyclic ureas as HIV protease inhibitors.

Mechanistic information and structure-based design methods have been used to design a series of nonpeptide cyclic ureas that are potent inhibitors of human immunodeficiency virus (HIV) protease and HIV replication. A fundamental feature of these inhibitors is the cyclic urea carbonyl oxygen that mimics the hydrogen-bonding features of a key structural water molecule. The success of the design in both displacing and mimicking the structural water molecule was confirmed by x-ray crystallographic studies. Highly selective, preorganized inhibitors with relatively low molecular weight and high oral bioavailability were synthesized.

Rapid purification of inositol monophosphate phosphatase from beef brain.

A procedure is described for preparation of homogeneous inositol monophosphate phosphatase (EC 3.1.3.25) from beef brain in less than 2 days with an overall recovery of 15-25%. This enzyme, an essential part of the inositol phospholipid cycle in brain, is a proposed site of action of lithium ions in manic-depressive disorders. The major purification steps are: a) removal of most interfering protein by heat denaturation at 75 degrees C for 1 h, b) separation by anion exchange at a pH (6.0) near the enzyme's pI (4.9), and c) adsorption of most remaining impurities on a Procion Red affinity column.

Demonstration of inositol 1,3,4,5-tetrakisphosphate receptor binding.

Inositol 1,3,4,5-tetrakisphosphate (InsP4) is produced rapidly upon stimulation of the phosphoinositide system and may serve as a second messenger in hormone and neurotransmitter action. In this report we demonstrate specific binding sites for [3H]InsP4 in rat tissue membranes. In cerebellar membranes, [3H]InsP4 binding sites are displaced both by InsP4 and inositol 1,4,5-trisphosphate (InsP3) with similar potency (IC50 approximately equal to 300 nM) whereas several other inositol phosphates are much weaker. We have distinguished the InsP4 binding site from the InsP3 receptor binding site by differences in brain regional and tissue distribution, affinity for InsP4 and InsP3, and sensitivity to calcium.

Inositol bis-, tris-, and tetrakis(phosphate)s: analysis in tissues by HPLC.

The concentration of isomers of inositol tris(phosphate) (InsP3) was measured in tissues of intact animals. The method employed involved anion-exchange HPLC with on-line enzymatic hydrolysis of the phosphate esters and detection of the inorganic phosphate formed. All seven organs tested from rats killed by decapitation contained Ins(1,4,5)P3 in concentrations of 13-40 nmol/g; a distribution that bears no resemblance to that reported for its precursor [phosphatidylinositol bis(phosphate)]. A second InsP3 isomer [probably Ins(1,3,4)P3] was also detectable in brain and salivary gland. The content of Ins(1,4,5)P3 in brain and salivary gland from rats killed by decapitation was 10-60 times greater than that from rats killed by focused microwave irradiation to block postmortem metabolism. Inositol bis(phosphate) concentrations also changed dramatically postmortem. A much smaller postmortem change was seen in the content of Ins(1,3,4)P3. Receptor stimulation by muscarinic cholinergic agonists increased the content not only of Ins(1,3,4)P3, but also its recently discovered probable precursor, inositol tetrakis-(phosphate).

Coupling of inositol phospholipid metabolism with excitatory amino acid recognition sites in rat hippocampus.

Ibotenate, a rigid structural analogue of glutamate, markedly enhances the hydrolysis of membrane inositol phospholipids, as reflected by the stimulation of [3H]inositol monophosphate formation in rat hippocampal slices prelabeled with [3H]inositol and treated with Li+. Quisqualate, homocysteate, L-glutamate, and L-aspartate also induce a significant (albeit weaker) increase in [3H]inositol monophosphate formation, whereas N-methyl-D-aspartate, kainate, quinolinate, and N-acetylaspartylglutamate are inactive. The increase in [3H]inositol monophosphate formation elicited by the above-mentioned excitatory amino acids is potently and selectively antagonized by DL-2-amino-4-phosphonobutyric acid, a dicarboxylic amino acid receptor antagonist. These results suggest that, in the hippocampus, a class of dicarboxylic amino acid recognition sites is coupled with phospholipase C, the enzyme that catalyzes the hydrolysis of membrane inositol phospholipids.

Estradiol benzoate decreases nigral GABAergic activity in male rats.

Repeated doses of estradiol benzoate (10 micrograms/kg, s.c., once a day for 2, 5 or 8 days) to male rats decreased gamma-aminobutyric acid (GABA) content and glutamate decarboxylase (GAD) activity in substantia nigra (SN) but failed to change these parameters in hippocampus, cerebral cortex, cerebellum, lateral septum and olfactory tubercle. In the caudate nucleus, estradiol benzoate decreased GABA concentration but did not modify GAD activity. A decrease in nigral GABA concentration and GAD activity was also observed 24 and 48 but not 3 h after a single injection of estradiol benzoate. These data are consistent with results on GAD activity reported by McGinnis et al. in ovariectomized rats. Kinetic analysis of nigral GAD activity revealed that repeated estradiol benzoate injection reduced the Vmax without affecting the Km of GAD. Estradiol benzoate also reduced the rate of nigral GABA accumulation resulting from local infusion of gabaculine, suggesting that the steroid decreases GABA turnover in male rat SN. Hypophysectomy decreased GABA content and GAD activity in SN and GABA content in striatum. Administration of estradiol benzoate for 8 days to hypophysectomized rats failed to decrease further these parameters. Taken together, these data suggest that estradiol benzoate decreases SN GABAergic activity and that the integrity of the pituitary gland is required for this effect.

Effects of repeated administration of estradiol benzoate on tubero-infundibular GABAergic activity in male rats.

Repeated (once a day for 8 days) but not single administration of estradiol benzoate (10 micrograms/kg, s.c.) induced a sevenfold increase in anterior pituitary gamma-aminobutyric acid (GABA) concentration in male rats. GABA concentration also increased in the median eminence whereas no changes or decreases were observed in other brain regions including hypothalamic arcuate nucleus, lateral septum, hippocampus, caudate nucleus, and substantia nigra. Eight-day estradiol benzoate injection also enhanced the Vmax of median eminence glutamate decarboxylase activity without affecting the Km of the enzyme for glutamic acid. Taken together, these results suggest that repeated administration of estradiol benzoate increases the activity of the tubero-infundibular GABAergic system in male rats.

Ornithine as a precursor of neurotransmitter glutamate: effect of canaline on ornithine aminotransferase activity and glutamate content in the septum of rat brain.

Local injections of L-canaline into the septum produce a rapid and almost complete inhibition of ornithine aminotransferase activity followed by a decrease in glutamate content in this region. The time-course of canaline action shows the existence of two glutamate pools with different sizes and half-life values. Surgical lesions of the hippocampal-septal glutamatergic pathway affected the site and kinetics of the small pool of glutamate in the septum, suggesting the participation of ornithine aminotransferase in the synthesis of this pool. This indicates a possible role of ornithine as a precursor of the transmitter glutamate. The localization of ornithine aminotransferase does not seem, however, to be specific for the nerve-terminal compartment. The data obtained allow estimation of the turnover rate of the specific pool of neurotransmitter glutamate.

Vasoactive intestinal peptide which coexists with acetylcholine decreases acetylcholine turnover in mouse salivary glands.

Acetylcholine (ACh) and vasoactive intestinal peptide (VIP) probably coexist in cholinergic neurons of rodent salivary glands. In this tissue, cholinergic drugs regulate release of both ACh and VIP from postganglionic cholinergic neurons. In the present study we investigated whether VIP could modulate the metabolism of ACh in mouse submandibular gland cholinergic neurons using ACh turnover rate (TRACh) as a parameter. The TRACh was estimated via measurement of the formation of [3H]ACh during constant rate infusion of [3H]choline. Choline and ACh were separated by reverse phase high-performance liquid chromatography and were detected electrochemically after enzymatic postcolumn reaction. We calculated that the TRACh was about 3 nmol/mg of protein per hr. Pilocarpine, a muscarinic agonist decreased the TRACh about 5-fold whereas atropine methyl Br, a muscarinic antagonist, caused a large increase in turnover. Turnover, therefore, appears to be regulated by a feedback mechanism triggered by occupancy of postsynaptic receptors. VIP infused i.v. (40 micrograms/kg/min) decreased the TRACh by about 50%. Atropine completely prevented the inhibition of the TRACh induced by VIP. These results suggest that, by changing postsynaptic or presynaptic muscarinic receptor function, VIP may participate in the control of ACh metabolism. Parasympathetic decentralization of salivary glands did not prevent the effect of either atropine or VIP on TRACh. This finding suggests that the central afferent input to the ganglionic cells is not required for the regulation of ACh metabolism and, therefore, the feedback loop probably acts via a postganglionic mechanism which is not elucidated by present experiments.

Sulfation of peptides and simple phenols by rat brain phenolsulfotransferase. Inhibition by dichloronitrophenol.

Brain phenolsulfotransferase (PST) is involved in the sulfation of simple phenols like dopamine and of precursors of biologically active peptides like cholecystokinin octapeptide (CCK-8). Therefore, inhibition of brain PST would provide a new approach to studying the sulfation of CCK-8 and other sulfated compounds. Since 2,6-dichloro-4-nitrophenol (DCNP) produces a prolonged and selective inhibition of the sulfoconjugation of exogenous phenols by the liver, we decided to examine the applicability of DCNP to studies of sulfation of CCK-8 and other compounds by brain. DCNP was capable of completely inhibiting PST activity in rat brain homogenates incubated with p-nitrophenol, phenol or dopamine as substrates. The IC50 values for p-nitrophenol and dopamine were 12 and 14 microM respectively. The concentrations of DCNP in brain cortex and plasma were measured by high pressure liquid chromatography (HPLC) after a dose of 100 mumoles/kg, i.p. Peak concentrations of 380 microM in plasma and 25 mumoles/kg in brain were achieved 30 min after injection. Subsequently, DCNP concentrations decreased with half-lives of 8 and 6 hr in plasma and brain cortex, respectively. To establish if DCNP can inhibit CCK sulfation in vivo, rats were injected with 100 micromoles/kg, i.p., of the drug 30 min before injection of 35SO4(2-) into the cerebral cortex and were killed 4.5 hr later. DCNP caused a 55% inhibition of [35S]CCK-8-SO4 formation as measured by HPLC. No change in the content of endogenous CCK-8-SO4 was detectable, however, in the brain cortex of rats treated with DCNP for up to 4 days, indicating that the PST which remained active was capable of maintaining CCK-8 content at steady state.

Enzymes adsorbed on an ion exchanger as a post-column reactor: application to acetylcholine measurement.

Enzymes can be used in high-performance liquid chromatography post-column reactors to improve sensitivity and specificity of detection for some compounds by converting the compounds to easily detectable products. The enzymes can be covalently bound to a post-column reactor, but a simpler approach is to bind them by adsorption to an ion exchanger or a hydrophobic interaction support. This technique has been applied to electrochemical detection of acetylcholine by using adsorption of choline oxidase and cholinesterase to a 3-cm long commercially available weak anion-exchange cartridge. Conversion of acetylcholine to peroxide is quantitative during the 10-sec residence time in the cartridge. Enzyme elution from the cartridge is negligible when low ionic strength mobile phases are used. Fresh enzyme needs to be added to the cartridge at only 1-2 week intervals.

Acetylcholine measurement by high-performance liquid chromatography using an enzyme-loaded postcolumn reactor.

Choline oxidase and cholinesterase were found to retain their activity for 1-2 weeks at room temperature while adsorbed to a commercially available anion-exchange cartridge. These enzymes convert acetylcholine to H2O2. Acetylcholine can be measured in tissue extracts by separation at pH 7 on a polymeric reverse-phase high-performance liquid chromatography column, conversion of acetylcholine to H2O2 on a postcolumn enzyme-loaded anion-exchange cartridge, and electrochemical detection of the H2O2 formed.

Enkephalin biosynthesis in adrenal medulla. Modulation of proenkephalin mRNA content of cultured chromaffin cells by 8-bromo-adenosine 3',5'-monophosphate.

Incubation of primary cultures of chromaffin cells from bovine adrenal medulla with 8-bromo-adenosine 3',5'-monophosphate (8-Br-cyclic AMP) resulted in an increase in proenkephalin mRNA content. The mRNA that increased was detected by hybridization analysis using a cDNA probe and migrated with an apparent size of approximately 1400 bases. The increase in proenkephalin mRNA following 8-Br-cyclic AMP treatment was apparent in 12 hr and continued over 2 days. Corresponding changes were detected in enkephalin-like immunoreactivity but with a 24-hr lag: the cellular content increased significantly after 2 days of treatment and continued to rise over the next 2 days, whereas changes in the amount released to the medium followed the same time course. Dose-response curves for the increase in the content of proenkephalin mRNA and of enkephalin-containing peptides were essentially identical. Chromatographic characterization of the enkephalin-like peptides demonstrated that 8-Br-cyclic AMP increased both the high molecular weight fraction and the low molecular weight fraction, which was shown by high-pressure liquid chromatography to contain Met5-enkephalin, Leu5-enkephalin, and Met5-enkephalin-Arg6-Phe7. Previous results in chromaffin cells have demonstrated that the synthesis of tyrosine hydroxylase is also regulated by cyclic AMP, with a similar time course. These results therefore suggest the possibility of coordinate regulation by cyclic AMP of the expression of the cotransmitters, catecholamines and enkephalin peptides, in the adrenal medulla.

Measurement of acetylcholine turnover rate in brain: an adjunct to a simple HPLC method for choline and acetylcholine.

An existing method for measuring acetylcholine (ACh) and choline (Ch) is shown to be useful for measuring the turnover rate of ACh in mouse brain. Methyl-[3H]Ch is injected into mice. They are killed at different times by microwave irradiation and Ch and ACh extracted and separated by reverse-phase HPLC. Ch and ACh are converted to hydrogen peroxide by a post-column enzyme reaction. Hydrogen peroxide, which is directly related to the tissue content of Ch or ACh, is determined electrochemically. The fractions that correspond to the detector response for Ch and ACh are collected for the measurement of radioactivity. In this way specific radioactivities of endogenous Ch and ACh are estimated in the same sample. We used the specific radioactivity values determined by this procedure to estimate the turnover of ACh for striatum, cerebral cortex, and hippocampus of the mouse.

Gamma-aminobutyric acid turnover in rat striatum: effects of glutamate and kainic acid.

The turnover rate of gamma-aminobutyric acid (GABA) in the rat striatum was estimated by measuring its accumulation after inhibition of GABA-transaminase (GABA-T) with gabaculine. Intrastriatal injections of 100 micrograms gabaculine induced a rapid and complete inhibition of GABA-T. GABA accumulation was linear with time for at least 60 min (estimated turnover rate = 25 nmol/mg protein/h). The accumulation of GABA after gabaculine administration in animals that had been treated with kainic acid (5 nmol intrastriatally, 7 days) was only 40% of the control value, indicating that a major fraction of the net increase in GABA content induced by gabaculine originates in kainic acid-sensitive neurons. Intrastriatal injection of a mixture of kainic acid (5 nmol) and gabaculine caused a net increase in striatal GABA content significantly greater than that observed in controls, suggesting that neuronal death induced by kainic acid is preceded by a period of increased neuronal activity. Glutamic acid, the putative neurotransmitter for the excitatory corticostriatal pathway, also produced a significant increase in striatal GABA accumulation when injected together with gabaculine. This effect was blocked by the administration of the glutamate receptor antagonist glutamic acid diethyl ester. The interactions between GABAergic neurons and other neurotransmitters present in the striatum were also analyzed.

Cholecystokinin turnover in brain.

Cholecystokinin octapeptide (CCK-8) occurs in relatively large amounts in some neurons of the cerebral cortex. Unlike most other mammalian neuropeptides, this compound contains a sulfate ester. We injected radiolabelled inorganic sulfate [( 35S]sulfate) into rat cerebral cortex and measured the formation and elimination of radiolabelled CCK-8 using HPLC. The data allow the first calculation of the turnover rate of a putative transmitter neuropeptide in brain. The turnover of CCK-8 (half-life = 16 h) is considerably slower than that of the biogenic amines and amino acid neurotransmitters (half-lives less than 4 h).