Robert Ferris

Potent and Selective Inhibition of Human Cytomegalovirus Replication by 1263W94, a Benzimidazole l-Riboside with a Unique Mode of Action

ABSTRACT Benzimidazole nucleosides have been shown to be potent inhibitors of human cytomegalovirus (HCMV) replication in vitro. As part of the exploration of structure-activity relationships within this series, we synthesized the 2-isopropylamino derivative (3322W93) of 1H-β-d-ribofuranoside-2-bromo-5,6-dichlorobenzimidazole (BDCRB) and the biologically unnatural l-sugars corresponding to both compounds. One of the l derivatives, 1H-β-l-ribofuranoside-2-isopropylamino-5,6-dichlorobenzimidazole (1263W94), showed significant antiviral potency in vitro against both laboratory HCMV strains and clinical HCMV isolates, including those resistant to ganciclovir (GCV), foscarnet, and BDCRB. 1263W94 inhibited viral replication in a dose-dependent manner, with a mean 50% inhibitory concentration (IC50) of 0.12 ± 0.01 μM compared to a mean IC50 for GCV of 0.53 ± 0.04 μM, as measured by a multicycle DNA hybridization assay. In a single replication cycle, 1263W94 treatment reduced viral DNA synthesis, as well as overall virus yield. HCMV mutants resistant to 1263W94 were isolated, establishing that the target of 1263W94 was a viral gene product. The resistance mutation was mapped to the UL97 open reading frame. The pUL97 protein kinase was strongly inhibited by 1263W94, with 50% inhibition occurring at 3 nM. Although HCMV DNA synthesis was inhibited by 1263W94, the inhibition was not mediated by the inhibition of viral DNA polymerase. The parent benzimidazole d-riboside BDCRB inhibits viral DNA maturation and processing, whereas 1263W94 does not. The mechanism of the antiviral effect of l-riboside 1263W94 is thus distinct from those of GCV and of BDCRB. In summary, 1263W94 inhibits viral replication by a novel mechanism that is not yet completely understood.

In Vitro Antiretroviral Properties of S/GSK1349572, a Next-Generation HIV Integrase Inhibitor

ABSTRACT S/GSK1349572 is a next-generation HIV integrase (IN) inhibitor designed to deliver potent antiviral activity with a low-milligram once-daily dose requiring no pharmacokinetic (PK) booster. In addition, S/GSK1349572 demonstrates activity against clinically relevant IN mutant viruses and has potential for a high genetic barrier to resistance. S/GSK1349572 is a two-metal-binding HIV integrase strand transfer inhibitor whose mechanism of action was established through in vitro integrase enzyme assays, resistance passage experiments, activity against viral strains resistant to other classes of anti-HIV agents, and mechanistic cellular assays. In a variety of cellular antiviral assays, S/GSK1349572 inhibited HIV replication with low-nanomolar or subnanomolar potency and with a selectivity index of 9,400. The protein-adjusted half-maximal effective concentration (PA-EC50) extrapolated to 100% human serum was 38 nM. When virus was passaged in the presence of S/GSK1349572, highly resistant mutants were not selected, but mutations that effected a low fold change (FC) in the EC50 (up to 4.1 fold) were identified in the vicinity of the integrase active site. S/GSK1349572 demonstrated activity against site-directed molecular clones containing the raltegravir-resistant signature mutations Y143R, Q148K, N155H, and G140S/Q148H (FCs, 1.4, 1.1, 1.2, and 2.6, respectively), while these mutants led to a high FC in the EC50 of raltegravir (11- to >130-fold). Either additive or synergistic effects were observed when S/GSK1349572 was tested in combination with representative approved antiretroviral agents; no antagonistic effects were seen. These findings demonstrate that S/GSK1349572 would be classified as a next-generation drug in the integrase inhibitor class, with a resistance profile markedly different from that of first-generation integrase inhibitors.

Evidence that the Acute Behavioral and Electrophysiological Effects of Bupropion (Wellbutrin®) Are Mediated by a Noradrenergic Mechanism

Bupropion (BW 323U66) has been considered a dopaminergic antidepressant based on its ability to inhibit the uptake of dopamine (DA) somewhat more selectively than it inhibits uptake of norepinephrine (NE) or serotonin (5-HT). This report describes new evidence that bupropion selectively inhibits firing rates of NE cells in the locus coeruleus (LC) at doses significantly lower than those that inhibit activity of midbrain DA cells or dorsal raphe 5-HT cells. The IC50 dose (13 mg/kg IP) for inhibition of LC firing produced plasma concentrations that were not significantly different from those generated by the ED50 in the Porsolt test (10 mg/kg IP). The fourfold higher dose needed to inhibit DA cell firing (IC50 = 42 mg/kg IP) was similar to the dose associated with locomotor stimulation in freely moving rats. Bupropion did not change the firing rates of 5-HT cells in the dorsal raphe nucleus at any dose. In both in vitro and in vivo tests, the metabolite 306U73 (hydroxy-bupropion), a weak inhibitor of NE uptake, was approximately equipotent to bupropion with regard to inhibition of LC cells. Another metabolite, 494U73, had no effect on LC firing rates over a wide range of doses. Because of species variation in metabolism, 306U73 was not detected in plasma of rats after IV doses of bupropion that inhibited LC firing. Only trace amounts of 306U73 were detected after bupropion dosing for the Porsolt test. Pretreatment with reserpine markedly depleted catecholamines and reduced (by 30-fold) the potency of bupropion to inhibit LC firing. The effects of clonidine, a direct acting α2 agonist, were not significantly changed by reserpine. Likewise, a reduction in the effect of bupropion on LC firing was observed in vitro after depletion of catecholamines with reserpine or tetrabenazine. These results suggest that bupropion preferentially affects NE neurons in locus coeruleus at doses that are active in animal antidepressant tests. The doses of bupropion required to inhibit DA cell firing were associated with inhibition of DA uptake and behavioral stimulation and were significantly higher than those selectively producing behavioral effects in animal antidepressant tests. The acute electrophysiological actions of bupropion on NE cells require a reserpine-sensitive store of NE and occur at doses having activity in antidepressant screening tests.

Evidence that the potential antipsychotic agent rimcazole (BW 234U) is a specific, competitive antagonist of sigma sites in brain

Rimcazole (BW 234U) is a potential antipsychotic agent which in open-clinical trials appears to be effective in acute schizophrenic patients. In the present study, rimcazole was found to block the specific binding of [3H]-(+)-SKF 10,047 to sigma sites in rat and guinea pig brain (IC50 = 5.0 X 10(-7) M). The compound was 100 times weaker as a blocker of phencyclidine sites (IC50 = 4.3 X 10(-5) M). At 1 X 10(-5) M, rimcazole had only weak effects on mu, delta, kappa and epsilon opioid receptors. Scatchard analysis of the binding data from guinea pig brain revealed an apparent KD for [3H]-(+)-SKF 10,047 of 85 +/- 5 nM and a Bmax of 824 +/- 27 fmole/mg protein. In the presence of 5 X 10(-7) M BW 234U, the apparent KD was 165 +/- 35 nM, but the Bmax (892 +/- 146 fmoles/mg protein) was not affected. This suggests that rimcazole is a competitive inhibitor of sigma sites. The agent was also capable of blocking sigma sites in vivo (ID50 = 6 mg/kg i.p., mice) as judged by an in vivo sigma receptor binding assay. Thus, if the antipsychotic activity of rimcazole is confirmed in double-blind, placebo-controlled trials, it would be the first compound whose mechanism of antipsychotic activity may best be explained by a direct blockade of sigma sites and not by a direct blockade of dopamine (D2) receptors in brain.

Bupropion hydrochloride ((±) α-t-butylamino-3-chloropropiophenone HCl): a novel antidepressant agent

Tricyclic compounds such as amitriptyline and related substances are considered to be clinically effective antidepressant agents (Kuhn, 1958; Hollister, 1972) with a mode of action dependent on the inhibition of the neuronal reuptake of one or more biogenic amine transmitters in the central nervous system (Glowinski & Axelrod, 1964). The tricyclics have not proved to be ideal agents in treating depression due to their slow OllSet of action, cholinolytic side effects, interactions with pressor amines and monoamine oxidase inhibitors and also due to a tendency to elicit cardiac arrhythmias Or standstill (Jefferson, 1975). Monoamine oxidase inhibitors are likewise considered to be effective antidepressant agents (American Psychiatric Association, 1974) which act by inhibiting intraneuronal monoamine Omdase in the central nervous system (cns). However, the currently available inhibitors also inhibit monow e oxidase in the liver and the ingestion of phenethylamine-type pressor substances in the diet, ‘WY an event made innocuous with the effective of the amines by liver monoamine oxidase, Correspondence. may lead to hypertensive crisis (Marley & Blackwell, 1970). We therefore sought an agent that would be active in antidepressant screening models, but differ chemically and pharmacologically from the tricyclics, and not be sympathomimetic, cholinolytic nor an inhibitor of monoamine oxidase. Bupropion (Wellbatrin) which was synthesized by one of the authors (N.B.M.) (Baltzly & Mehta, 1968; Mehta, 1974, 1975) meets these criteria and has the following structure:

BW234U,(cis-9-[3–(3,5-dimethyl-1-piperazinyl)propyl]carbazole dihydrochloride): a novel antipsychotic agent

The capacity of neuroleptic drugs to antagonize the behavioural effects of the dopamine agonist , apomorphine, in animals has been used as a basis for identifying new compounds with potential antipsychotic actions in man (Fielding & La1 1974). Experience has shown that neuroleptic drugs which are potent antagonists of apomorphine-induced stereotyped behaviour, produce a high incidence of extrapyramidal side effects and tardive dyskinesias in man (Niemegeers 1974; Berger et a1 1978). In addition to stereotyped behaviour, however, apomorphine produces other behavioural effects. Thus, McKenzie (1971) reported that apomorphine elicited fighting in paired male rats and Costall et a1 (1979) reported that apomorphine elicited climbing behaviour in mice. Both of these behavioural effects can be antagonized by neuroleptic drugs. These considerations led to the formulation of a research program which had as its goal the development of a compound that would (1) block apomorphine-induced aggression in rats and apomorphine-induced climbing in mice, but (2) not block stereotyped behaviour in either species. From a theoretical point of view climbing and aggressiveness produced by apomorphine probably reflect stimulation of dopamine receptors in the limbic system whereas stereotyped behaviour reflects stimulation of dopamine receptors in the striatum (Fielding & La1 1974; Costall et a1 1979; Costall & Naylor 1981). Overactivity of the limbic system has been implicated in the etiology of psychotic behaviour in man (Hokfelt et a1 1974). It was reasoned that a compound exhibiting selective blockade of climbing and aggressive behaviour would share with neuroleptic drugs the ability to influence cognitive and affective changes characteristic of psychosis, but unlike neuroleptic agents would not produce extrapyramidal side effects. The pharmacology of compound, BW 234U which evolved from this program, is presented below.

Structural insights into mechanisms of non-nucleoside drug resistance for HIV-1 reverse transcriptases mutated at codons 101 or 138

Lys101Glu is a drug resistance mutation in reverse transcriptase clinically observed in HIV‐1 from infected patients treated with the non‐nucleoside inhibitor (NNRTI) drugs nevirapine and efavirenz. In contrast to many NNRTI resistance mutations, Lys101(p66 subunit) is positioned at the surface of the NNRTI pocket where it interacts across the reverse transcriptase (RT) subunit interface with Glu138(p51 subunit). However, nevirapine contacts Lys101 and Glu138 only indirectly, via water molecules, thus the structural basis of drug resistance induced by Lys101Glu is unclear. We have determined crystal structures of RT(Glu138Lys) and RT(Lys101Glu) in complexes with nevirapine to 2.5 Å, allowing the determination of water structure within the NNRTI‐binding pocket, essential for an understanding of nevirapine binding. Both RT(Glu138Lys) and RT(Lys101Glu) have remarkably similar protein conformations to wild‐type RT, except for significant movement of the mutated side‐chains away from the NNRTI pocket induced by charge inversion. There are also small shifts in the position of nevirapine for both mutant structures which may influence ring stacking interactions with Tyr181. However, the reduction in hydrogen bonds in the drug‐water‐side‐chain network resulting from the mutated side‐chain movement appears to be the most significant contribution to nevirapine resistance for RT(Lys101Glu). The movement of Glu101 away from the NNRTI pocket can also explain the resistance of RT(Lys101Glu) to efavirenz but in this case is due to a loss of side‐chain contacts with the drug. RT(Lys101Glu) is thus a distinctive NNRTI resistance mutant in that it can give rise to both direct and indirect mechanisms of drug resistance, which are inhibitor‐dependent.

Pharmacological Properties of 403U76, a New Chemical Class of 5-Hydroxytryptamine- and Noradrenaline-reuptake Inhibitor

403U76 (5‐chloro‐[[2‐[(dimethylamino)methyl]phenyl]thio]benzene‐methanol hydrochloride) is a potent, competitive, inhibitor of 5‐hydroxytryptamine (5‐HT) and noradenaline reuptake into rat brain synaptosomes. Inhibition of 5‐HT uptake in‐vivo by 403U76 was demonstrated by potentiation of the behavioural effects of 5‐hydroxytryptophan in rats and mice and blockade of p‐induced depletion of 5‐HT in rats.

In Vitro Antiviral Activity of the Novel, Tyrosyl-Based Human Immunodeficiency Virus (HIV) Type 1 Protease Inhibitor Brecanavir (GW640385) in Combination with Other Antiretrovirals and against a Panel of Protease Inhibitor-Resistant HIV

ABSTRACT Brecanavir, a novel tyrosyl-based arylsulfonamide, high-affinity, human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), has been evaluated for anti-HIV activity in several in vitro assays. Preclinical assessment of brecanavir indicated that this compound potently inhibited HIV-1 in cell culture assays with 50% effective concentrations (EC50s) of 0.2 to 0.53 nM and was equally active against HIV strains utilizing either the CXCR4 or CCR5 coreceptor, as was found with other PIs. The presence of up to 40% human serum decreased the anti-HIV-1 activity of brecanavir by 5.2-fold, but under these conditions the compound retained single-digit nanomolar EC50s. When brecanavir was tested in combination with nucleoside reverse transcriptase inhibitors, the antiviral activity of brecanavir was synergistic with the effects of stavudine and additive to the effects of zidovudine, tenofovir, dideoxycytidine, didanosine, adefovir, abacavir, lamivudine, and emtricitabine. Brecanavir was synergistic with the nonnucleoside reverse transcriptase inhibitor nevirapine or delavirdine and was additive to the effects of efavirenz. In combination with other PIs, brecanavir was additive to the activities of indinavir, lopinavir, nelfinavir, ritonavir, amprenavir, saquinavir, and atazanavir. Clinical HIV isolates from PI-experienced patients were evaluated for sensitivity to brecanavir and other PIs in a recombinant virus assay. Brecanavir had a <5-fold increase in EC50s against 80% of patient isolates tested and had a greater mean in vitro potency than amprenavir, indinavir, lopinavir, atazanavir, tipranavir, and darunavir. Brecanavir is by a substantial margin the most potent and broadly active antiviral agent among the PIs tested in vitro.

ENANTIOSELECTIVITY IN THE BINDING OF (+)- AND (−)-2-AMINO-6,7-DIHYDROXY-1,2,3,4-TETRAHYDRONAPHTHALENE AND RELATED AGONISTS TO DOPAMINE RECEPTORS

ABSTRACT The potent DA agonist 5 was resolved into enantiomers. The absolute configurations of the enantiomers of 3 and 5 were determined by chemical degradation. These compounds were tested as inhibitors of 3H-DA binding to membrane fragments from rat striatum. It was found that the configuration of the more potent enantiomer of 5 is opposite to that of 3 and APO. This is relevant to the mode of binding of such agonists to DA receptors.