Effect of forskolin on voltage-gated K+ channels is independent of adenylate cyclase activation.

Forskolin is commonly used to stimulate adenylate cyclase in the study of modulation of ion channels and other proteins by adenosine 3',5'-monophosphate (cAMP)-dependent second messenger systems. In addition to its action on adenylate cyclase, forskolin directly alters the gating of a single class of voltage-dependent potassium channels from a clonal pheochromocytoma (PC12) cell line. This alteration occurred in isolated cell-free patches independent of soluble cytoplasmic enzymes. The effect of forskolin was distinct from those of other agents that raise intracellular cAMP levels. The 1,9-dideoxy derivative of forskolin, which is unable to activate the cyclase, was also effective in altering the potassium channel activity. This direct action of forskolin can lead to misinterpretation of results in experiments in which forskolin is assumed to selectively activate adenylate cyclase.

Auditory brainstem anomalies in human albinos.

Brainstem auditory evoked potentials recorded from human albinos indicate significant hemispheric asymmetry. The asymmetry is symptomatic of differences between decussated and nondecussated auditory pathways in albino and pigmented humans at approximately the level of the superior olivary nuclei. Abnormal decussation of auditory pathways in albinos probably coincides with known visual system anomalies.

Properties of ShB A-type potassium channels expressed in Shaker mutant Drosophila by germline transformation.

We have used P element-mediated germline transformation to express ShB channels in Shaker mutant Drosophila and have examined their properties by patch-clamp of embryonic myotubes. The transformed ShB cDNA was placed under the transcriptional control of a heat shock promoter (hsp70). Northern blots revealed that transformed DNA is efficiently transcribed in response to heat shock. Cultured myotubes from the transformants produced large A-type potassium currents in response to heat shock. Although qualitatively similar to native Shaker A-currents in wild-type myotubes, transformant A-current inactivates more rapidly and recovers from inactivation more rapidly, similar to ShB channels expressed in Xenopus oocytes. Unlike the channels in oocytes, however, the transformant A-current is insensitive to 50 nM charybdotoxin.

Modulation of volume regulated anion current by I(Cln).

I(Cln), a cytosolic protein associated with a nucleotide-sensitive chloride current, may be involved in the regulation of a volume-regulated anion current (VRAC) associated with hypotonic cell swelling. We have determined the nucleic acid sequences of I(Cln) from human tsA201a, colonic (T84) and myeloma (RPMI 8826) cell lines. The amino acid sequences are highly homologous (>/=99%) to each other but less homologous to I(Cln) protein from other species. Using the whole-cell patch clamp technique, we examined the effect of I(Cln) protein expression levels on VRAC properties during a hyposmotic challenge. Overexpression of T84 or RPMI 8226-derived I(Cln) protein in tsA201a cells results in a more than 9-fold increase in the rate of VRAC activation over control values, while having no effect on VRAC inactivation properties. Underexpression of endogenous I(Cln) protein in tsA201a cells using antisense oligonucleotides results in a more than 180-fold decrease in VRAC activation rate as compared to control values. These results indicate that I(Cln) protein expression modulates VRAC activation but not inactivation.

Single Na+ channels activated by veratridine and batrachotoxin.

Voltage-sensitive Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers in the presence of either of the alkaloid toxins veratridine (VT) or batrachotoxin (BTX). Both of these toxins are known to cause persistent activation of Na+ channels. With BTX as the channel activator, single channels remain open nearly all the time. Channels activated with VT open and close on a time scale of 1-10 s. Increasing the VT concentration enhances the probability of channel opening, primarily by increasing the rate constant of opening. The kinetics and voltage dependence of channel block by 21-sulfo-11-alpha-hydroxysaxitoxin are identical for VT and BTX, as is the ionic selectivity sequence determined by bi-ionic reversal potential (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). However, there are striking quantitative differences in open channel conduction for channels in the presence of the two activators. Under symmetrical solution conditions, the single channel conductance for Na+ is about twice as high with BTX as with VT. Furthermore, the symmetrical solution single channel conductances show a different selectivity for BTX (Na+ greater than Li+ greater than K+) than for VT (Na+ greater than K+ greater than Li+). Open channel current-voltage curves in symmetrical Na+ and Li+ are roughly linear, while those in symmetrical K+ are inwardly rectifying. Na+ currents are blocked asymmetrically by K+ with both BTX and VT, but the voltage dependence of K+ block is stronger with BTX than with VT. The results show that the alkaloid neurotoxins not only alter the gating process of the Na+ channel, but also affect the structure of the open channel. We further conclude that the rate-determining step for conduction by Na+ does not occur at the channel's "selectivity filter," where poorly permeating ions like K+ are excluded.

Batrachotoxin-activated Na+ channels in planar lipid bilayers. Competition of tetrodotoxin block by Na+.

Single Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers formed from neutral phospholipids and were observed in the presence of batrachotoxin. The batrachotoxin-modified channel activates in the voltage range -120 to -80 mV and remains open almost all the time at voltages positive to -60 mV. Low levels of tetrodotoxin (TTX) induce slow fluctuations of channel current, which represent the binding and dissociation of single TTX molecules to single channels. The rates of association and dissociation of TTX are both voltage dependent, and the association rate is competitively inhibited by Na+. This inhibition is observed only when Na+ is increased on the TTX binding side of the channel. The results suggest that the TTX receptor site is located at the channel's outer mouth, and that the Na+ competition site is not located deeply within the channel's conduction pathway.

Voltage-dependent blockade of muscle Na+ channels by guanidinium toxins.

Na+ channels from rat muscle plasma membrane vesicles were inserted into neutral planar phospholipid bilayers and were activated by batrachotoxin. Single channel blocking events induced by the addition of various guanidinium toxins were analyzed to derive the rates of channel-toxin association and dissociation. Blocking by tetrodotoxin, saxitoxin, and six natural saxitoxin derivatives containing sulfate or hydroxyl groups were studied. Although the binding affinities vary over 2,000-fold, all of the toxins exhibit identical voltage dependence of the blocking reactions, regardless of the toxin's net charge. The results suggest that the voltage dependence of toxin binding is due to a voltage-dependent conformational equilibrium of the toxin receptor, rather than to direct entry of the charged toxin molecule into the applied transmembrane electric field.

Drug design with a new transition state analog of the hydrated carbonyl: silicon-based inhibitors of the HIV protease.

BACKGROUND: Silicon is the element most similar to carbon, and bioactive organosilanes have therefore been of longstanding interest. Design of bioactive organosilanes has often involved a systematic replacement of a bioactive molecule's stable carbon atoms with silicon. Silanediols, which are best known as unstable precursors of the robust and ubiquitous silicone polymers, have the potential to mimic an unstable carbon, the hydrated carbonyl. As a bioisostere of the tetrahedral intermediate of amide hydrolysis, a silanediol could act as a transition state analog inhibitor of protease enzymes. RESULTS: Silanediol analogs of a carbinol-based inhibitor of the HIV protease were prepared as single enantiomers, with up to six stereogenic centers. As inhibitors of this aspartic protease, the silanediols were nearly equivalent to both their carbinol analogs and indinavir, a current treatment for AIDS, with low nanomolar K(i) values. IC(90) data from a cell culture assay mirrored the K(i) data, demonstrating that the silanediols can also cross cell membranes and deliver their antiviral effects. CONCLUSIONS: In their first evaluation as inhibitors of an aspartic protease, silanediol peptidomimetics have been found to be nearly as potent as currently available pharmaceutical agents, in enzyme and cell protection assays. These neutral, cell-permeable transition state analogs therefore provide a novel foundation for the design of therapeutic agents.

Design and selection of DMP 850 and DMP 851: the next generation of cyclic urea HIV protease inhibitors.

BACKGROUND: Recent clinical trials have demonstrated that HIV protease inhibitors are useful in the treatment of AIDS. It is necessary, however, to use HIV protease inhibitors in combination with other antiviral agents to inhibit the development of resistance. The daunting ability of the virus to rapidly generate resistant mutants suggests that there is an ongoing need for new HIV protease inhibitors with superior pharmacokinetic and efficacy profiles. In our attempts to design and select improved cyclic urea HIV protease inhibitors, we have simultaneously optimized potency, resistance profile, protein binding and oral bioavailability. RESULTS: We have discovered that nonsymmetrical cyclic ureas containing a 3-aminoindazole P2 group are potent inhibitors of HIV protease with excellent oral bioavailability. Furthermore, the 3-aminoindazole group forms four hydrogen bonds with the enzyme and imparts a good resistance profile. The nonsymmetrical 3-aminoindazoles DMP 850 and DMP 851 were selected as our next generation of cyclic urea HIV protease inhibitors because they achieve 8 h trough blood levels in dog, with a 10 mg/kg dose, at or above the protein-binding-adjusted IC90 value for the worst single mutant--that containing the Ile84-->Val mutation. CONCLUSIONS: In selecting our next generation of cyclic urea HIV protease inhibitors, we established a rigorous set of criteria designed to maximize chances for a sustained antiviral effect in HIV-infected individuals. As DMP 850 and DMP 851 provide plasma levels of free drug that are sufficient to inhibit wild-type HIV and several mutant forms of HIV, they could show improved ability to decrease viral load for clinically significant time periods. The ultimate success of DMP 850 and DMP 851 in clinical trials might depend on achieving or exceeding the oral bioavailability seen in dog.

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.

Selection conditions affect the evolution of specific mutations in the reverse transcriptase gene associated with resistance to DMP 266.

OBJECTIVE: To monitor the appearance of HIV-1 variants resistant to inhibition by DMP 266, a benzoxazinone non-nucleoside reverse transcriptase inhibitor using two different protocols for applying drug selective pressure in tissue culture. To compare the phenotype and genotype of viral isolates selected by each method. METHODS: MT-2 cells and fresh donor peripheral blood mononuclear cells (PBMC) were infected with HIV-1 strain RF. The MT-2 cells were infected in the presence of a 50% inhibitory concentration (IC50) of DMP 266 and the concentration was slowly increased during the selection period. The PBMC were infected for 1 week in the absence of inhibitor and then a single concentration was maintained throughout the selection period. Both cultures were passaged for approximately 4 months. Virus and cell pellets were harvested over this in vitro selection period, the RT genes amplified by polymerase chain reaction from the cell pellets, and the proviral DNAs sequenced. Isolated virus was tested for DMP 266 susceptibility in either the AIDS Clinical Trials Group/Department of Defense consensus assay or MT-2 yield reduction assay. RESULTS: Passage in MT-2 cells resulted in accumulation of three substitutions in RT (V179D, L1001, Y181C) after 24 passages associated with 1000-fold reduced susceptibility to DMP 266. In PBMC cultures treated with 0.96 microM DMP 266, virus replication was completely suppressed after 2 weeks; no regrowth occurred in the presence of compound after 10 weeks or in the absence of compound for 3 additional weeks. The 0.096 microM treated cultures had an initial 2.5-log reduction in infectious virus titre followed by rapid regrowth. Virus obtained at week 6 displayed a 28-fold reduction in susceptibility with an L1001 substitution in RT, and by week 11 displayed a 1000-fold reduction in susceptibility with an additional V1081 substitution. CONCLUSIONS: High-level resistance to DMP 266 may develop by at least two pathways and experimental conditions influence the genotype selected. The continued absence of detectable virus in the PBMC cultures grown at 0.96 microM is supportive evidence that maintaining trough plasma levels of DMP 266 which result in sustained antiviral activity in vivo may delay emergence of highly resistant viral variants. Confirmation of this hypothesis will require clinical trials.

Staphylococcal meningitis: a complication of psoas abscess.

A previously healthy 59-year-old woman presented with fever, neck stiffness, and localized back tenderness. Spinal fluid and blood cultures grew Staphylococcus aureus. A diagnosis of pyogenic meningitis was made, but further investigation revealed that the meningitis arose from a clinically occult psoas abscess.

Cyclic urea amides: HIV-1 protease inhibitors with low nanomolar potency against both wild type and protease inhibitor resistant mutants of HIV.

Cyclic urea amides, a novel series of HIV-1 protease (HIV PR) inhibitors, have increased activity against drug-resistant mutants of the HIV PR. The design strategy for these inhibitors is based on the hypotheses that (i) the hydrogen-bonding interactions between the inhibitor and the protease backbone will remain constant for wild-type and mutant enzymes and (ii) inhibitors which are capable of forming many nonbonded interactions, distributed throughout the active site, will experience a lower percent change in binding energy as a result of mutation in the target enzyme than those that form fewer interactions by partial occupation of the active site. The cyclic urea amide, SD146, forms 14 hydrogen bonds and 191 van der Waals contacts to HIV PR. SD146 is a very potent antiviral agent (IC90 = 5.1 nM) against wild-type HIV and maintains the same or improved level of high potency against a range of mutant strains of HIV with resistance to a wide variety of HIV protease inhibitors.

Improved P1/P1' substituents for cyclic urea based HIV-1 protease inhibitors: synthesis, structure-activity relationship, and X-ray crystal structure analysis.

We present several novel P1/P1' substituents that can replace the characteristic benzyl P1/P1' moiety of the cyclic urea based HIV protease inhibitor series. These substituents typically provide 5-10-fold improvements in binding affinity compared to the unsubstituted benzyl analogs. The best substituent was the 3,4-(ethylenedioxy)benzyl group. Proper balancing of the molecule's lipophilicity facilitated the transfer of this improved binding affinity into a superior cellular antiviral activity profile. Several analogs were evaluated further for protein binding and resistance liabilities. Compound 18 (IC90 = 8.7 nM) was chosen for oral bioavailability studies based on its log P and solubility profile. A 10 mg/kg dose in dogs provided modest bioavailability with Cmax = 0.22 microg/mL. X-ray crystallographic analysis of two analogs revealed several interesting features responsible for the 3,4-(ethylenedioxy)benzyl-substituted analog's potency: (1) Comparing the two complexes revealed two distinct binding modes for each P1/P1' substituent; (2) The ethylenedioxy moieties are within 3.6 A of Pro 81 providing additional van der Waals contacts missing from the parent structure; (3) The enzyme's Arg 8 side chain moves away from the P1 substituent to accommodate the increased steric volume while maintaining a favorable hydrogen bond distance between the para oxygen substituent and the guanidine NH.

Inhibition of clinically relevant mutant variants of HIV-1 by quinazolinone non-nucleoside reverse transcriptase inhibitors.

A series of 4-alkenyl and 4-alkynyl-3, 4-dihydro-4-(trifluoromethyl)-2-(1H)-quinazolinones were found to be potent non-nucleoside reverse transcriptase inhibitors (NNRTIs) of human immunodeficiency virus type-1 (HIV-1). The 4-alkenyl-3, 4-dihydro-4-(trifluoromethyl)-2-(1H)-quinazolinones DPC 082 and DPC 083 and the 4-alkynyl-3, 4-dihydro-4-(trifluoromethyl)-2-(1H)-quinazolinones DPC 961 and DPC 963 were found to exhibit low nanomolar potency toward wild-type RF virus (IC(90) = 2.0, 2.1, 2.0, and 1.3 nM, respectively) and various single and many multiple amino acid substituted HIV-1 mutant viruses. The increased potency is combined with favorable plasma serum protein binding as demonstrated by improvements in the percent free drug in human plasma when compared to efavirenz: 3.0%, 2.0%, 1.5%, 2. 8%, and 0.2-0.5% for DPC 082, DPC 083, DPC 961, DPC 963, and efavirenz, respectively.

Nonsymmetric P2/P2' cyclic urea HIV protease inhibitors. Structure-activity relationship, bioavailability, and resistance profile of monoindazole-substituted P2 analogues.

Using the structural information gathered from the X-ray structures of various cyclic urea/HIVPR complexes, we designed and synthesized many nonsymmetrical P2/P2'-substituted cyclic urea analogues. Our efforts concentrated on using an indazole as one of the P2 substituents since this group imparted enzyme (Ki) potency as well as translation into excellent antiviral (IC90) potency. The second P2 substituent was used to adjust the physical and chemical properties in order to maximize oral bioavailability. Using this approach several very potent (IC90 11 nM) and orally bioavailable (F% 93-100%) compounds were discovered (21, 22). However, the resistance profiles of these compounds were inadequate, especially against the double (I84V/V82F) and ritonavir-selected mutant viruses. Further modification of the second P2 substituent in order to increase H-bonding interactions with the backbone atoms of residues Asp 29, Asp 30, and Gly 48 led to analogues with much better resistance profiles. However, these larger analogues were incompatible with the apparent molecular weight requirements for good oral bioavailability of the cyclic urea class of HIVPR inhibitors (MW < 610).

Evolution of experimentally induced papillary necrosis to focal segmental glomerulosclerosis and nephrotic proteinuria.

Bromoethylamine (BEA)-induced papillary necrosis is a reproducible model for analgesic nephropathy. We induced this lesion in groups of male Sprague-Dawley rats and followed the functional and histological changes for 1 year. We found that by 1 month, necrosis of the papilla was complete, glomerular filtration rate was depressed, and urine albumin excretion was increased. There was an extensive interstitial fibrosis characterized by a mononuclear cell infiltrate and patchy tubular atrophy. By 6 months, there was re-epithelialization of the papillary stump accompanied by a marked increase in albuminuria and an improvement in concentrating ability. Changes seen at 9 months were more advanced. There was extensive cortical fibrosis manifested by pitting of the surface of the kidney. At 1 year, renal function remained impaired (creatinine clearance reduced by 65% to 0.26 mL/min/100 g), and the animals were now markedly nephrotic, with albuminuria of 254 mg of albumin/24 h. In the BEA rats, there was selective destruction of the deep nephrons leading to an increase in the volume-ratio of superficial to deep nephrons. Glomerular changes, affecting approximately 60% of the glomeruli, were characteristic of focal segmental glomerular sclerosis. This model of papillary necrosis/interstitial fibrosis is associated with chronic renal insufficiency and leads to the development of focal glomerular sclerosis and nephrotic proteinuria by 6 to 12 months after its induction.

Persistence of the immunoprotective effects of leukemia X fibroblast hybrid cells toward leukemia in histocompatible mice.

Hybrids of ASL-1 murine leukemia cells and LM(TK-) cells, a cultured line of mouse fibroblast origin, stimulate partial immunity toward ASL-1 cells in (A/J X C3H/HeJ)F1 mice (F1 mice). Such mice ordinarily exhibit no resistance to the malignant proliferation of ASL-1 cells. Unprotected animals invariably die within 14-18 days after receiving as few as 200 ASL-1 cells. The hybrid cells, the mice used in the experimental studies and the leukemia cells used for challenge all share the same histocompatibility antigens. ASL-1 cells are H-2a; LM(TK-) cells are H-2k, both ASL-1 X LM(TK-) hybrid cells and A/J X C3H/HeJ)F1 mice are H-2a/k. The long-term persistence of the immunoprotective properties of the hybrid cells toward murine leukemia was investigated by using cells that had been in continuous culture for approx. 36 months. (A/J X C3H/HeJ)F1 mice injected previously with hybrid cells in continuous culture and then challenged with up to 10(7) ASL-1 cells survived longer (p less than 0.001) than mice who had not received hybrid cells previously. Some mice challenged with lesser number of ASL-1 cells survived indefinitely (greater than 200 days). The median survival time of F1 mice injected simultaneously with 10(7) hybrid cells and 200 or 2000 ASL-1 cells was significantly (p less than 0.001) prolonged as well, although the differences between experimental and control groups are less pronounced than if the hybrid cells were injected before challenge with ASL-1 cells. The hybrid cells like those freshly prepared continue to be rejected by histocompatible precipients. In no instance has there been evidence of a progressively growing tumor of hybrid cells in immunocompetent F1 mice. Hybrid cells like those investigated previously do form rapidly growing metastasizing tumors in immunodeficient nu/nu (BALB/c) or X-irradiated (550 R) F1 mice. The cells possess approx. 70 chromosomes (reduced from 85) including chromosomes identified as having originated in each parental source. Like (A/J X C3H/HeJ)F1 animals, they continue to express both H-2a and H-2k antigenic determinants.

Identification of a clinical isolate of HIV-1 with an isoleucine at position 82 of the protease which retains susceptibility to protease inhibitors.

The HIV-1 protease (PR) is essential for the production of mature virions. As such, it has become a target for the development of anti-HIV chemotherapeutics. Multiple passages of virus in cell culture in the presence of PR inhibitors have resulted in the selection of variants with decreased sensitivity to inhibitors of the PR. The most common alteration observed is a single amino acid change at position 82. This particular position has been well characterized by several laboratories as being important for the susceptibility of the virus to inhibitors of PR function. Mutations which result in the substitution of the wild-type valine with alanine, phenylalanine, threonine or isoleucine at position 82 of the PR have been associated with decreased sensitivity to several PR inhibitors. We describe here a clinical strain of HIV-1 that contains an isoleucine at position 82 of the PR instead of the usual valine. This strain is unique in that it was isolated from a patient that was anti-retroviral naive, and in the past, variants at position 82 of the PR have only been found after treatment of patients or cell culture with PR inhibitors. Moreover, this virus remains sensitive to PR inhibitors of the cyclic urea and C-2 symmetrical diol classes.