3-Arylpiperidines as potentiators of existing antibacterial agents.

Important resistance patterns in Gram-negative pathogens include active efflux of antibiotics out of the cell via a cellular pump and decreased membrane permeability. A 3-arylpiperidine derivative (1) has been identified by high-throughput assay as a potentiator with an IC(50) approximately 90 microM. This report details the evaluation of the tether length, aryl substitution and the importance of the fluorine on antibiotic accumulation. Evaluation of various tether lengths demonstrated that the two-carbon tethered analogues are optimal. Removal of the fluorine has a modest effect on antibiotic accumulation and the defluorinated analogue 17 is equally potent to the original lead 1.

Identification of novel potent hydroxamic acid inhibitors of peptidyl deformylase and the importance of the hydroxamic acid functionality on inhibition.

Peptidyl deformylase (PDF) is a metallo protease that catalyzes the removal of a formyl group from the N-termini of prokaryotic prepared polypeptides, an essential step in bacterial protein synthesis. Screening of our compound collection using Staphylococcus aureus PDF afforded a very potent inhibitor with an IC(50) in the low nanomolar range. Unfortunately, the compound that contains a hydroxamic acid did not exhibit antibacterial activity (MIC). In order to address the lack of activity in the MIC assay and to determine what portion of the molecule was responsible for binding to PDF, we prepared several analogues. This paper describes our findings that the hydroxamic acid functionality found in 1 is mainly responsible for the high affinity to PDF. In addition, we identified an alternative class of PDF inhibitors, the N-hydroxy urea 18, which has both PDF and antibacterial activity.

Tipranavir (PNU-140690): a potent, orally bioavailable nonpeptidic HIV protease inhibitor of the 5,6-dihydro-4-hydroxy-2-pyrone sulfonamide class.

A broad screening program previously identified phenprocoumon (1) as a small molecule template for inhibition of HIV protease. Subsequent modification of this lead through iterative cycles of structure-based design led to the activity enhancements of pyrone and dihydropyrone ring systems (II and V) and amide-based substitution (III). Incorporation of sulfonamide substitution within the dihydropyrone template provided a series of highly potent HIV protease inhibitors, with structure-activity relationships described in this paper. Crystallographic studies provided further information on important binding interactions responsible for high enzymatic binding. These studies culminated in compound VI, which inhibits HIV protease with a Ki value of 8 pM and shows an IC90 value of 100 nM in antiviral cell culture. Clinical trials of this compound (PNU-140690, Tipranavir) for treatment of HIV infection are currently underway.

Development of activity assays for high-volume evaluation of human immunodeficiency virus (HIV) protease inhibitors in rat serum: results with ditekiren.

We showed previously that a commercially available synthetic tetradecapeptide, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser, produces authentic angiotensin I (Ang I) upon incubation with the HIV-1 protease (S. K. Sharma et al., Anal. Biochem. 198:363, 1991). Therefore, we developed an Ang-I based activity assay for HIV protease inhibitors based on the technology developed earlier (M. J. Ruwart et al., Pharm. Res. 7:407, 1990; S. K. Sharma et al., Anal. Biochem. 186:24, 1990) for tracking renin inhibitors in rat sera. Ditekiren was either extracted from sera with ethyl acetate or assayed after the interfering substances in sera were precipitated with acetonitrile. Purified recombinant HIV-1 protease was added to extracted rat serum and the enzymatic reaction was initiated in the presence of the tetradecapeptide substrate. The inhibition of Ang I production was measured by a commercially available RIA kit. The cleanup methodology also enabled a commercially available Proteinase Scintillation Proximity Assay (SPA, Amersham) to quantify ditekiren in rat serum through the addition of recombinant HIV-1 protease and cleavage of substrate from SPA beads. Results were confirmed by HPLC or by the renin assay for ditekiren, which inhibits both aspartyl proteases. These technologies should prove useful for assessing serum levels of HIV protease inhibitors in rat.

Soluble CD4-PE40 is cytotoxic for a transfected mammalian cell line stably expressing the envelope protein of human immunodeficiency virus (HIV-1), and cytotoxicity is variably inhibited by the sera of HIV-1-infected patients.

Sera were obtained from 50 individuals infected with human immunodeficiency virus type 1 or from HIV-1-uninfected individuals before or after vaccination with recombinant gp160. These sera were evaluated for activity antagonistic to the cell-killing activity of the chimeric Pseudomonas exotoxin hybrid protein, sCD4-PE40. For these studies, Chinese hamster ovary (CHO) cells were transfected with a chimeric plasmid encoding the tat, rev, and envelope genes of HIV-1 and a cell line was selected for stable expression of the envelope glycoproteins at the cell surface (CHO-env). Cytotoxicity of sCD4-PE40 for CHO-env in the presence or absence of added human serum was quantitated spectrophometrically following enzymatic reduction of a tetrazolium bromide within the mitochondria of viable cells (MTT assay). Several HIV+ sera inhibited the cytotoxic activity of sCD4-PE40; the antagonist had properties consistent with those of immunoglobulins in that it was heat stable, absorbed by protein A, and reversible by increasing the concentration of sCD4-PE40. Of 15 HIV+ sera which strongly reacted with gp120, 11 (73%) also potently inhibited sCD4-PE40 cytotoxicity, and cytotoxicity was inhibited by sera from some HIV- individuals after, but not before, immunization with gp160. These data suggested a role for antibody to gp120 in the antagonistic activity. However, not all sera with antibody to gp120 antagonized sCD4-PE40 cytotoxicity and high levels of antagonist activity were frequently (40%) found in HIV+ sera lacking immunoblot-detectable antibody to gp120, or antibody to either CD4 or PE40. Grouping of the HIV+ sera according to the patients' absolute number of CD4+ cells revealed that the degree of inhibition of sCD4-PE40 cytotoxicity approached a Gaussian distribution, suggesting that persons with CD4+ cell counts between 200 and 700/mm3 may be more likely to possess significant levels of serum antagonist. This data have implications for the clinical development of sCD4-PE40 or other sCD4-based therapeutics in the management of HIV-1 infection.

Localization of Separate Genetic Loci for Reduced Sensitivity towards Small Isometric-Headed Bacteriophage sk1 and Prolate-Headed Bacteriophage c2 on pGBK17 from Lactococcus lactis subsp. lactis KR2.

The mechanism of reduced sensitivity to the small isometric-headed bacteriophage sk1 encoded on a 19-kilobase (kb) HpaII fragment subcloned from pKR223 of Lactococcus lactis subsp. lactis KR2 was examined. The reduced sensitivity to phage sk1 was due to a modest restriction/modification (R/M) system that was not active against prolate-headed phage c2. The genetic loci for the R/M system against sk1 and the abortive phage infection (Abi) mechanism effective against phage c2 were then localized by restriction mapping, subcloning, and deletion analysis. The restriction gene was localized to a region of a 2.7-kb EcoRV fragment and included an EcoRI site within that fragment. The modification gene was found to be physically separable from the restriction gene and was present on a 1.75-kb BstEII-XbaI fragment. The genetic locus for the Abi phenotype against phage c2 was localized to a region containing a 1.3-kb EcoRI fragment. Attempts to clone the c2 Abi mechanism independent of the sk1 R/M system were unsuccessful, suggesting that expression of the abi genes required sequences upstream of the modification gene. Some pGBK17 (vector pGB301 plus a 19-kb HpaII insert fragment) transformants exhibited the R/M system against phage sk1 but lost the Abi mechanism against phage c2. These transformants contained a 1.2- to 1.3-kb insertion in the Abi region. The data identified genetic loci on a cloned 19-kb HpaII fragment responsible for restriction activity and for modification activity against a small isometric-headed phage and for Abi activity against prolate-headed phage c2. A putative insertion element was also found to inactivate the abi gene(s).

Procedure for intrapleural implantation in mice by thoracotomy.

An intrapleural implantation procedure by thoracotomy was developed for application in mice. Animal survival was found to depend on the following: (A) slow induction and quick reversal of a pneumothorax by controlled pleural air injection and withdrawal, (B) keeping the pleura open for the shortest possible time by quick insertion of the implant and immediate tight closure with prepositioned sutures, and (C) use of the right, not the left, pleural cavity especially for maximum size implants which for a 30-gram mouse was 12 X 5 X 4 mm. Surgical and postsurgical mortality was below 5% (n = over 300).