Through computationally directed broad screening, a novel 1, 5-diphenylpyrazole (DPP) class of HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs) has been discovered. Compound 2 (PNU-32945) was found to have good activity versus wild-type (IC(50) = 2.3 microM) and delavirdine-resistant P236L (IC(50) = 1.1 microM) reverse transcriptase (RT). Also, PNU-32945 has an ED(50) for inhibition of viral replication in cell cultures of 0.1 microM and was shown to be noncytotoxic with a CC(50) > 10 microM. Structure-activity relationship studies on the 3- and 4-positions of PNU-32945 led to interesting selectivity and activity within the class. In particular, the 3-hydroxyethyl-4-ethyl congener 29 is a potent inhibitor of the P236L mutant (IC(50) = 0.65 microM), whereas it is essentially inactive versus the wild-type enzyme (IC(50) > 50 microM). Furthermore, this compound was significantly more active versus the P236L mutant than delavirdine. The synthesis and RT inhibitory activity of various 3- and 4-substituted analogues are discussed.
Recipient strains of Streptococcus faecalis excrete multiple, peptide sex pheromones that induce mating responses in donors harboring certain conjugative plasmids. Acquisition of plasmid DNA leads to a "shutting off" of pheromone excretion, and such cells become responsive to exogenous pheromone. Data are presented showing that donors excrete low levels of a modified, inactive form of the pheromone. This substance, when mixed in excess with active pheromone, inhibits pheromone activity (probably by competition for a receptor site on the donor). Modified forms of both cPDI and cADI were revealed, and each appeared to have a mass about 350-400 daltons larger than the active pheromone. In both cases, pheromone activity could be regenerated by treatment with phosphodiesterase II.Recipient strains of Streptococcusfaecalis excrete multiple, heatstable, peptide sex pheromones specific for donor strains harboring certain conjugative plasmids (1-4). Donors respond by synthesizing an adherent surface structure, which facilitates the formation of mating aggregates upon contact (random collisions) with recipients. The response involves the appearance of a unique antigen which uniformly coats the donor surface (5, 6). In addition, an undefined "preparation for plasmid transfer" takes place (3). If exposed to a culture filtrate of recipients, donors are induced to self clump; for this reason pheromone is also referred to as "clumping inducing agent." The clumping response provides a basis for quantitating (by dilution) pheromone activity (2).When plasmid DNA is acquired by the recipient bacterium, production of the related pheromone ceases and the strain becomes responsive to exogenous pheromone. However, the cell continues to produce other pheromones specific for donors harboring different conjugative plasmids (2, 4). The pheromones are designated by their relationship to the plasmid originally used to detect them. Thus, cADi refers to the pheromone to which strains harboring pADI respond, whereas cPD1 refers to the pheromone related to pPD1.In this paper we address the mechanism by which plasmid acquisition leads to the "shutting off" of endogenous pheromone production. We present evidence that donor cells harboring pADi [a 37.8-megadalton Table 1. The construction of plasmid-bearing strains was by mixing overnight cultures of appropriate donors and recipients (0.05 ml of donors with 0.5 ml of recipients with 4.5 ml of fresh broth) and allowing the mixture to incubate for 2-4 hr prior to plating on an appropriate selective medium. Insertions of Tn916 (determines resistance to tetracycline) and Tn917 (determines resistance to erythromycin) into plasmid DNA were derived as described (8,9,11,12). Drug concentrations in selective media were as described (2). Unless otherwise noted, the medium used throughout this study was N2GT (Oxoid Nutrient Broth no. 2 supplemented with 0.2% glucose and 0.1 M Tris-HCI, pH 7.7). Turbidity was monitored by using a KlettSummerson colorimeter with a no. 54 (green) filter.The mutant d...
Three plasmids designated α, β, and γ, distinguishable by their molecular weights (6, 17, and 34 million, respectively) were isolated from
Streptococcus faecalis
strain DS-5 (ATCC 14508). Derivatives of this strain “cured” for erythromycin resistance lacked the β-plasmid. In the parent strain the β-plasmid was estimated to be present to the extent of one to two copies per chromosomal genome equivalent whereas the α- and γ-plasmids were about nine and five copies, respectively.
Streptococcus faecalis 39-5 is a haemolytic, bacteriocinogenic strain harbouring six plasmids. One of these plasmids, pPD1 (36.4 MDal) determines a bacteriocin and encodes a conjugative response to the sex pheromone cPD1 excreted by recipient (plasmid-free) strains. The pheromone response is characterized by the formation of mating aggregates of donors (responders) with recipients. Aggregation required the presence of phosphate and divalent cations and was inhibited by agents or conditions that destroy protein structure. Aggregation was postulated to be due to synthesis of a new proteinaceous molecule on the donor cell surface. Referred to as 'aggregation substance', such a material was identified and found to exhibit antigenic properties not associated with uninduced cells; it could be detected by immunoelectron microscopy. Aggregation substance could be extracted from induced cells but not uninduced cells as demonstrated by crossed immunoelectrophoresis. Antibody raised against the aggregation substance controlled by pPD1 cross-reacted with aggregation substance determined by other plasmid systems which respond to pheromones unrelated to cPD1.
A novel class of bis(heteroaryl)piperazine (BHAP) analogs which possesses the ability to inhibit NNRTI (non-nucleoside reverse transcriptase inhibitor) resistant recombinant HIV-1 reverse transcriptase (RT) and NNRTI resistant variants of HIV-1 has been identified via targeted screening. Further investigation of the structure-activity relationships of close congeners of these novel (alkylamino)piperidine BHAPs (AAP-BHAPs) led to the synthesis of several compounds possessing the desired phenotype (e.g., activity against recombinant RTs carrying the Y181C and P236L substitutions). Further structural modifications were required to inhibit metabolism and modulate solubility in order to obtain compounds with the desired biological profile as well as appropriate pharmaceutical properties. The AAP-BHAPs with the most suitable characteristics were compounds 7, 15, and 36.
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