Discovery of aminopyridine-based inhibitors of bacterial enoyl-ACP reductase (FabI)

Bacterial enoyl-ACP reductase (FabI) catalyzes the final step in each cycle of bacterial fatty acid biosynthesis and is an attractive target for the development of new antibacterial agents. Our efforts to identify potent, selective FabI inhibitors began with screening of the GlaxoSmithKline proprietary compound collection, which identified several small-molecule inhibitors of Staphylococcus aureus FabI. Through a combination of iterative medicinal chemistry and X-ray crystal structure based design, one of these leads was developed into the novel aminopyridine derivative 9, a low micromolar inhibitor of FabI from S. aureus (IC(50) = 2.4 microM) and Haemophilus influenzae (IC(50) = 4.2 microM). Compound 9 has good in vitro antibacterial activity against several organisms, including S. aureus (MIC = 0.5 microg/mL), and is effective in vivo in a S. aureus groin abscess infection model in rats. Through FabI overexpressor and macromolecular synthesis studies, the mode of action of 9 has been confirmed to be inhibition of fatty acid biosynthesis via inhibition of FabI. Taken together, these results support FabI as a valid antibacterial target and demonstrate the potential of small-molecule FabI inhibitors for the treatment of bacterial infections.     

IgA With altered carbohydrate structure as a tumor-associated marker in serum of cancer patients

Serum IgA₁ contains O-linked carbohydrate structures, whereas other immuno-globulins contain only N-linked structures. An assay was developed which detects IgA₁ by measuring the DGalβ(1–3) DGalNAc structures O-linked to the IgA₁ hinge region. Fifty percent of the serum specimens from cancer patients tested were elevated compared to 1% of the total normal serum specimens tested by this assay. The assay combines antibody (anti-human IgA) and lectin and measures the DGalβ(1–3)DGalNAc structure in the IgA₁ molecule accessible to lectin binding. Three lectins were compared–the lectins from Bauhinia purpurea alba, Maclura pomifera, and peanut agglutinin (PNA). PNA in the assay discriminated best between serum specimens from normal subjects and cancer patients and was chosen as the lectin component in the assay. The anti-IgA-PNA enzyme immunoassay was evaluated in a retrospective study of cancer patients. A panel of 845 serum specimens was tested and the results analyzed at three cutoff R values. The R value of a specimen is the ratio of the assay absorbance obtained for that test specimen with respect to the assay absorbance obtained for a pool of normal human sera. At a cutoff R value of 2.38, 1% normals and 26% benigns were elevated. The following cancer specimens had elevated R values: 52% lung, 60% colon, 43% head and neck, 35% breast, 64% kidney, 38% bladder, 43% prostate, 8% ovarian, and 67% pancreas. Further testing showed a significant percentage of serum specimens from patients with cirrhosis, pancreatitis, or ulcerative colitis also had elevated R values. No correlation of this assay was observed with CEA levels. The results suggest that the carbohydrate structure of serum IgA₁ is altered by malignancies and that measurement of this altered IgA may supplement CEA testing.     

Evaluation of precursor prostate-specific antigen isoform ratios in the detection of prostate cancer

OBJECTIVE: Disease-associated isoforms of the prostate-specific antigen (PSA) have recently been identified. We evaluated the efficacy of using precursor isoforms of PSA (pPSA) and their ratios for the detection of prostate cancer. METHODS: Serum concentrations of [-2], [-4], and [-7]pPSA, BPSA, and free PSA (fPSA) were retrospectively measured in 43 selected men. Of the 43 men, 15 had clinical T2 prostate cancer with ultrasound-estimated prostate volumes (PVs) of >50 cm(3), 13 had clinical T2 prostate cancer with (PVs) <25 cm(3), and 15 were prostate cancer-free with PV >50 cm(3). We calculated sum pPSA ([-2]+[-4]+[-7]pPSA). We also compared the ratios of: free/total PSA, [-2]pPSA/fPSA, [-2]pPSA/BPSA, [-2]pPSA/(fPSA-BPSA), [-2]pPSA/(fPSA-sum pPSA), and [-2]pPSA/{fPSA-(sum pPSA+BPSA)} among these three groups. RESULTS: The median [-2]pPSA/(fPSA-sum pPSA) ratio was significantly higher in men with prostate cancer with or without large PV compared with men with large PV without prostate cancer. Values for median [-2]pPSA/free PSA ratio were higher in men with prostate cancer with or without large PV compared with men with large PV, and without prostate cancer, but the differences were not statistically significant. CONCLUSIONS: In this preliminary study, [-2]pPSA/(fPSA-sum pPSA) ratio was not associated with prostate gland volume but was associated with prostate cancer. This ratio may be useful in the detection of prostate cancer, particularly in men with larger glands.     

Comparison of predictive accuracy for pathologically organ confined clinical stage T1c prostate cancer using human glandular kallikrein 2 and prostate specific antigen combined with clinical stage and Gleason grade

PURPOSE: Previously human glandular kallikrein 2 (hK2) has been implicated to predict pathologically organ confined prostate cancer (PCa) in patients with stage T2 disease. Now we evaluated the usefulness of hK2, as measured by 2 entirely different immunoassay designs, to enhance the discrimination of pathologically organ from nonorgan confined clinical stage T1c PCa. MATERIALS AND METHODS: A consecutive series of pretreatment serum from 148 men with clinical stage T1c PCa was used in 2 equally sensitive and specific methods to measure total hK2 with independent reagents and entirely different assay designs. Total prostate specific antigen (tPSA) and free PSA (fPSA) were measured and percent fPSA was calculated. We determined the algorithm, hK2*tPSA/fPSA, from data generated by each hK2 assay, calculated means, medians and ranges for each analyte and algorithm, and calculated the significance of differences on univariate analysis. Using pretreatment PSA, clinical stage and biopsy Gleason grade we then developed a multivariate logistic regression base model to predict organ confined cancer and we compared predictions of the base model supplemented by the different hK2 measurements. RESULTS: hK2 and hK2 based algorithms obtained by each hK2 assay were significantly different for pT2a/b vs pT3a or greater PCa (p = 0.034 to 0.0001) compared to tPSA (p = 0.06), fPSA (p = 0.90) or percent fPSA (p = 0.059). However, AUC (0.67 to 0.70) calculated by ROC analysis of the 4 models containing hK2 derived information was not significantly larger than that of the base model (AUC = 0.64, p = 0.52). CONCLUSIONS: The current data confirm that hK2 alone or hK2*tPSA/fPSA measured by 2 immunoassays is significantly lower in men with pT2a/b vs pT3a or greater PCa compared to tPSA, fPSA or percent fPSA on univariate analysis of a validation set of clinical stage T1c prostate cancer treated at an American center of excellence for prostate cancer surgery. However, the incorporation of preoperative hK2 into multiparameter predictive models for pT2 cancers did not increase predictive accuracy in this cohort of men.     

The preclotting of porous arterial prostheses.

A four-step preclotting method is presented for use with porous filamentous Dacron prostheses in the fully-heparinized patient. The method employs controlled fibrin formation within graft interstices, heparin neutralization of all thrombin remaining in the graft wall, and delay of systemic heparin neutralization until 15--20 minutes after clamp release. The resulting flow surface is impervious, smooth and hypothrombogenic. Experimental data are presented which support the rationale of this four-step preclotting method. Four years of clinical experience with the method are summarized, involving 300 prosthesis limbs used in aortic bifurcation, aortofemoral, femorofemoral, axillary-femoral and femoropopliteal positions in 192 patients. A clinical perspective of preclotting techniques is presented in which the proper use of this new method is suggested.     

Variable sensitivity to bacterial methionyl-tRNA synthetase inhibitors reveals subpopulations of Streptococcus pneumoniae with two distinct methionyl-tRNA synthetase genes

As reported previously (J. R. Jarvest et al., J. Med. Chem. 45:1952-1962, 2002), potent inhibitors (at nanomolar concentrations) of Staphylococcus aureus methionyl-tRNA synthetase (MetS; encoded by metS1) have been derived from a high-throughput screening assay hit. Optimized compounds showed excellent activities against staphylococcal and enterococcal pathogens. We report on the bimodal susceptibilities of S. pneumoniae strains, a significant fraction of which was found to be resistant (MIC, > or =8 mg/liter) to these inhibitors. Using molecular genetic techniques, we have found that the mechanism of resistance is the presence of a second, distantly related MetS enzyme, MetS2, encoded by metS2. We present evidence that the metS2 gene is necessary and sufficient for resistance to MetS inhibitors. PCR analysis for the presence of metS2 among a large sample (n = 315) of S. pneumoniae isolates revealed that it is widespread geographically and chronologically, occurring at a frequency of about 46%. All isolates tested also contained the metS1 gene. Searches of public sequence databases revealed that S. pneumoniae MetS2 was most similar to MetS in Bacillus anthracis, followed by MetS in various non-gram-positive bacterial, archaeal, and eukaryotic species, with streptococcal MetS being considerably less similar. We propose that the presence of metS2 in specific strains of S. pneumoniae is the result of horizontal gene transfer which has been driven by selection for resistance to some unknown class of naturally occurring antibiotics with similarities to recently reported synthetic MetS inhibitors.