Virulence blockers as alternatives to antibiotics: type III secretion inhibitors against Gram-negative bacteria.

In recent years mounting problems related to antibiotic-resistant bacteria have resulted in the prediction that we are entering the preantibiotic era. A way of preventing such a development would be to introduce novel antibacterial medicines with modes of action distinct from conventional antibiotics. Recent studies of bacterial virulence factors and toxins have resulted in increased understanding of the way in which pathogenic bacteria manipulate host cellular processes. This knowledge may now be used to develop novel antibacterial medicines that disarm pathogenic bacteria. The type III secretion system (T3SS) is known to be a potent virulence mechanism shared by a broad spectrum of pathogenic Gram-negative bacteria that interact with human, animal and plant hosts by injecting effector proteins into the cytosol of host cells. Diseases, such as bubonic plague, shigellosis, salmonellosis, typhoid fever, pulmonary infections, sexually transmitted chlamydia and diarrhoea largely depend on the bacterial proteins injected by the T3SS machinery. Recently a number of T3SS inhibitors have been identified using screening-based approaches. One class of inhibitors, the salicylidene acylhydrazides, has been subjected to chemical optimization and evaluation in several in vitro and ex vivo assays in multiple bacterial species including Yersinia spp., Chlamydia spp., Salmonella spp. and Pseudotuberculosis aeruginosa. Reports published up to date indicate that T3SS inhibitors have the potential to be developed into novel antibacterial therapeutics.

[An entire rain forest can be screened at pharmaceutical industry's laboratories]. / Hel regnskog kan screenas på läkemedelsbolagets laboratorium.

The pharmaceutical industry has long been heavily reliant on natural products, and today more than half of the twenty best-selling pharmaceuticals are derived from natural sources. Hitherto sample collection from fauna and flora has been based on an ethnobotanical approach involving traditional healers and oral histories of indigenous peoples as sources of information on folk uses of plants and organisms. In the future, however, random sampling combined with automated high-throughput screening (HTS) may come to the forefront in drug design, enabling extensive libraries of active compounds to be built up, and rendering local knowledge largely irrelevant. New technological advances in chemistry, molecular biology and data processing are combined in automated systems whereby HTS, based on bioassay-guided fractionation procedures, is used to isolate active compounds, and enabling the chemical structure of isolated compounds to be determined within 24 hours. The combination of the speed of HTS, and a remarkable decrease in the amount of sample required for the isolation and structure determination of natural compounds, has improved our ability to find unique natural products for drug development. Moreover, it has already been demonstrated that, owing to the relatively minute amounts of material required for HTS, this random sampling approach to bioprospecting for the purposes of drug will benefit efforts to conserve such sensitive biosystems as the rain forests and marine ecosystems. Thus, this technology would seem to be more compatible with the needs of modern drug design in the pharmaceutical industry that the ethnobotanical approach.

Increased potency of antagonists of substance P having asparagine in position 6.

The general structure of antagonists of substance P (SP) which was found with the development of Spantide and analogs based on Spantide served for further refinement. The antagonistic potency was tested in vitro on guinea pig ileum and taenia coli. It was unexpectedly found that introduction of Asn6 gave rise to a considerable increase in potency. The exchange of Gln6 for Asn6 entails the shortening of the side chain by one CH2 unit and seems slight for steric advantages and potency increase. The analog [D-Arg1,D-Cl2Phe5,Asn6,D-Trp7,9,Nle11]SP had pA2 values of 7.4 (ileum) and 8.0 (taenia coli). We then used this sequence as a new lead to introduce new changes, which were made in positions 1, 3, 5, 7 and 9. It was found that Arg1 is important, but Lys3 can be exchanged. The Pal3 derivative had pA2 values of 8.1 and 8.0 and the Nle3 counterpart had 7.7 and 7.4 D-Cl2Phe is an effective substituent in position 5. D-Trp in positions 7 and 9 were superior to other alternatives.

Design and synthesis of antagonists of substance P.

Synthesis and bioassay of about 65 analogs of substance P (SP) over five years yielded the antagonist [D-Arg1,D-Trp7,9,Leu11]-SP, which was named Spantide, and which was used by many investigators as a "tool". Spantide served as a reference antagonist for the design of 47 new peptides toward the goal of more potent inhibitors. Designs emphasized analogs with D-Trp7, D-Trp9, D-Trp10, D-pClPhe10, Nle11, Leu11, Ile11 and Met11, etc. Twenty-one/47 antagonists were superior in potency to that of Spantide, the best was [D-Arg1,D-Na1(5), D-Trp7,9,Nle11]-SP which required a 255-fold increase in SP concentration to give 50% of the maximum response at a concentration of 10(-5)M of the antagonist; this potency is ca. 5 times that of Spantide. For certain, but not all pairs of undecapeptides and truncated analogs, the undecapeptides may be significantly more potent than the truncated counterparts.

Tachykinin multiplicity in rat central nervous system as studied using antisera raised against substance P and neurokinin A.

Antisera were raised in rabbits against the tachykinins neurokinin A (NKA) and substance P (SP). All NKA-antisera tested cross-reacted markedly with NKB, kassinin and eledoisin in radioimmunoassay (RIA), but virtually not with SP and physalaemin. Also when used for immunohistochemistry, one of the NKA-antisera was found to be virtually without cross-reactivity with SP. The most specific SP-antiserum did not cross-react with NKA but to some extent with NKB at the immunohistochemical level. Using these two antisera, the same distribution pattern of immunoreactivity was seen in both the rat substantia nigra and dorsal spinal cord. In neutral extracts of the substantia nigra, all NKA-antisera used for RIA detected a major component which eluted at the position of NKA in reverse phase high performance liquid chromatography, while no or only little immunoreactivity was detected at the position of NKB. A major component of substance P-like immunoreactivity (SPLI) co-eluting with SP and one or two minor SPLI-components were also detected in these extracts. An SP-antiserum, which cross-reacted markedly with physalaemin, detected an additional rather prominent component. In neutral water extracts of dorsal spinal cord the component detected with the NKA-antisera at the position of NKB, as well as one of the SPLI-components not eluting in the position of SP, were much more prominent than in the corresponding extracts of substantia nigra. In acetic acid extracts of both tissues, only one major SPLI-component co-eluting with SP could be detected, while only very small amounts of immunoreactivity eluting at the position of NKA and NKB (dorsal spinal cord only) could be detected using the NKA-antisera. The present results illustrate the importance of the extraction method used in immunochemical studies and demonstrate that the relative proportions of various tachykinins are markedly different in the rat substantia nigra and dorsal spinal cord.

Neurotensin increases colonic motility.

The effects of neurotensin on colonic motility were investigated in 6 healthy volunteers (rectosigmoid area) and 7 patients (ascending colon and at the splenic flexure). Neurotensin (12 pmol/kg X min) infused intravenously for 30 min increased the duration of the contractions to 76% in the ascending colon and 46% in the rectosigmoid area. In the postinfusion period, the values were 42% and 67%, respectively. The motor activity did not change significantly at the splenic flexure. During the infusion period, the motility index increased from 870 to 4500 in the ascending colon and from 332 to 1700 in the rectosigmoid area. In the rectosigmoid area, however, a statistically significant increase was recorded first after cessation of the infusion. All subjects reported increased sensation of intestinal movement after intravenous infusion of neurotensin, and the patients discharged a median volume of 600 ml of bowel contents 20 min after cessation of the infusion. The data show that neurotensin causes an increase in colonic motility in the ascending colon of patients and also, after a latent period, in the rectosigmoid area of healthy subjects.

The effect of nephrectomy and ureter ligation on plasma neurotensin-like immunoreactivity levels in the conscious rat.

The effect of nephrectomy and ureter ligation on the concentration of endogenous plasma neurotensin-like immunoreactivity (p-NTLI) was studied in conscious rats by means of antiserum 17-8201 which detects NT (1-13) only, and antiserum 0-7709 which detects NT(1-13) and NT(1-8). The unstimulated p-NTLI concentration did not change significantly during a 25-h observation period following nephrectomy in comparison with sham operation. However, stimulation of the release of NTLI by intraduodenal administration of oleic acid (0.2 ml) resulted in significantly higher p-NTLI levels in the nephrectomized rats than in the sham operated rats. Ureter ligation did not significantly affect basal or stimulated p-NTLI. The data indicate that the kidneys play an important part in the elimination of p-NTLI released after fat ingestion.

Dexamethasone reduces fat-induced release of neurotensin in the rat.

The effect of glucocorticoids on the release of neurotensin (NT) was studied in the rat. Oleic acid (200 microliters) was instilled into the duodenum of fasted rats given saline or 1 mg . kg-1 dexamethasone subcutaneously 16 h prior to the experiment. Neurotensin-like immunoreactivity (p-NTLI) was analysed in unextracted plasma with the C-terminal directed NT antiserum 6-8206. Pretreatment with dexamethasone almost totally abolished the fat-induced increase in p-NTLI. The data suggest that glucocorticoids may modulate the post-prandial release of NT. Since NT is known to constrict the vascular beds of subcutaneous adipose tissue, this effect of glucocorticoids may be one of the factors responsible for the abnormal distribution of fat caused by high levels of glucocorticoids, e.g. in Cushing's syndrome.

Antisera raised against eledoisin and kassinin detect elevated levels of immunoreactive material in plasma and tumor tissues from patients with carcinoid tumors.

Radioimmunoassays based on antisera raised against the tachykinins eledoisin (antiserum E7) and kassinin (antiserum K12) were used to measure the concentration of tachykinin-like immunoreactivity (TKLI) in plasma from 52 healthy subjects. 65 patients with carcinoid tumors (of which 46 had symptoms of both flushing and diarrhoea), and 6 patients with endocrine pancreatic tumors. The antisera did not crossreact with substance P (SP). Elevated concentrations of TKLI, as compared with healthy subjects, were found in 75% of the carcinoid patients, but in none of the patients with pancreatic tumors. Tumor metastases from 8 of the carcinoid patients all contained TKLI. Ion-exchange chromatography of plasma samples and tumor tissue extracts indicated the presence of several immunoreactive molecular forms. The elution patterns of the immunoreactivity detected by antisera E7 and K12 were similar, indicating that the same molecular species are measured by these antisera. None of the components coeluted with synthetic SP. One of the immunoreactive components in carcinoid tumor extracts coeluted with synthetic NKA. The major immunoreactive components in plasma from the patients eluted in a position different from that of all currently known mammalian tachykinins. Tachykinin immunoreactive material detected in tumor tissue and plasma of patients with carcinoid tumor may play a role in the symptomatology of the carcinoid syndrome.

Antisera raised against eledoisin and kassinin detect immunoreactive material in rat tissue extracts: tissue distribution and chromatographic characterization.

Radioimmunoassays were developed for the tachykinins eledoisin (ELE) and kassinin (KAS) using antisera raised in rabbits. The antisera exhibited low (less than 0.1%) cross-reactivities to substance P (SP) and physalaemin (PHY), but crossreacted (with one exception, antiserum K7) to varying extents with neurokinin A (NKA) and neurokinin B (NKB). In the rat, the tissue distribution of the immunoreactive material detected by antiserum (E7) raised against ELE and by another antiserum (K1) raised against KAS both resembled that previously described for SP. Using the highly KAS-specific antiserum K7, no or only very low levels of immunoreactivity could be detected in extracts of various rat tissues. Gel permeation chromatography and ion-exchange chromatography of tissue extracts indicated that all antisera (except K7) detected the same population of immunoreactive molecules. One of the components was chromatographically indistinguishable from NKA. The tissue distribution of this component also resembled that of SP. Another immunoreactive component co-chromatographed with NKB at cation exchange chromatography. Acid tissue extracts, but not neutral tissue extracts, were found to contain immunoreactive components which appeared more basic than NKA and NKB. The total levels of immunoreactivity were higher in neutral than in acid tissue extracts. However, the ratio between the amounts of immunoreactivities in the two types of extracts varied considerably between tissues, indicating that tachykinin immunoreactive components may be present in different relative proportions in various tissues.

Nervous control of the release of neurotensin-like immunoreactivity from the small intestine of the rat.

Intraduodenal administration of oleic acid increased plasma neurotensin-like immunoreactivity (p-NTLI). The integrated responses to saline and oleic acid were 5.7 and 9.7 nM0-180 min, respectively. The integrated response was not significantly altered by i.v. administration of atropine, guanethidine, mepyramine, cimetidine, methysergide or a substance P antagonist, but it was abolished by hexamethonium and morphine (5.9 and 6.3 nM0-180 min, respectively). An exogenous supply of bile and pancreatic juice did not alter the integrated response in morphine- and hexamethonium-treated rats. Haloperidol significantly increased the p-NTLI response to oleic acid (13 nM0-180 min). The results suggest that the release of neurotensin is influenced by nervous pathways involving nicotinic and opioid receptors. Catecholamines and 5-HT receptors may exert an inhibitory influence on the release of NTLI.

Cigarette smoking potentiates fat-induced elevation of neurotensin-like immunoreactivity in human plasma.

The aim of the present study in man was to investigate the influence of cigarette smoking on basal plasma concentrations of neurotensin-like immunoreactivity (p-NTLI), as well as on fat-induced elevation of p-NTLI levels. On three separate occasions, habitual tobacco smokers either ingested fat (55 ml Intralipid 20%) or smoked three cigarettes, or ingested fat and smoked. The subjects were fasted overnight and told no to smoke during the fasting period. Smoking of one cigarette at one hour intervals (0, 60, and 120 min) did not change the p-NTLI levels (n = 6). Ingestion of fat caused a significant increase (n = 16) and when these two procedures were combined (n = 16), the mean integrated p-NTLI response (nM0-180 min) was 56% larger than the response after fat ingestion alone. The results show that cigarette smoking potentiates the fat-induced elevation of p-NTLI levels, whereas cigarette smoking per se has no effect.

Influence of fasting and bombesin-induced myoelectric activity on the transit of small-intestinal contents in the rat.

The relation between the occurrence of the migrating myoelectric complexes (MMC) and the transit of small-intestinal contents was studied in fasted, conscious rats. MMC were monitored by means of three bipolar electrodes implanted along the small intestine 5, 20, and 35 cm distal to the pylorus. In the presence of an MMC, the radioactive marker was transported along the small intestine as one main peak and recovered aboral to the activity front. Intravenous infusion of bombesin, 3 pmol X kg-1 X min-1, disrupted the MMC and induced irregular spiking activity at all recording levels. Furthermore, during bombesin infusion the radioactive marker was propelled a considerable distance, although the transit was significantly retarded in comparison to that in the presence of an MMC (p less than 0.01). The results indicate that the small-intestinal contents are propelled aboral to an activity front. Continuous irregular spiking activity during bombesin infusion contributes less to the transit of the small-intestinal contents than the activity front of MMC observed during fasting.

Effect of intravenous and intrajejunal fat infusion on gastric acid secretion and plasma neurotensin-like immunoreactivity in man.

The effects of intrajejunal and intravenous infusion of fat (100 ml Intralipid 20%) on pentagastrin-stimulated gastric acid secretion and on the concentrations of plasma neurotensin-like immunoreactivity (p-NTLI) were studied in eight healthy subjects. Gastric acid secretion was studied for a 1.5-h period at 15-min intervals after fat administration. Acid secretion was reduced by 58% and 36%, respectively, after intrajejunal and intravenous administration. The concentration of p-NTLI rose significantly from 29 to 177 pmol/l at 45 min after intrajejunal administration but did not change after intravenous infusion. The results indicate that large volumes of fat inhibit gastric acid secretion by a neurotensin-independent postabsorptive mechanism and by an intestinal mechanism that may in part be mediated by neurotensin.

Synthesis and biological activities of neurokinin alpha and beta.

Two novel neuropeptides, neurokinin alpha and beta isolated from porcine spinal cord, were announced. We have synthesized neurokinin alpha as Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2, which were 98-99% pure by HPLC. Assays on the isolated guinea pig ilium showed neurokinin alpha to have 81% and neurokinin beta to have 65% of the activity of Substance P. Knowledge of these three related peptides having a common activity opens new considerations of their intrinsic physiological roles in neurotransmission versus pharmacological activities, and reappraisal of the diverse activities of Substance P including that in the inflammatory response of the eye.