Late stage antibacterial drugs in the clinical pipeline

Bacterial resistance to antimicrobial agents is a growing problem worldwide. Not only is issue compounded by the fact that there are fewer pharmaceutical companies conducting research to discover novel antimicrobials than in the past but development time lines have stretched so that a dozen years from discovery to the market is now the standard. Eleven antibacterial drugs in late stage clinical development are discussed. Whereas many of these may successfully deal with resistant strains of Gram-positive pathogens, there is very little in development to address the gorwing unmet medical need of multi-drug resistant Gram-negative infections.     

Specific lysis of GABAergic synaptosomes by an antiserum to glutamate decarboxylase

An antiserum to pure glutamate decarboxylase (GAD) when incubated with rat cortical synaptosomes in the presence of complement caused release of 33-53% of lactate dehydrogenase (LDH) and 22-41% of total GAD. In addition most of the gamma-aminobutyrate (GABA) present was released. Anti-GAD antiserum alone, or complement alone, were without action. The antiserum plus complement had no effect on noradrenaline or choline uptake, and did not release choline acetylase (ChAT). Anti-ChAT serum plus complement released 30-37% of ChAT and 10-13% of LDH. It prevented choline uptake. This serum did not produce GAD release or prevent GABA, choline or noradrenaline uptake. When cortical synaptosomes were exposed to both antisera plus complement, their actions were strictly additive. The data indicate specific lysis of GABAergic and cholinergic synaptosomal sub-populations.     

The two SAMP repeats and their phosphorylation state in Drosophila Adenomatous polyposis coli-2 play mechanistically distinct roles in negatively regulating Wnt signaling

The tumor suppressor Adenomatous polyposis coli (APC) plays a key role in regulating the canonical Wnt signaling pathway as an essential component of the β-catenin destruction complex. C-terminal truncations of APC are strongly implicated in both sporadic and familial forms of colorectal cancer. However, many questions remain as to how these mutations interfere with APC’s tumor suppressor activity. One set of motifs frequently lost in these cancer-associated truncations is the SAMP repeats that mediate interactions between APC and Axin. APC proteins in both vertebrates and Drosophila contain multiple SAMP repeats that lack high sequence conservation outside of the Axin-binding motif. In this study, we tested the functional redundancy between different SAMPs and how these domains are regulated, using Drosophila APC2 and its two SAMP repeats as our model. Consistent with sequence conservation–based predictions, we show that SAMP2 has stronger binding activity to Axin in vitro, but SAMP1 also plays an essential role in the Wnt destruction complex in vivo. In addition, we demonstrate that the phosphorylation of SAMP repeats is a potential mechanism to regulate their activity. Overall our findings support a model in which each SAMP repeat plays a mechanistically distinct role but they cooperate for maximal destruction complex function.     

Invariant chain transmembrane domain trimerization: a step in MHC class II assembly

The transmembrane (TM) domain of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) has long been implicated in both correct folding and function of the MHC class II complex. To function correctly, Ii must form a trimer, and the TM domain is one of the domains thought to stabilize the trimeric state. Specific mutations in the TM domain have been shown previously to disrupt MHC class II functions such as mature complex formation and antigen presentation, possibly due to disruption of Ii TM helix-helix interactions. Although this hypothesis has been reported several times in the literature, thus far no experimental measurements have been made to explore the relationship between TM domain structure and TM mutations that affect Ii function. We have applied biophysical and computational methods to study the folding and assembly of the Ii TM domain in isolation and find that the TM domain strongly self-associates. According to analytical ultracentrifugation analyses, the primary oligomeric state for this TM domain is a strongly associated trimer with a dissociation constant of approximately 120 nM in DPC micelles. We have also examined the effect of functionally important mutations of glutamine and threonine residues in the TM domain on its structure, providing results that now link the disruption of TM helix interactions to previously reported losses of Ii function.     

Extracellular release of the glycosylphosphatidylinositol (GPI)-linked Leishmania surface metalloprotease, gp63, is independent of GPI phospholipolysis: implications for parasite virulence.

The major zinc metalloprotease of Leishmania (gp63), an important determinant of parasite virulence, is attached to the parasite surface via a glycosylphosphatidylinositol anchor. Here we report the spontaneous release of proteolytically active gp63 from a number of Leishmania isolates, causing cutaneous and visceral disease. To investigate the mechanism(s) of gp63 release, we transfected a gp63-deficient variant of Leishmania amazonensis with constructs expressing gp63 and various mutants thereof. Surprisingly, approximately half of wild type gp63 was found in the culture supernatant 12 h post-synthesis. Biochemical analysis of the extracellular gp63 revealed two forms of the protein, one that is released from the cell surface, and another, that apparently is directly secreted. Release of cell surface gp63 was significantly reduced when the proteolytic activity of the protein was inactivated by site-specific mutagenesis or inhibited by zinc chelation, suggesting that release involves autoproteolysis. The extracellular gp63 does not contain a glycosylphosphatidylinositol moiety or ethanolamine, indicating that phospholipolysis is not involved in the release process. Release of gp63 is also independent of glycosylation. The finding of proteolytically active, extracellular gp63 produced by multiple Leishmania isolates suggests a potential role of the extracellular enzyme in substrate degradation relevant to their survival in both the mammalian host and the insect vector.     

Glycogen concentrations and endurance capacity of rats with chronic heart failure

The endurance capacities of rats with myocardial infarctions (MI) and of rats having undergone sham operations (SHAM) were tested during a submaximal exercise regimen that consisted of swimming to exhaustion. During this test, a decrement in the endurance capacity of the MI rat was demonstrated as the SHAM rat swam 25% longer than the MI rat (65 +/- 4 vs. 52 +/- 4 min). Glycogen concentrations were measured in the liver and the white gastrocnemius, plantaris, and soleus muscles of SHAM and MI rats that were randomly divided into four subgroups, which consisted of resting control, swim to exhaustion, swim to exhaustion + 24 h recovery, and swim to exhaustion + 24 h recovery + a second swim to exhaustion. The results demonstrated that the glycogen concentrations found in the liver, white gastrocnemius, plantaris, and soleus muscles of the SHAM and MI rats belonging to the resting control groups were similar. After swimming to exhaustion the glycogen concentrations in these tissues were significantly reduced compared with those found in the resting control groups of rats, and after 24 h of recovery the glycogen concentrations in these tissues were again similar to those found in the resting control groups of rats. Since the magnitude of the glycogen depletion in the liver and the white gastrocnemius, plantaris, and soleus muscles was similar in the SHAM and MI rats and because the SHAM rats consistently swam for longer periods of time in each of the experimental groups, it would be logical to assume that the rates of glycogen utilization for the various tissues may have been greater in the MI rat during exercise.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exposure of sink drain microcosms to triclosan: population dynamics and antimicrobial susceptibility

Recent concern that the increased use of triclosan (TCS) in consumer products may contribute to the emergence of antibiotic resistance has led us to examine the effects of TCS dosing on domestic-drain biofilm microcosms. TCS-containing domestic detergent (TCSD) markedly lowered biofouling at 50% (wt/vol) but was poorly effective at use levels. Long-term microcosms were established and stabilized for 6 months before one was subjected to successive 3-month exposures to TCSD at sublethal concentrations (0.2 and 0.4% [wt/vol]). Culturable bacteria were identified by 16S rDNA sequence analysis, and their susceptibilities to four biocides and six antibiotics were determined. Microcosms harbored ca. 10 log(10) CFU/g of biofilm, representing at least 27 species, mainly gamma proteobacteria, and maintained dynamic stability. Viable cell counts were largely unaffected by TCSD exposure, but species diversity was decreased, as corroborated by denaturing gradient gel electrophoresis analysis. TCS susceptibilities ranged widely within bacterial groups, and TCS-tolerant strains (including aeromonads, pseudomonads, stenotrophomonads, and Alcaligenes spp.) were isolated before and after TCSD exposure. Several TCS-tolerant bacteria related to Achromobacter xylosoxidans became clonally expanded during dosing. TCSD addition did not significantly affect the community profiles of susceptibility to the test biocides or antibiotics. Several microcosm isolates, as well as reference bacteria, caused clearing of particulate TCS in solid media. Incubations of consortia and isolates with particulate TCS in liquid led to putative TCS degradation by the consortia and TCS solubilization by the reference strains. Our results support the view that low-level exposure of environmental microcosms to TCS does not affect antimicrobial susceptibility and that TCS is degradable by common domestic biofilms.     

Range maps and species richness patterns: errors of commission and estimates of uncertainty

Range maps are often combined into “range overlap maps” to estimate spatial variation in species richness. Range maps are, in most cases, designed to represent a species’ maximum geographical extent and not patterns of occupancy within the range. As a consequence, range maps overestimate occupancy by presenting false occupancy (errors of commission) within the interior of the range. To assess the implications of errors of commission when developing and applying range overlap maps, we used neutral landscapes to simulate range maps and patterns of occupancy within ranges. We explored several scenarios based on combinations of six parameters defining biogeographical and cartographic factors typically encountered by investigators. Our results suggest that, in general, uncertainty is lowest when map resolutions are moderately fine, the majority of species have geographically restricted ranges, species occur throughout their range, patterns of occupancy within the range are not correlated among species, and geographically local and widespread species tend to occupy different regions. Several of these outcomes are associated with broad geographical extents, the scale at which range overlap maps are typically applied. Thus, under most circumstances, reasonably accurate and precise representation of species richness patterns can be achieved. However, these representations can be improved by enhancing occupancy data for widespread species – a primary source of uncertainty – and selecting a map resolution that captures relevant biological and environmental heterogeneity. Hence, by determining where a study is situated within the scenarios examined in our simulations, uncertainty and its sources and implications can be ascertained. With this knowledge, research goals, methods, and data sources can be reassessed and refined and, in the end, conclusions and recommendations can be qualified. Alternatively, unique regional, taxonomic, or cartographic factors could be included in future simulations to provide direct assessments of uncertainty.