Turning promiscuous kinase inhibitors into safer drugs

Burgeoning interest in multi-target drugs to treat complex diseases and malignancies has motivated a reassessment of the therapeutic value of promiscuity. Although drug efficacy might not correlate with specificity, it would be risky to welcome promiscuous compounds without a rational strategy to control therapeutic impact. This situation might change as novel selectivity filters are incorporated into drug design. For example, cardiotoxic side effects of the cancer drug imatinib might be curbed by applying such premises. Here, we survey approaches to control the therapeutic impact of cross-reactive kinase inhibitors and advocate the application of a novel selectivity filter by illustrating its cleaning efficacy. Finally, we evaluate the possibility of turning multi-target kinase inhibitors into clinical opportunities through judicious chemical modifications.     

CylA is a sequence-specific protease involved in toxin biosynthesis

Journal of Industrial Microbiology & Biotechnology - CylA is a subtilisin-like protein belonging to a recently expanded serine protease family related to class II lanthipeptide biosynthesis. As...     

Investigations into,and development of,a lyophilized and formulated recombinant human factor IX produced from CHO cells

Objectives

To develop a recombinant human factor IX (rFIX) formulation equivalent to commercially available products in terms of cake appearance, residual moisture, proportion of soluble aggregates and activity maintenance for 3 months at 4–8 °C.

Results

NaCl and low bulking agent/cryoprotectant mass ratio had a negative impact on cake quality upon lyophilisation for a wide range of formulations tested. Particular devised formulations maintained rFIX activity after lyophilization with a similar performance when compared with the rFIX formulated using the excipients reported for a commercially available FIX formulation (Benefix). rFIX remained active after 3 months when stored at 4 °C, though this was not the case with samples stored at 40 °C. Interestingly, particular formulations had an increase in residual moisture after 3 months storage, but not above a 3% threshold. All four formulations tested were equivalent to the Benefix formulation in terms of particle size distribution and cake appearance.

Conclusions

Three specific formulations, consisting of surfactant polysorbate-80, sucrose or trehalose as cryoprotectant, mannitol or glycine as bulking agent, l-histidine as buffering agent, and NaCl added in the reconstitution liquid at 0.234% (w/v) were suitable for use with a CHO cell-derived recombinant FIX.

     

A promiscuous aminoacyl-tRNA synthetase that incorporates cysteine, methionine, and alanine homologs into proteins

A mutant Escherichia coli leucyl-tRNA synthetase has been evolved for the selective incorporation of the methionine homolog 1 into proteins in yeast. This single aminoacyl-tRNA synthetase is capable of charging an amber suppressor EctRNA(CUA)(Leu) with at least eight different amino acids including methionine and cysteine homologs, as well as straight chain aliphatic amino acids. In addition we show that incorporation yields for these amino acids can be increased substantially by mutations in the editing CP1 domain of the E. coli leucyl-tRNA synthetase.     

Molecular analysis of a promiscuous, fluctuating mating system

The Soay sheep population of St. Kilda fluctuates widely in population size and sex ratio, so diat the level of male-male competition for mates varies from one rut to the next. In this paper we investigate variation in individual male breeding success in relation to age and population size at the rut, and its outcome in terms of lifetime breeding success. Since both sexes are promiscuous, and census-based behavioural data do not predict paternity, we conducted the whole analysis on breeding success derived by molecular techniques. We assumed that every male living in our study area during the rut (N= 68–294 in different years) was a candidate father for each subsequent lamb, and used the parentage inference software CERVUS 1.0, applied to up to 17 allozyme and microsatellite loci, to infer paternity at 95% and 80% confidence. Using 945 paternities assigned at 80% confidence, we show that juvenile rams (aged 7 months) and yearling rams (aged 19 months) regularly obtained paternities and that mean individual breeding success varied inversely with levels of competition in the rut for all age classes of ram. The proportion of young (juvenile and yearling) and adult rams gaining one or more paternities showed similar variation with population size, but the sibship size sired by young and adult breeders showed different patterns: adult rams sired larger sibships at low population size, while the size of sibships sired by young rams was small across all population sizes. Variable breeding success by young rams approximately halved the estimated coefficient of variation in lifetime breeding success of Soay rams.     

Investigations into rabies II

Antonie van Leeuwenhoek -     

Ficin E,a serine-centred protease from Ficus elastica

A protease of M, 50 000 was purified from the latex of Ficus elastica. The enzyme has a pI of 3.7 and pH optimum at 6.0. Its activity is dependent on serine and histidine residues. The amino acid composition is reported.     

Euphorbain p,a serine protease from euphorbia pulcherrima

A serine protease named euphorbain p has been isolated in a homogeneous state from the latex of Euphorbia pulcherrima. This multi-chain enzyme, MW 74000, is similar in composition to one in E. lathyris, but is larger in size and has a more restricted activity. It has a pI of 4.7, and displays maximum activity at pH 7.0.     

Ingensin, a high-molecular-mass alkaline protease from rabbit reticulocyte

A high-molecular-mass protease, ingensin, was purified to homogeneity from rabbit reticulocytes by DEAE-cellulose, HPLC gel filtration, and hydroxyapatite chromatographies. By these procedures, ingensin activity was separated from the activities of two other unique aminopeptidases, one of which is activated by ATP. Ingensin had the following properties: the optimum activity was seen around pH 9.0 and at 50 degrees C; addition of 0.04% SDS and 1 mg/ml linoleic acid resulted in 8- and 4-fold increases in peptide-hydrolyzing activity, respectively. The molecular mass was found to be 700,000 +/- 100,000 daltons on gel filtration, but SDS electrophoresis revealed that the enzyme is composed of several subunits with molecular weights of less than 35,000. The N-terminal-blocked tyrosine- and arginine-MCA derivatives, but not Arg-MCA, were hydrolyzed rapidly by ingensin. The approximate Km values for the reaction of ingensin with Suc-Leu-Leu-Val-Tyr-MCA and Z-Ala-Arg-Arg-MCA were 0.32 and 0.12 mM, respectively. The degradation of several proteins in the reticulocyte extract was stimulated by the addition of SDS and linoleic acid. The activator concentrations necessary for stimulation of the protein hydrolysis are similar to those of the purified reticulocyte ingensin for synthetic substrates. Ingensin did not associate with either right-side-out or inside-out red cell membranes. These results suggest that ingensin is a cytosolic fatty acid-stimulated protease, which is involved in the protein turnover in reticulocyte extracts.     

Effects of a protease inhibitor on biosynthesis of Escherichia coli proteins.

A protease inhibitor, tosyl-L-lysine chloromethylketone, altered the composition of newly synthesized protein in Escherichia coli; a significant portion of the proteins synthesized sedimented rapidly, presumably forming aggregates of the abnormal proteins. Protein I of the outer membrane was only slightly synthesized under these conditions.     

Insights into protein biosynthesis from structures of bacterial ribosomes

Understanding the structural basis of protein biosynthesis on the ribosome remains a challenging problem for cryo-electron microscopy and X-ray crystallography. Recent high-resolution structures of the Escherichia coli 70S ribosome without ligands, and of the Thermus thermophilus and E. coli 70S ribosomes with bound mRNA and tRNAs, reveal many new features of ribosome dynamics and ribosome-ligand interactions. In addition, the first high-resolution structures of the L7/L12 stalk of the ribosome, responsible for translation factor binding and GTPase activation, reveal the structural basis of the high degree of flexibility in this region of the ribosome. These structures provide groundbreaking insights into the mechanism of protein synthesis at the level of ribosome architecture, ligand binding and ribosome dynamics.     

Bioengineering heterodimeric cytokines: turning promiscuous proteins into therapeutic agents

The interleukin 12 (IL-12) family comprises a group of heterodimeric cytokines that can cope with a great variety of immune conditions as the microenvironment demands. By sharing cytokine and receptor subunits, IL-12 (comprised of p40/p35 subunits), IL-23 (p40/p19), IL-27 (p28/EBI3), and IL-35 (p35/EBI3) represent, as a whole, a highly versatile system participating in controlling the continuum from inflammation to tolerance. Promiscuity, a peculiar feature of those cytokines, is a powerful and economic means of producing individual factors with distinct activities via different combinations of a single set of subunits. Whereas IL-12 and IL-23 have a clearly dominant immunostimulatory functional profile and IL-35 is a potent immunosuppressive agent, IL-27 can exert both adjuvant and regulatory effects, depending on the cytokine milieu. Promiscuity itself, however, may significantly hamper the therapeutic use of heterodimeric cytokines. The subunits of a recombinant cytokine, when administered in its native form, will rapidly dissociate in vivo and reassociate with alternative partners, thus generating different heterodimeric or even homodimeric molecules (i.e., p40/p40) with unwanted effects. As in other areas, bioengineering has provided a formidable tool to overcome the constraints associated with the potential use of IL-12 family cytokines. The generation of several gene constructs expressing IL-12, IL-23, IL-27, IL-35, or even the homodimer p40/p40, in their monomerized, single-chain form has allowed us to unveil the efficacy of those molecules in several experimental settings, including neoplasia, viral infection, chronic inflammation, allergy and autoimmunity. Although work is still needed to obtain an overall picture of therapeutic vs. adverse effects of individual molecules before any use in humans, the new frontiers of bioengineering are now driving the production of completely new combinations of cytokine subunits that may further extend the potential clinical use of such eclectic proteins.     

Extracellular protease from Bacillus coagulans,a psychrotrophic,Antarctic bacterium

Bacillus coagulans, when grown on casein at 20°C, produced an inducible, metalloprotease of 28 kDa at 1.6 U/mg cell protein. (NH₄)₂SO₄ at 2 g/l decreased enzyme production irrespective of carbon source.The authors are with the Defence R & D Establishment, Tansen Road, Gwalior-474 002, India.     

Sexually transmitted disease in a promiscuous insect, Adalia bipunctata

Abstract.

• 1 Sexually transmitted diseases (STDs) have rarely been reported in insects and other invertebrates. The majority of those reported involve organisms where sexual transmission is augmented by either vertical (i.e. inherited) transmission, or horizontal transmission, independent of host sexual activity.
• 2 We here demonstrate the existence of an STD in the coccinellid beetle Adalia bipunctata. This species bears a parasitic mite of the genus Coccipolipus. We show that, like many other podapolipid mites, this mite is transmitted between host individuals at a high rate during copulation. It also appears to be transmitted at a low rate between non-copulating individuals.
• 3 We show that infected female A.bipunctata produced eggs at a reduced rate, and that the eggs produced by infected females have highly decreased viability. However, no effect of infection upon host longevity was observed.
• 4 The results are discussed in relation to the incidences of sexually transmitted disease in invertebrates in general, the causes of disease symptoms, and the importance of this disease in the evolution of A.bipunctata.

     

Biosynthesis of Astacus protease, a digestive enzyme from crayfish

For the first time, the site of biosynthesis of a well characterized invertebrate digestive enzyme is localized. The enzyme chosen, Astacus protease, is a zinc-metalloenzyme occuring in high concentration in the gastric fluid of the freshwater crayfish Astacus astacus. Enzyme production was stimulated in adult crayfish either by feeding or by removal of the gastric fluid. Immunohistochemistry, cytology and investigation with radioactive tracers demonstrate that in the hours following stimulation, new enzyme was produced in the F-cells of the midgut gland and subsequently discharged into the midgut gland lumen. The enzyme was then accumulated and stored extracellularly in the cardiac stomach in active form. The mechanism of enzyme production observed in Astacus differs considerably from vertebrates suggesting an alternative model for synthesis and storage of digestive enzymes.     

Biosynthesis of Astacus protease,a digestive enzyme from crayfish

Summary For the first time, the site of biosynthesis of a well characterized invertebrate digestive enzyme is localized. The enzyme chosen, Astacus protease, is a zinc-metalfoenzyme occuring in high concentration in the gastric fluid of the freshwater crayfish Astacus astacus. Enzyme production was stimulated in adult crayfish either by feeding or by removal of the gastric fluid. Immunohistochemistry, cytology and investigation with radioactive tracers demonstrate that in the hours following stimulation, new enzyme was produced in the F-cells of the midgut gland and subsequently discharged into the midgut gland lumen. The enzyme was then accumulated and stored extracellularly in the cardiac stomach in active form. The mechanism of enzyme production observed in Astacus differs considerably from vertebrates suggesting an alternative model for synthesis and storage of digestive enzymes.Supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to V.S./R.Z. (Sto 75/10) and to W.S./R.Z. (Sto 185/1-1)