Clindamycin binding to ribosomes revisited: foot printing and computational detection of two binding sites within the peptidyl transferase center

Clindamycin is a semi-synthetic lincosamide, active against most Gram-positive bacteria and some protozoa. It binds to the 50S ribosomal subunit and inhibits early peptide chain elongation. By kinetic analysis it has been shown that clindamycin (I) competitively interacts with the A-site of translating ribosomes (C) to form the encounter complex Cl, which then slowly isomerizes to a tighter complex, termed C*I. As the final complex is capable of synthesizing peptide bonds with decreased velocity, it was assumed that in C*I complex the drug is fixed near the P-site of the ribosome. In the present study, two series of chemical foot printing experiments were carried out. In the first series, clindamycin and ribosomal complex C were incubated for 1 s and then DMS or kethoxal was added (Cl probing). In the second series, complex C was preincubated with clindamycin for 1 min before the addition of DMS or kethoxal (C*I probing). It was found that clindamycin in Cl complex protects A2451 and A2602 from chemical probing, both located within the A-site of the catalytic center. In contrast, it strongly protects G2505 in C*I complex, which is a discrete foot print of peptidyl-tRNA bound to the P-site. In both Cl and C*I complexes, clindamycin also protects nucleotides A2058 and A2059, located next to the entrance of the exit-tunnel where the nascent peptide leaves the ribosome. Polyamines negatively affect the protection of G2505, but favor the protection of A2451 and A2602 nucleotides. Structure modeling confirms the kinetic and chemical foot printing results and suggests that clindamycin mode of action is more complex than a simple competitive inhibition of peptide bond formation

Conjugation with polyamines enhances the antibacterial and anticancer activity of chloramphenicol

Chloramphenicol (CAM) is a broad-spectrum antibiotic, limited to occasional only use in developed countries because of its potential toxicity. To explore the influence of polyamines on the uptake and activity of CAM into cells, a series of polyamine-CAM conjugates were synthesized. Both polyamine architecture and the position of CAM-scaffold substitution were crucial in augmenting the antibacterial and anticancer potency of the synthesized conjugates. Compounds 4 and 5, prepared by replacement of dichloro-acetyl group of CAM with succinic acid attached to N4 and N1 positions of N-8,N-8-dibenzylspermidine, respectively, exhibited higher activity than CAM in inhibiting the puromycin reaction in a bacterial cell-free system. Kinetic and footprinting analysis revealed that whereas the CAM-scaffold preserved its role in competing with the binding of aminoacyl-tRNA 3'-terminus to ribosomal A-site, the polyamine-tail could interfere with the rotatory motion of aminoacyl-tRNA 3'-terminus toward the P-site. Compared to CAM, compounds 4 and 5 exhibited comparable or improved antibacterial activity, particularly against CAM-resistant strains. Compound 4 also possessed enhanced toxicity against human cancer cells, and lower toxicity against healthy human cells. Thus, the designed conjugates proved to be suitable tools in investigating the ribosomal catalytic center plasticity and some of them exhibited greater efficacy than CAM itself

Research into Teaching and Learning of Tertiary Mathematics and Statistics

Reviewed in this chapter is the growing depth and variety of research being undertaken in the tertiary mathematics field. In particular, the scope of the research and issues being examined have been highlighted in sections: Tertiary mathematics education. The focus is strategies for teaching and learning in mathematics education and the professional learning of the lecturers and tutors. Mathematical content. Papers reviewed are those that focus on certain content areas in tertiary mathematics, both practical and theoretical. Tertiary statistics education. The authors reviewed the small but growing area of research that examines the teaching and learning of statistics, content areas of statistics education and some of the innovations being used in this area of teaching and learning. Transitions and support. The focus is school to university transition and the support structures being implemented to provide academic support for undergraduate students of mathematics. Service teaching. The authors consider papers that link mathematics into service areas such as engineering and the health sciences