Edoardo Sarubbi

Peptide aldehydes as inhibitors of HIV protease

We have recently shown that α‐MAPI, a peptidic aldehyde of microbial origin, inhibits the HIV protease with a potency comparable to pepstatin, having, differently from pepstatin, no activity on other aspartic proteases. In this study different peptide derivatives containing a C‐terminal aldehyde have been tested to assess the potential of this function for the inhibition of HIV protease. The results of our analysis correspond with the recently published subsite preferences of the viral enzyme, indicating that aldehydes bind to the active site of the HIV protease. Our data suggest that peptide aldehydes can act in their hydrated forms as transition state analogues with the most potent inhibitor having an IC50 of 0.9 μM.

Evaluation of phenylboronate agarose for industrial-scale purification of erythropoietin from mammalian cell cultures

The search for novel, cost-effective ways to produce erythropoietin (Epo), the world top-selling biopharmaceutical, is a major challenge for todays biotechnology industry. However, Epos high glycosylation content (almost 40% of total mass) and the requirement for sialic acid for optimal in vivo activity still make mammalian cells the expression system of choice. In contrast to the abundance of reports on Epo production, robust, cost-effective methods for large-scale Epo purification can hardly be found in literature. To fill this gap, we describe here a process specifically studied for industrial-scale purification of the protein. Our method is based on the ability of phenylboronate agarose (PBA) to form reversible complexes with 1,2-cis-diol-containing molecules, like sugars in glycoproteins. Finding that additional factors (i.e., ionic and hydrophobic interactions) contribute to the Epo-PBA binding reaction, chromatography conditions have been optimized in scale-down experiments to improve selectivity and yield. As a result, the high performance of affinity chromatography has been achieved using a support possessing the robustness, chemical stability and low cost of a small synthetic ligand. By adding an anion exchange chromatography step and gel filtration for polishing, a pure and active product can easily be obtained by an integrated, start-to-end process optimized for industrial-scale operations.

Human IL-1 receptor antagonist from Escherichia coli: Large-scale microbial growth and protein purification

Interleukin-1 receptor antagonist (IL-1ra) is a recently discovered cytokine which specifically inhibits IL-1 pro-inflammatory activities in various experimental conditions. In this work, the growth conditions of a recombinant E. coli strain which in laboratory studies expressed human IL-1ra mostly in insoluble form, have been optimized at the level of 6-1 bioreactors and then scaled up to a 50-1 process. As a result, a high amount (0.43 g l-1 of microbial culture) of soluble, active IL-1ra has been directly obtained in the large-scale cell lysate with no need for protein solubilization. Also, an efficient purification procedure has been developed for the soluble protein, based on cation exchange expanded bed adsorption directly followed by anion exchange chromatography. This process, which does not include any intermediate dialysis step or gradient elutions, can be easily scaled up to larger production volumes and is therefore well-suited for manufacturing. As a result of the overall optimization study, more than 12 g of pure IL-1ra have been obtained from a single 50-1 fermentation run, without any denaturation/renaturation process. The final product, whose identity and purity have been checked also by MALDI-TOF and ESI-MS, shows full biological activity both in cellular assays and in in vivo experiments with Cynomolgus monkeys.

A HIGH THROUGHPUT ASSAY FOR INHIBITORS OF HIV-1 PROTEASE. SCREENING OF MICROBIAL METABOLITES

A novel method for discovery of HIV‐1 protease inhibitors in complex biological samples has been developed. The assay is based on two specific reagents: a recombinant protein constituted by a portion of the HIV‐1 Gag polyprotein comprising the p17–p24 cleavage site, fused to E. coli β‐galactoxidase, and a monoclonal antibody which binds the fusion protein in the Gag region. Binding occurs only if the fusion protein has not been cleaved by the HIV‐1 protease. The assay has been adapted for the screening of large numbers of samples in standard 96‐well microliter plates. Using this method about 12000 microbial fermentation broths have been tested and several HIV‐1 protease inhibitory activities have been detected. One of these has been studied in detail.

CRYSTALS OF SOLUBLE INTERLEUKIN-1 RECEPTOR COMPLEXED WITH ITS NATURAL ANTAGONIST REVEAL A 1:1 RECEPTOR-LIGAND COMPLEX

Interleukin‐1 is a cytokine involved in the acute phase response against infection and injury. We obtained crystals of a complex of soluble, recombinant human interleukin‐1 receptor and recombinant human interleukin‐1 receptor antagonist, a naturally occurring antagonist. The crystals are suitable for X‐ray analysis and diffract to 2.7 Å resolution. Solvent content calculations indicate that the crystals contain one receptor and one antagonist molecule per asymmetric unit. Other receptor to antagonist ratios are highly unlikely. These results suggest that the interleukin‐1 antagonist binds a single receptor molecule and does not cause receptor aggregation.