Yannis D. Clonis

The recovery of products from fermentor broths is both difficult and expensive; therefore, downstream processing has long been regarded as the critical factor in the commercial development of biotechnology. For example, 60 to 70% of the selling price of enzymes corresponds to downstream processing costs. Product cost increases with the number of steps involved in the process, not only because of the capital and operating costs but also because the overall yield decreases with the number of steps (Fig. 1). Therefore, it is evident why high-grade, high-purity products are expensive. The latter is also a consequence of the practical problems encountered in protein and enzyme isolation as these biomolecules are labile to degradation and denaturation. One way of overcoming this problem is by marketing partially pure enzyme preparations which also contain other activities that do not interfere with the product or its applications. Furthermore, recombinant DNA technology among its other benefits, provides great commercial incentives for substantial reduction of the downstream processing costs. However, the stringent quality criteria imposed on biomolecules intended for human use has necessitated the use of sophisticated downstream processes which involve primarily chromatographic techniques.

A series of 2,2′‐dihydroxybenzophenones and their carbonyl N‐analogues were studied as potential inhibitors against human glutathione transferase M1‐1 (hGSTM1‐1) purified from recombinant E. coli. Their screening revealed an inhibition against hGSTM1‐1 within a range of 0‐42% (25 μM). The IC₅₀ values for the two stronger ones, 16 and 13, were 53.5 ± 5.6 μΜ and 28.5 ± 2.5 μΜ, respectively. The results were compared with earlier ones for isoenzymes hGSTP1‐1 and hGSTA1‐1 involved in MDR. All but one bind more strongly to A1‐1, than M1‐1 and P1‐1, the latter being a poor binder. An order of potency A1‐1 > > M1‐1 > P1‐1 meritted 13, 14 and 16 as the most potent inhibitors with hGSTM1‐1. Enzyme kinetics with hGSTM1‐1 (K_m(CDNB) 213 ± 10 μΜ and K_m(GSH) 303 ± 11 μΜ) revealed a competitive modality for 16 (K_i(16) = 22.3 ± 1.1 μΜ) and a mixed one for 13 versus CDNB (K_i(13) = 33.3 ± 1.6 μM for the free enzyme and K_i(13)′ = 17.7 ± 1.7 μM for the enzyme‐CDNB complex). 5‐ or 5′‐Bromo‐ or phenyl‐substituted (but not in combination) inhibitors, having a H‐bonded oxime weakly acidic group of a small volume, are optimal candidates for binding hGSTM1‐1. The outcome of the isoenzyme trilogy identified good binder leads for the investigated GSTs involved in MDR.

Glutathione (GSH) structure‐guided tripeptide analogues were designed and synthesized by solid phase technology, purified (≥95%) by RP and/or GF column chromatography, to identify those that, compared with GSH, exhibited similar or higher binding and catalytic efficiency toward the MDR‐involved human GSTP1‐1 isoenzyme, and could discriminate between the allozymic expression products of the polymorphic human GSTP1 gene locus, designated as hGSTP1*A (Ile¹⁰⁴/Ala¹¹³), hGSTP1*B (Val¹⁰⁴/Ala¹¹³), and hGSTP1*C (Val¹⁰⁴/Val¹¹³). The analogues bear single amino acid alterations as well as alterations in more than one position. Some analogues showed remarkable allozyme selectivity, binding catalytically to A (I, II, IV, XII), to C (V and XVI), to A and C (III, VII, XIV) or to all three allozymes (XV). A heterocyclic substituent at positions 1 or 2 of GSH favors inhibition of A, whereas a small hydrophobic/hydrophilic amide substituent at position 2 (Cys) favors inhibition of B and C. Heterocyclic substituents at position 1, only, produce catalytic analogues for A, whereas less bulky and more flexible hydrophobic/hydrophilic substituents, at positions 1 or 3, lead to effective substrates with C. When such substituents were introduced simultaneously at positions 1 and 3, the analogues produced have no catalytic potential but showed appreciable inhibitory effects, instead, with all allozymes. It is anticipated that when GSH analogues with selective inhibitory or catalytic binding, were conjugated to allozyme‐selective inhibitors of hGSTP1‐1, the derived leads would be useful for the designing of novel chimeric inhibitors against the MDR‐involved hGSTP1‐1 allozymes. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 330–344, 2016.

The selectivity of certain benzophenones and their carbonyl N‐analogues was investigated towards the human GSTP1‐1 allozymes A, B and C involved in MDR. The allozymes were purified from extracts derived from E. coli harbouring the plasmids pEXP5‐CT/TOPO‐TA‐hGSTP1*A, pOXO4‐hGSTP1*B or pOXO4‐hGSTP1*C. Compound screening with each allozyme activity indicated three compounds with appreciable inhibitory potencies, 12 and 13 with P1‐1A 62% and 67%, 11 and 12 with P1‐1C 51% and 70%, whereas that of 15 fell behind with P1‐1B (41%). These findings were confirmed by IC₅₀ values (74–125 μm). Enzyme inhibition kinetics, aided by molecular modelling and docking, revealed that there is competition with the substrate CDNB for the same binding site on the allozyme (K_i(13/A) = 63.6 ± 3.0 μm, K_i(15/B) = 198.6 ± 14.3 μm, and K_i(11/C) = 16.5 ± 2.7 μm). These data were brought into context by an in silico structural comparative analysis of the targeted proteins. Although the screened compounds showed moderate inhibitory potency against hGSTP1‐1, remarkably, some of them demonstrated absolute isoenzyme and/or allozyme selectivity.

Adenosine detection in human serum is important because this ribonucleoside has established clinical applications, modulating many physiological processes. Furthermore, a simple and cheap detection method is useful in adenosine production processes. Adenosine can be determined enzymatically using either S‐adenosyl‐homocysteine hydrolase and ³[H]‐adenosine, or adenosine kinase combined with GTP and luciferase, or an amperometric biosensor carrying adenosine deaminase (ADA), purine nucleoside phosphorylase, and xanthine oxidase. We developed a simple and cheap method relying on a transparent biostrip bearing ADA and the indicator phenol red (PR), co‐immobilized to polyacrylamide, itself chemically adhered to a derivatized glass strip. The ADA‐catalyzed conversion of adenosine to inosine and ammonia leads to a local pH alteration, changing the absorbance maximum of PR (from 425 to 567 nm), which is measured optically. The biostrip shows an analytical range 0.05–1.5 mM adenosine and is reusable when stored at 4 °C. When the biostrip was tested with serum, spiked with adenosine (70 and 100 μM), and filtered for protein and adenosine phosphates depletion, it showed good adenosine recovery. In summary, we show the proof‐of‐concept that adenosine can be determined reagent‐free, at moderate sensitivity on an easy to construct, cheap, and reusable biostrip, based on commercially available molecular entities.

Glutathione transferases (GSTs) are enzymes that contribute to cellular detoxification by catalysing the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of xenobiotic compounds, including several chemotherapeutic drugs. In the present work we investigated the interaction of the chemotherapeutic drug chlorambucil (CBL) with human GSTA1-1 (hGSTA1-1) using kinetic analysis, protein crystallography and molecular dynamics. In the presence of GSH, CBL behaves as an efficient substrate for hGSTA1-1. The rate-limiting step of the catalytic reaction between CBL and GSH is viscosity-dependent and kinetic data suggest that product release is rate-limiting. The crystal structure of the hGSTA1-1/CBL-GSH complex was solved at 2.1 Å resolution by molecular replacement. CBL is bound at the H-site attached to the thiol group of GSH, is partially ordered and exposed to the solvent, making specific interactions with the enzyme. Molecular dynamics simulations based on the crystal structure indicated high mobility of the CBL moiety and stabilization of the C-terminal helix due to the presence of the adduct. In the absence of GSH, CBL is shown to be an alkylating irreversible inhibitor for hGSTA1-1. Inactivation of the enzyme by CBL followed a biphasic pseudo-first-order saturation kinetics with approximately 1 mol of CBL per mol of dimeric enzyme being incorporated. Structural analysis suggested that the modifying residue is Cys112 which is located at the entrance of the H-site. The results are indicative of a structural communication between the subunits on the basis of mutually exclusive modification of Cys112, indicating that the two enzyme active sites are presumably coordinated.

Overexpression of human GSTA1-1 in tumor cells is part of MDR mechanisms. We report on the synthesis of 11 pyrrole derivatives as hGSTA1-1 inhibitors starting from 1-methyl-2-[(2-nitrobenzylsulfanyl]-1H-pyrrole. Molecular modeling revealed two locations in the enzyme H binding site: the catalytic primary one accommodating shorter and longer derivatives and the secondary one, where shorter derivatives can occupy. Derivative 9, displaying the highest inhibition and bearing a p-nitroarylimino moiety, and derivative 4, lacking this moiety, were studied kinetically. Derivative 9 binds (K_i(9) = 71 ± 4 μM) at the primary site competitively vs CDNB. Derivative 4 binds (K_i(4) = 135 ± 27 μM) at the primary and secondary sites, allowing the binding of a second molecule (4 or CDNB) leading to formation of unreactive and reactive complexes, respectively. The arylmethylsulfonylpyrrole core structure is a new pharmacophore for hGSTA1-1, whereas its derivative 9 may serve as a lead structure.

Reverse transcriptase (RT) catalyzes the formation of dsDNA from single-stranded retroviral RNA genome. This enzyme is unique among DNA polymerases in its ability to use either RNA or DNA as a template. Moloney Murine Leukemia virus reverse transcriptase lacking RNase H activity (M-MLVH-RT) especially holds particular interest because of its ability to eliminate the deleterious effect of RNase H, which results in more efficient synthesis of full-length cDNA from mRNA. Therefore, the development of a simple purification method attracts the attention of retroviral drug and enzyme researchers and manufacturers. The present work is the first purification example of a non-tagged (native) RT by affinity chromatography using synthetic affinity ligands. In this study, the ligand was selected from a structure-biased combinatorial library of dNTP-mimetic ligands, and it was evaluated for its ability to bind and purify M-MLVH-RT from inclusion bodies of recombinant E. coli. The selected ligand (AEAd), bearing 9-aminoethyladenine and 1,6-diamine-hexane both linked on the same triazine scaffold, displayed the highest enzyme purifying ability after applying mild desorption conditions (6 mM MnCl₂ in 20 mM Tris-HCl buffer, pH 7.5). The binding capacity of immobilized AEAd with M-MLVH-RT was determined to be equal to approximately 1 mg enzyme/g moist weight gel. Adsorption studies with immobilized AEAd and soluble M-MLVH-RT demonstrated that the formation of the respective complex was perturbed by ATP. Quality control tests of the purified M-MLVH-RT essentially showed a single band (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and absence of nucleic acids and contaminating nuclease activities.