Using protease inhibitors to improve protein stability in the presence of skin: A case study on the stability of insulin like growth factor 1.

Insulin-like growth factor 1 (IGF-1) is indicated for growth failure in pediatric patients with primary IGF-1 deficiency and for patients with neutralizing antibodies to growth hormone. IGF-1 was cloned, expressed and purified in-house. Preliminary stability studies prior to the transdermal delivery experiments showed that although stable in contact with stratum corneum, the solution concentration of IGF-1 decreased to 23.63 ± 2.48 and 21.58 ± 2.62% of the initial value upon exposure for 8 h to porcine dermis of 250 and 750 µm thickness. This led to an investigation into how it might be possible to improve the stability of IGF-1 in the presence of porcine/human skin. The stability of IGF-1 in the presence of dermis improved upon heating the skin samples at 60 °C for 2 min suggesting that IGF-1 was subject to enzymatic degradation. Although addition of the protease inhibitor, phenylmethanesulfonyl fluoride (PMSF) alone, did not improve stability, the use of a protease inhibitor cocktail completely blocked proteolytic degradation of IGF-1; the solution concentration after an 8 h exposure to porcine skin was equivalent to the initial level (103.87 ± 9.15%). The results obtained with porcine skin were confirmed with human skin (IGF-1 recovery was 99.31 ± 9.98%). These findings suggest that the inclusion of protease inhibitor cocktails may be useful in limiting the degradation of therapeutic proteins during iontophoresis and transdermal delivery in general - this could be of particular interest for local delivery of peptide/protein therapeutics for dermatological applications.

Erratum: Withdrawal notice to 'WITHDRAWN: Specific protein-protein interactions limit the cutaneous iontophoretic transport of interferon beta-1B and a poly-ARG interferon beta-1B analogue' [International Journal of Pharmaceutics 589 (2020) 119913].

[This corrects the article DOI: 10.1016/j.ijpx.2020.100051.].

Specific protein-protein interactions limit the cutaneous iontophoretic transport of interferon beta-1b and a poly-Arg interferon beta-1b analogue.

The first objective was to investigate the transdermal iontophoresis of interferon beta 1b (IFN); the second was to determine whether the addition of 10 Arg residues at the N-terminus, creating a highly charged poly-Arg analogue (Arg10-IFN), increased delivery. Cumulative permeation of IFN and Arg10-IFN after iontophoresis at 0.5 mA/cm2 for 8 h was 6.97 ± 4.82 and 9.55 ± 1.63 ng/cm2, respectively - i.e. >1000-fold less than that of ribonuclease A, cytochrome c and human basic fibroblast growth factor. Co-iontophoresis of acetaminophen showed that, in contrast to lysozyme, neither IFN nor Arg10-IFN interacted with skin to decrease convective solvent flow. Furthermore, there was no statistically significant difference between (i) iontophoretic delivery of IFN across intact or laser porated skin and (ii) passive or iontophoretic delivery of IFN across laser porated skin. Chromatographic characterisation supported the hypothesis that IFN was bound strongly to albumin. The formation of a ~86 kDa complex with albumin was probably responsible for the poor cutaneous delivery of IFN/Arg10-IFN despite the use of iontophoresis and/or laser microporation. Biopharmaceuticals might interact with specific proteins during iontophoretic transport and so decrease their (per)cutaneous delivery without affecting electroosmotic solvent flow, which is usually considered as a reliable marker to report on permeant binding during electrotransport across the skin.

Specific protein-protein interactions limit the cutaneous iontophoretic transport of interferon beta-1B and a poly-ARG interferon beta-1B analogue.

The first objective was to investigate the transdermal iontophoresis of interferon beta 1b (IFN); the second was to determine whether the addition of 10 Arg residues at the N-terminus, creating a highly charged poly-Arg analogue (Arg10-IFN), increased delivery. Cumulative permeation of IFN and Arg10-IFN after iontophoresis at 0.5 mA/cm2 for 8 h was 6.97 ± 4.82 and 9.55 ± 1.63 ng/cm2, respectively - i.e. >1000-fold less than that of ribonuclease A, cytochrome c and human basic fibroblast growth factor. Co-iontophoresis of acetaminophen showed that, in contrast to lysozyme, neither IFN nor Arg10-IFN interacted with skin to decrease convective solvent flow. Furthermore, there was no statistically significant difference between (i) iontophoretic delivery of IFN across intact or laser porated skin and (ii) passive or iontophoretic delivery of IFN across laser porated skin. Chromatographic characterisation supported the hypothesis that IFN was bound strongly to albumin. The formation of a ~ 86 kDa complex with albumin was probably responsible for the poor cutaneous delivery of IFN/Arg10-IFN despite the use of iontophoresis and/or laser microporation. Biopharmaceuticals might interact with specific proteins during iontophoretic transport and so decrease their (per)cutaneous delivery without affecting electroosmotic solvent flow, which is usually considered as a reliable marker to report on permeant binding during electrotransport across the skin.

Inhibition of iPLA2 ß and of stretch-activated channels by doxorubicin alters dystrophic muscle function.

BACKGROUND AND PURPOSE: Chronic elevation in intracellular Ca(2+) concentration participates in death of skeletal muscle from mdx mice, a model for Duchenne muscular dystrophy (DMD). Candidate pathways mediating this Ca(2+) overload involve store-operated channels (SOCs) and stretch-activated channels (SACs), which are modulated by the Ca(2+) -independent form of PL A2 (iPLA2 ). We investigated the effect of doxorubicin (Dox), a chemotherapeutic agent reported to inhibit iPLA2 in other systems, on the activity of this enzyme and on the consequences on Ca(2+) handling and muscle function in mdx mice. EXPERIMENTAL APPROACH: Effects of Dox on iPLA2 activity, reactive oxygen species production and on Ca(2+) influx were investigated in C2C12 and mdx myotubes. The mechanism of Dox-mediated iPLA2 inhibition was evaluated using purified 6x histidine-tagged enzyme. Aequorin technology was used to assess Ca(2+) concentrations underneath the plasma membrane. Isolated muscles were exposed to fatigue protocols and eccentric contractions to evaluate the effects of Dox on muscle function. KEY RESULTS: Dox at 1-30 µM inhibited iPLA2 activity in cells and in the purified enzyme. Dox also inhibited SAC- but not SOC-mediated Ca(2+) influx in myotubes. Stimulated elevations of Ca(2+) concentrations below the plasmalemma were also blocked. Exposure of excised muscle to Dox was not deleterious to force production and promoted recovery from eccentric contractions. CONCLUSIONS AND IMPLICATIONS: Dox showed efficacy against targets known to play a role in the pathology of DMD, namely iPLA2 and SAC. The potent SAC inhibitory effect of Dox is a novel finding that can explain partly the cardiomyopathy seen in chronic anthracycline treatment.

Noninvasive transdermal iontophoretic delivery of biologically active human basic fibroblast growth factor.

Human basic fibroblast growth factor (hbFGF; 17.4 kDa) has shown promise in the treatment of several dermatological conditions; symptomatic improvement was also observed in patients with peripheral arterial disease after arterial infusion. The objective of this study was to demonstrate the feasibility of using transdermal iontophoresis to deliver biologically active hbFGF noninvasively into and across the skin. The protein was cloned, expressed and purified in-house. Porcine skin was used to investigate transdermal iontophoretic transport of hbFGF as a function of current density (0.15, 0.3, and 0.5 mA/cm(2)); results were subsequently confirmed using human skin. Cumulative hbFGF permeation and skin deposition were quantified by ELISA. The absence of proteolytic degradation during skin transit was confirmed by SDS-PAGE. Biological activity postdelivery was determined using cell proliferation assays in human foreskin fibroblast (HFF) and NIH 3T3 cell lines. Confocal laser scanning microscopy (CLSM) was used to visualize the distribution of rhodamine-tagged hbFGF in the skin. Cumulative iontophoretic permeation at 0.3 mA/cm(2) was statistically superior to that at 0.15 mA/cm(2); however, there was no further improvement at 0.5 mA/cm(2). Significant skin deposition of hbFGF was observed, and this dominated transport; for example, after iontophoresis for 8 h at 0.5 mA/cm(2), skin deposition (77.74 ± 37.36 µg/cm(2)) was 4.4-fold higher than cumulative permeation (17.64 ± 5.18 µg/cm(2)). The superior skin deposition may be advantageous for dermatological applications. The HFF and NIH 3T3 cell proliferation assays confirmed that biological activity of hbFGF was retained postdelivery. Coiontophoresis of acetaminophen showed that the dominant transport mechanism switched from electroosmosis to electromigration upon increasing current density from 0.15 to 0.3 mA/cm(2). Experiments using human skin confirmed that iontophoretic permeation of hbFGF across porcine and human membranes was statistically equivalent. CLSM images of rhodamine-tagged hbFGF postiontophoresis indicated that the protein was evenly distributed throughout the epidermis and dermis. In conclusion, the results confirmed that transdermal iontophoresis was indeed able to deliver structurally intact, functional hbFGF noninvasively into and across the skin. The amounts of protein delivered were similar to those in reports from preclinical and clinical studies.

Turkish freshwater and marine macrophyte extracts show in vitro antiprotozoal activity and inhibit FabI, a key enzyme of Plasmodium falciparum fatty acid biosynthesis.

The ethanolic extracts of a number of Turkish freshwater macrophytes (Potamogeton perfoliatus, Ranunculus tricophyllus and Cladophora glomerata) and marine macroalgae (Dictyota dichotoma, Halopteris scoparia, Posidonia oceanica, Scinaia furcellata, Sargassum natans and Ulva lactuca) were assayed for their in vitro antiprotozoal activity. Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum were used as test organisms. The cytotoxicity of the extracts was also assessed against primary rat skeletal myoblasts (L6 cells). Whereas none of the extracts were active against T. cruzi, all crude extracts displayed appreciable trypanocidal activity against T. brucei rhodesiense, with S. natans being the most active one (IC(50) 7.4microg/ml). Except for the marine alga H. scoparia, all extracts also possessed leishmanicidal potential. The best antileishmanial activity was exerted by U. lactuca and P. oceanica (IC(50)'s 5.9 and 8.0microg/ml, respectively). Five extracts that demonstrated inhibitory activity towards P. falciparum (IC(50)'s 18.1-48.8microg/ml) were simultaneously assayed against FabI, a crucial enzyme of the fatty acid system of P. falciparum, to find out whether FabI was their target. The extracts of C. glomerata and U. lactuca efficiently inhibited the FabI enzyme with IC(50) values of 1.0 and 4.0microg/ml, respectively. None of the extracts were cytotoxic towards mammalian L6 cells. This work reports for the first time antiprotozoal activity of some Turkish marine and freshwater algae, as well as a target-based antiplasmodial screening for the identification of P. falciparum FabI inhibitors from aquatic and marine macrophytes.

Evaluation of antiprotozoal and plasmodial enoyl-ACP reductase inhibition potential of turkish medicinal plants.

A total of 58 extracts of different polarity were prepared from various organs of 16 species of Turkish plants and screened for their antitrypanosomal, antileishmanial and antiplasmodial activities. No significant activity was observed against Trypanosoma cruzi, whereas many extracts showed appreciable trypanocidal potential against T. brucei rhodesiense, with the CHCl(3)-soluble portion of Phlomis kurdica being the most active (IC(50) 2.7 microg[sol ]mL). Almost all extracts, particularly the CHCl(3) phases, exhibited growth inhibition activity against Leishmania donovani amastigotes. The CHCl(3)-solubles of Putoria calabrica roots (IC(50) 1.9 microg[sol ]mL), Wendlandia ligustroides leaves (IC(50) 2.1 microg[sol ]mL) and Rhododendron luteum leaves (IC(50) 2.3 microg[sol ]mL) displayed the highest leishmanicidal potential. The majority of the extracts also possessed antiplasmodial activity against the multi-drug resistant K1 Plasmodium falciparum strain. The most potent antiplasmodial activity was observed with the CHCl(3) extracts of Phlomis kurdica (IC(50) 1.5 microg[sol ]mL), P. leucophracta (IC(50) 1.6 microg[sol ]mL), Scrophularia cryptophila (IC(50) 1.8 microg[sol ]mL), Morina persica (IC(50) 1.9 microg[sol ]mL) and the aqueous root extract of Asperula nitida subsp. subcapitellata (IC(50) 1.6 microg[sol ]mL). Twenty-one extracts with significant antimalarial activity (IC(50) < 5 microg[sol ]mL) were also tested for their ability to inhibit the purified enoyl-ACP reductase (FabI), a crucial enzyme in the fatty acid biosynthesis of P. falciparum. The CHCl(3) extract of Rhododendron ungernii leaves (IC(50) 10 microg[sol ]mL) and the H(2)O-soluble portion of Rhododendron smirnovii leaves (IC(50) 0.4 microg[sol ]mL) strongly inhibited the FabI enzyme. The preliminary data indicate that some (poly)phenolic compounds are responsible for the FabI inhibition potential of these extracts. The presented work reports for the first time the antiprotozoal activity of nine different genera as well as a target specific antimalarial screening for the identification of P. falciparum FabI inhibitors from medicinal plant extracts.

Nucleoside binding site of herpes simplex type 1 thymidine kinase analyzed by X-ray crystallography.

The crystal structures of the full-length Herpes simplex virus type 1 thymidine kinase in its unligated form and in a complex with an adenine analogue have been determined at 1.9 A resolution. The unligated enzyme contains four water molecules in the thymidine pocket and reveals a small induced fit on substrate binding. The structure of the ligated enzyme shows for the first time a bound adenine analogue after numerous complexes with thymine and guanine analogues have been reported. The adenine analogue constitutes a new lead compound for enzyme-prodrug gene therapy. In addition, the structure of mutant Q125N modifying the binding site of the natural substrate thymidine in complex with this substrate has been established at 2.5 A resolution. It reveals that neither the binding mode of thymidine nor the polypeptide backbone conformation is altered, except that the two major hydrogen bonds to thymidine are replaced by a single water-mediated hydrogen bond, which improves the relative acceptance of the prodrugs aciclovir and ganciclovir compared with the natural substrate. Accordingly, the mutant structure represents a first step toward improving the virus-directed enzyme-prodrug gene therapy by enzyme engineering.

Kinetics and crystal structure of the wild-type and the engineered Y101F mutant of Herpes simplex virus type 1 thymidine kinase interacting with (North)-methanocarba-thymidine.

Kinetic and crystallographic analyses of wild-type Herpes simplex virus type 1 thymidine kinase (TK(HSV1)) and its Y101F-mutant [TK(HSV1)(Y101F)] acting on the potent antiviral drug 2'-exo-methanocarba-thymidine (MCT) have been performed. The kinetic study reveals a 12-fold K(M) increase for thymidine processed with Y101F as compared to the wild-type TK(HSV1). Furthermore, MCT is a substrate for both wild-type and mutant TK(HSV1). Its binding affinity for TK(HSV1) and TK(HSV1)(Y101F), expressed as K(i), is 11 microM and 51 microM, respectively, whereas the K(i) for human cytosolic thymidine kinase is as high as 1.6 mM, rendering TK(HSV1) a selectivity filter for antiviral activity. Moreover, TK(HSV1)(Y101F) shows a decrease in the quotient of the catalytic efficiency (k(cat)/K(M)) of dT over MCT corresponding to an increased specificity for MCT when compared to the wild-type enzyme. Crystal structures of wild-type and mutant TK(HSV1) in complex with MCT have been determined to resolutions of 1.7 and 2.4 A, respectively. The thymine moiety of MCT binds like the base of dT while the conformationally restricted bicyclo[3.1.0]hexane, mimicking the sugar moiety, assumes a 2'-exo envelope conformation that is flatter than the one observed for the free compound. The hydrogen bond pattern around the sugar-like moiety differs from that of thymidine, revealing the importance of the rigid conformation of MCT with respect to hydrogen bonds. These findings make MCT a lead compound in the design of resistance-repellent drugs for antiviral therapy, and mutant Y101F, in combination with MCT, opens new possibilities for gene therapy.

Compulsory order of substrate binding to herpes simplex virus type 1 thymidine kinase. A calorimetric study.

Isothermal titration calorimetry has been used to investigate the thermodynamic parameters of the binding of thymidine (dT) and ATP to herpes simplex virus type 1 thymidine kinase (HSV1 TK). Binding follows a sequential pathway in which dT binds first and ATP second. The free enzyme does not bind ATP, whose binding site becomes only accessible in the HSV1 TK.dT complex. At pH 7.5 and 25 degrees C, the binding constants are 1.9 x 10(5) m(-1) for dT and 3.9 x 10(6) m(-1) for ATP binding to the binary HSV1 TK.dT complex. Binding of both substrates is enthalpy-driven and opposed by a large negative entropy change. The heat capacity change (DeltaCp) obtained from DeltaH in the range of 10-25 degrees C is -360 cal K(-1) mol(-1) for dT binding and -140 cal K(-1) mol(-1) for ATP binding. These large DeltaCp values are incompatible with a rigid body binding model in which the dT and ATP binding sites pre-exist in the free enzyme. Values of DeltaCp and TDeltaS strongly indicate large scale conformational adaptation of the active site in sequential substrate binding. The conformational changes seem to be more pronounced in dT binding than in the subsequent ATP binding. Considering the crystal structure of the ternary HSV1 TK.dT.ATP complex, a large movement in the dT binding domain and a smaller but substantial movement in the LID domain are proposed to take place when the enzyme changes from the substrate-free, presumably more open and less ordered conformation to the closed and compact conformation of the ternary enzyme-substrate complex.

Substrate diversity of herpes simplex virus thymidine kinase. Impact Of the kinematics of the enzyme.

Herpes simplex virus type 1 (HSV 1) thymidine kinase (TK) exhibits an extensive substrate diversity for nucleobases and sugar moieties, in contrast to other TKs. This substrate diversity is the crucial molecular basis of selective antiviral and suicide gene therapy. The mechanisms of substrate binding of HSV 1 TK were studied by means of site-directed mutagenesis combined with isothermal calorimetric measurements and guided by theoretical calculations and sequence comparison. The results show the link between the exceptionally broad substrate diversity of HSV 1 TK and the presence of structural features such as the residue triad His-58/Met-128/Tyr-172. The mutation of Met-128 into a Phe and the double mutant M128F/Y172F result in mutants that have lost their activity. However, by exchanging His to form the triple mutant H58L/M128F/Y172F, the enzyme regains activity. Strikingly, this triple mutant becomes resistant toward acyclovir. Furthermore, we give evidence for the importance of Glu-225 of the flexible LID region for the catalytic reaction. The data presented give new insights to understand mechanisms ruling substrate diversity and thus are crucial for both the development of new antiviral drugs and engineering of mutant TKs apt to accept novel substrate analogs for gene therapeutic approaches.

The periplasmic domain of the histidine autokinase CitA functions as a highly specific citrate receptor.

The two-component regulatory system CitA/CitB is essential for induction of the citrate fermentation genes in Klebsiella pneumoniae. CitA represents a membrane-bound sensor kinase consisting of a periplasmic domain flanked by two transmembrane helices, a linker domain and the conserved kinase or transmitter domain. A fusion protein (MalE-CitAC) composed of the maltose-binding protein and the CitA kinase domain (amino acids 327-547) showed constitutive autokinase activity and transferred the gamma-phosphate group of ATP to its cognate response regulator CitB. The autokinase activity of CitA was abolished by an H350L exchange, and phosphorylation of CitB was inhibited by a D56N exchange, indicating that H-350 and D-56 represent the phosphorylation sites of CitA and CitB respectively. In the presence of ATP, CitB-D56N formed a stable complex with MalE-CitAC. To analyse the sensory properties of CitA, the periplasmic domain (amino acids 45-176) was overproduced as a soluble, cytoplasmic protein with a C-terminally attached histidine tag (CitAPHis). Purified CitAPHis bound citrate, but none of the other tri- and dicarboxylates tested, with high affinity (KD approximately 5 microM at pH 7) in a 1:1 stoichiometry. As shown by isothermal titration calorimetry, the binding reaction was driven by the enthalpy change (DeltaH = -76.3 kJ mol-1), whereas the entropy change was opposed (-TDeltaS = + 46.3 kJ mol-1). The pH dependency of the binding reaction indicated that the dianionic form H-citrate2- is the citrate species recognized by CitAPHis. In the presence of Mg2+ ions, the dissociation constant increased significantly, suggesting that the Mg-citrate complex is not bound by CitAPHis. This work defines the periplasmic domain of CitA as a highly specific citrate receptor and elucidates the binding characteristics of CitAPHis.