For 70 years antibiotics have saved countless lives and enabled the development of modern medicine, but it is becoming clear that the success of antibiotics may have only been temporary and we now anticipate a long-term, generational and perhaps never-ending challenge to find new therapies to combat antibiotic-resistant bacteria. As the search for new conventional antibiotics has become less productive and there are no clear strategies to improve success, a broader approach to address bacterial infection is needed. This review of potential alternatives to antibiotics (A2As) was commissioned by the Wellcome Trust, jointly funded by the Department of Health, and involved scientists and physicians from academia and industry. For the purpose of this review, A2As were defined as non-compound approaches (that is, products other than classical antibacterial agents) that target bacteria or approaches that target the host. In addition, the review was limited to agents that had potential to be administered orally, by inhalation or by injection for treatment of systemic/invasive infection. Within these criteria, the review has identified 19 A2A approaches now being actively progressed. The feasibility and potential clinical impact of each approach was considered. The most advanced approaches (and the only ones likely to deliver new treatments by 2025) are antibodies, probiotics, and vaccines now in Phase II and Phase III trials. These new agents will target infections caused by P. aeruginosa, C. difficile and S. aureus. However, other than probiotics for C. difficile, this first wave will likely best serve as adjunctive or preventive therapies. This suggests that conventional antibiotics will still be needed. The economics of pathogen-specific therapies must improve to encourage innovation, and greater investment into A2As with broad-spectrum activity (e.g. antimicrobial-, host defense-and, anti-biofilm peptides) is needed. Increased funding, estimated at >£1.5 bn over 10 years is required to validate and then develop these A2As. Investment needs to be partnered with translational expertise and targeted to support the validation of these approaches at Clinical Phase II proof of concept. Such an approach could transform our understanding of A2As as effective new therapies and should provide the catalyst required for both active engagement and investment by the pharma/biotech industry. Only a sustained, concerted and coordinated international effort will provide the solutions needed for the next decade.

Recombinant human phenylalanine hydroxylase (hPAH) was produced in high yields in Escherichia coli using the pET and pMAL expression vectors. In the pMAL system, hPAH was fused through the target sequences of the restriction protease factor Xa (IEGR) or enterokinase (D4K) to the C-terminal end of the highly expressed E. coli maltose-binding protein (MBP). The recombinant hPAH, recovered in soluble forms, revealed a high specific activity even in crude extracts and was detected as a homogeneous band by Western-blot analysis using affinity-purified polyclonal rabbit anti-(rat PAH) antibodies. The enzyme expressed in the pET system was subject to limited proteolysis by host cell proteases and was difficult to purify with a satisfactory yield. By contrast, when expressed as a fusion protein in the pMAL system, hPAH was resistant to cleavage by host cell proteases and was conveniently purified by affinity chromatography on an amylose resin. Catalytically active tetramer-dimer (in equilibrium) forms of the fusion protein were separated from inactive, aggregated forms by size-exclusion h.p.l.c. After cleavage by restriction protease, factor Xa or enterokinase, hPAH was separated from uncleaved fusion protein, MBP and restriction proteases by hydroxylapatite or ion-exchange (DEAE) chromatography. The yield of highly purified hPAH was approx. 10 mg/l of culture. The specific activity of the isolated recombinant enzyme was high (i.e. 1440 nmol of tyrosine.min-1.mg-1 with tetrahydrobiopterin as the cofactor) and its catalytic and physicochemical properties are essentially the same as those reported for the enzyme isolated from human liver. The recombinant enzyme, both as a fusion protein and as purified full-length hPAH, was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. The phosphorylated from of hPAH electrophoretically displayed an apparently higher molecular mass (approximately 51 kDa) than the non-phosphorylated (approximately 50 kDa) form.

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The effect of the paramagnetic high-spin Fe(II1) ion in phenylalanine 4-monooxygenase (phenylalanine hydroxylase, EC 1.14.16.1) on the water proton longitudinal relaxation rate has been used to study the environment of the iron center. The relaxation rate was measured as a function of the concentration of enzyme, substrate (phenylalanine), inhibitor (noradrenaline) and activator (lysolecithin), asrwell as of the temperature (1 8 -40 "C) and the external magnetic field strength (100 -600 MHz). From the frequency dependence of the relaxation rate, an effective correlation time (7,) of 4.2( f0.5) x 10-l o s was calculated for the enzyme-substrate complex, which most likely represents the electron spin relaxation rate (zs) for Fe(II1) ( S = 5/2) in this complex. The relaxation rate was proportional to the concentration of enzyme (0.04 -1 mM) both in the absence and presence of phenylalanine, but the paramagnetic molar relaxivity at 400 MHz and 22 "C decreased from 2.2(+0.05) x lo3 s-l . M-' i n the enzyme as isolated to 1.2(+0.06) x lo3 s-l . M-' in the presence of saturating concentrations of the substrate. The activation energy of the relaxation rate also decreased from 11.3 k 0.8 kJ/mol to -1.5 k 0.2 kJ/mol upon incubation of the enzyme with 5 mM phenylalanine. The results obtained can be interpreted in terms of a slowly exchanging water molecule coordinated to the catalytic paramagnetic Fe(II1) in the native and resting enzyme, and that this water molecule seems to be displaced from coordination on the binding of substrate or inhibitor. Moreover, the effect of increasing concentrations of phenylalanine and noradrenaline on the water proton relaxation rate and on the hydrophobic surface properties of the enzyme indicate that substrate and inhibitor induce a similar cooperative conformational change upon binding at the active site. By contrast, the activator lysolecithin does not seem to affect the interaction of water with the catalytic Fe(II1).Phenylalanine 4-monooxygenase (phenylalanine hydroxylase) catalyzes the hydroxylation of L-phenylalanine to L-tyrosine in the liver, using (6R)-l-erythro-tetrahydrobiopterin (BH,) as the electron donor (Kaufman and Fisher, 1974

Three isoforms of human tyrosine hydroxylase were expressed in Escherichiu coli and purified to homogeneity as the apoenzymes (metal-free). The apoenzymes exhibit typical tryptophan fluorescence emission spectra when excited at 250 -300 nm. The emission maximum (342 nm) was not shifted by the addition of metal ions, but reconstitution of the apoenzymes with Fe(I1) at pH 7-9 reduced the fluorescence intensity by about 35%, with an end point at 1.0 iron atom/enzyme subunit. The fluorescence intensity of purified bovine adrenal tyrosine hydroxylase, containing 0.78 mol tightly bound iron/mol subunit, was reduced by only 6% on addition of an excess amount of Fe(I1). Other divalent metal ions [Zn(II), Co(II), Mn(II), Cu(11) and Ni(II)] also reduced the fluorescence intensity of the human enzyme by 12-30% when added in stoichiometric amounts. The binding of Co(I1) at pH 7.2 was also found to affect its 'H-NMR spectrum and this effect was reversed by lowering the pH to 6.1. The quenching of the intrinsic fluorescence of the human isoenzymes by Fe(I1) was reversed by the addition of metal chelators. However, the addition of stoichiometric amounts of catecholamines, which are potent feedback inhibitors of tyrosine hydroxylase, to the iron-reconstituted enzyme, prevented the release of iron by the metal chelators. Fluorescence quenching, nuclear magnetic relaxation measurements and EPR spectroscopy all indicate that the reconstitution of an active holoenzyme from the isolated apoenzyme, with stoichiometric amounts of Fe(I1) at neutral pH, occurs without a measurable change in the redox state of the metal. However, on addition of dopamine or suprastoichiometric amounts of iron, the enzyme-bound iron is oxidized to a high-spin Fe(II1) ( S = 5/2) form in an environment of nearly axial symmetry, thus providing an explanation for the inhibitory action of the catecholamines Tyrosine hydroxylase is an iron-and tetrahydropterindependent enzyme which catalyses the rate-limiting reaction in the biosynthesis of catecholamines [l]. The human enzyme is present as four isoforms, generated by alternative splicing of pre-mRNA, and is a tetramer composed of four identical subunits (the mass of the subunits ranging over 55553-58 521 Da for the different isoforms) [2 -41. We have recently expressed three of these isozymes (hTH1, hTH2 and hTH4) in Escherichiu coli and shown that the purified apoenzymes (metal-free) bind stoichiometric amounts of iron and zinc with relatively high affinity at pH 5.4-6.5 [S]. The incorporation of Fe(1I) results in a rapid and up to 40-fold increase in activity Correspondence to J . Haavik, Department of Biochemistry, UniFax: f 4 7 5 20 64 00. Abbreviations. hTHl -hTH4, human tyrosine hydroxylase isozymes 1-4; apo-hTH1 -apo-hTH4, apoenzymes of the human tyrosine hydroxylase isozymes.Enzyrne.r. Tyrosine 3-monooxygenase or tyrosine hydroxylase (EC 1 .14.16.2), phenylalanine 4-monooxygenase or phenylalanine hydroxylase (EC 1.14.16.1). versity of Bergen, N-5009 Bergen, Norway [ 51. However, the kinetics and stoich...