International Journal of Molecular Sciences Review Arginase as a Potential Biomarker of Disease Progression: A Molecular Imaging Perspective Gonçalo S. Clemente 1 , Aren van Waarde 1, Inês F. Antunes 1, Alexander Dömling 2 and Philip H. Elsinga 1,* 1 Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands; [email protected] (G.S.C.); [email protected] (A.v.W.); [email protected] (I.F.A.) 2 Department of Drug Design, Groningen Research Institute of Pharmacy, University of Groningen, 9713 AV Groningen, The Netherlands; [email protected] * Correspondence: [email protected]; Tel.: +31-50-361-3247 Received: 2 July 2020; Accepted: 23 July 2020; Published: 25 July 2020 Abstract: Arginase is a widely known enzyme of the urea cycle that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. The action of arginase goes beyond the boundaries of hepatic ureogenic function, being widespread through most tissues. Two arginase isoforms coexist, the type I (Arg1) predominantly expressed in the liver and the type II (Arg2) expressed throughout extrahepatic tissues. By producing L-ornithine while competing with nitric oxide synthase (NOS) for the same substrate (L-arginine), arginase can influence the endogenous levels of polyamines, proline, and NO•. Several pathophysiological processes may deregulate arginase/NOS balance, disturbing the homeostasis and functionality of the organism. Upregulated arginase expression is associated with several pathological processes that can range from cardiovascular, immune-mediated, and tumorigenic conditions to neurodegenerative disorders. Thus, arginase is a potential biomarker of disease progression and severity and has recently been the subject of research studies regarding the therapeutic efficacy of arginase inhibitors. This review gives a comprehensive overview of the pathophysiological role of arginase and the current state of development of arginase inhibitors, discussing the potential of arginase as a molecular imaging biomarker and stimulating the development of novel specific and high-affinity arginase imaging probes. Keywords: arginase; nitric oxide; arginase inhibitors; molecular imaging; positron emission tomography (PET) 1. Introduction The identification of the Krebs–Henseleit urea cycle in the early 1930s highlighted the importance of arginase, a manganese-containing enzyme that catalyzes the conversion of L-arginine to urea and L-ornithine. The individual hydrolytic function of arginase was previously known, but the interdependence of arginase and other biochemical mechanisms, as evidenced in the urea cycle (Figure1), triggered scientific interest. Despite the early findings showing that arginase was mostly expressed in the mammalian liver [1], and to a lesser extent in kidneys [2], this enzyme was also identified in organs where the urea cycle is not present [3–5]. Thus, investigations on the L-arginine metabolism in non-ureagenic tissues revealed a parallel role of arginase beyond ureagenesis: to regulate L-ornithine levels and subsequent polyamine and proline biosynthesis. With the isolation of arginase from different rodent tissues, and comparing the physicochemical properties, it became evident that different isoforms exist. Extending these studies to human tissues led to similar results, and the co-existence of two arginase isoforms became widely accepted by the Int. J. Mol. Sci. 2020, 21, 5291; doi:10.3390/ijms21155291 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 5291 2 of 36 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 39 scientific community since the early 1980s [6]. Due to the abundance in subcellular compartments, arginase type I (predominantly expressed, but not exclusively, in the liver and with a prominent role in arginase type I (predominantly expressed, but not exclusively, in the liver and with a prominent role the urea cycle) became described as cytosolic, and type II (widely expressed in extrahepatic tissues and in the urea cycle) became described as cytosolic, and type II (widely expressed in extrahepatic tissues mainly involved in the production of L-ornithine outside the urea cycle) as mitochondrial. and mainly involved in the production of L-ornithine outside the urea cycle) as mitochondrial. Figure 1.1. Scheme of the urea cycle, including the overall role ofof L-ornithineL-ornithine withinwithin (highly(highly restrictedrestricted toto recycling)recycling) andand outsideoutside ofof thisthis cyclecycle (regulation(regulation ofof proteinprotein synthesis).synthesis). TheThe competitivecompetitive L-arginineL-arginine metabolism between arginase and nitricnitric oxideoxide synthasesynthase (NOS),(NOS), occurringoccurring in manymany non-hepaticnon-hepatic cellcell types,types, isis alsoalso represented.represented. Not all the outlined processes occur in every cell type, and theirtheir expression andand extentextent maymay dependdepend onon severalseveral physiologicalphysiological oror pathologicalpathological processes.processes. 1.1. Arginase Isoforms InIn mammals,mammals, Arg1 genesgenes are mostlymostly expressed in the liver, andand toto aa muchmuch lesserlesser extentextent inin bonebone marrow, whereaswhereas Arg2Arg2 genesgenes are present in virtually all tissues (with predominance in the kidney, kidney, prostate,prostate, digestivedigestive andand gastrointestinalgastrointestinal tract,tract, muscle,muscle, andand endocrineendocrine tissues)tissues) [[7]7].. Despite keeping the samesame enzymaticenzymatic function, function, the dithefferent different biochemical biochemical context incontext these tissues in these favors tissues two complementary favors two roles.complemen Hepatictary cytosolic roles. Hepatic Arg1 plays cytosolic a primary Arg1 role plays in the a primary net production role in ofthe urea net (forproduction ammonia of clearance) urea (for andammonia in the clearance) biosynthesis and of in L-ornithine, the biosynthesis which of is L usually-ornithine, recycled which within is usually the urea recycled cycle. within Mitochondrial the urea Arg2cycle. mainly Mitochondrial regulates Arg2 the physiological mainly regulates biosynthesis the physiological of L-ornithine bio insynthesis several otherof L- tissues.ornithine L-Ornithine in several isother a precursor tissues. L of-Ornithine polyamines is a (putrescine, precursor of spermidine, polyamines spermine), (putrescine, proline, spermidine, and glutamate, spermine), which proline, are essentialand glutamate, for collagen which synthesis,are essential tissue for collagen repair, cell synthesis, proliferation, tissue repair, growth cell and proliferation, viability, and growth neuronal and development,viability, and neuronal and in the development, regulation of and immune in the and regulation inflammatory of immune responses and inflammatory [8]. responses [8]. High-resolution crystallography techniques allowed characterizing the structure of human arginaseHigh and-resolution detailing crystallography the differences techniques between the allowed isoforms. characteriz Humaning Arg1 the (105structure kDa) of and human Arg2 (129arginase kDa) and exist detail primarilying the as differences homotrimeric between metalloenzymes the isoforms. encoding Human 322 Arg1 and (105 354 aminokDa) and acid Arg2 residues, (129 respectivelykDa) exist primarily [9,10]. Despiteas homotrimeric being encoded metalloenzymes by different encoding genes, approximately322 and 354 amino 61% acid of the residues, amino acidrespectively sequence [9,10] identity. Despite is shared being by encoded both isoforms, by different and genes, all active-site approximately residues 6 involved1% of the in amino substrate acid sequence identity is shared by both isoforms, and all active-site residues involved in substrate binding, as well as the binuclear Mn2+ cluster core, are strictly conserved (Figure 2A). These Mn2+ ions are approximately 3.3 Å apart, bridged by an OH− ion, and mainly surrounded by negatively charged Int. J. Mol. Sci. 2020, 21, 5291 3 of 36 binding, as well as the binuclear Mn2+ cluster core, are strictly conserved (Figure2A). These Mn 2+ Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 39 ions are approximately 3.3 Å apart, bridged by an OH− ion, and mainly surrounded by negatively charged amino acid residues (Figure2B), which form an electron paramagnetic resonance spin-coupled amino acid residues (Figure 2B), which form an electron paramagnetic resonance spin-coupled binuclear center located at the bottom of a 15 Å deep cleft in each of the three identical subunits [11]. binuclear center located at the bottom of a 15 Å deep cleft in each of the three identical subunits [11]. FigureFigure 2. (A 2.) Superposition(A) Superposition of humanof human Arg1 Arg1 and and Arg2 Arg2 (blue (blue and and green green color, color, respectively) respectively) subunitssubunits and active-sitesand active and-sites amino and acidamino sequence acid sequence alignment alignment showing showing the sharedthe shared homology homology percentage percentage by byboth both isoforms (molecular graphics and analyses performed with UCSF Chimera [12] using PDB isoforms (molecular graphics and analyses performed with UCSF Chimera [12] using PDB accession accession codes 2ZAV [13] and 1PQ3 [9]). (B) Schematic overview of the most relevant active-site codes 2ZAV [13] and 1PQ3 [9]). (B) Schematic overview of the most relevant active-site amino acid amino acid residues involved in catalytic plasticity,