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biomolecules Article 0 P1 Residue-Oriented Virtual Screening for Potent and Selective Phosphinic (Dehydro) Dipeptide Inhibitors of Metallo-Aminopeptidases Michał Talma and Artur Mucha * Department of Bioorganic Chemistry, Wrocław University of Science and Technology, Wybrze˙zeWyspia´nskiego 27, 50-370 Wrocław, Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-71-320-3446 Received: 20 March 2020; Accepted: 20 April 2020; Published: 24 April 2020 Abstract: Designing side chain substituents complementary to enzyme binding pockets is of great importance in the construction of potent and selective phosphinic dipeptide inhibitors of metallo-aminopeptidases. Proper structure selection makes inhibitor construction more economic, as the development process typically consists of multiple iterative preparation/bioassay steps. On the basis of these principles, using noncomplex computation and modeling methodologies, we comprehensively screened 900 commercial precursors of the P10 residues of phosphinic dipeptide and dehydrodipeptide analogs to identify the most promising ligands of 52 metallo-dependent aminopeptidases with known crystal structures. The results revealed several nonproteinogenic residues with an improved energy of binding compared with the best known inhibitors. The data are discussed taking into account the selectivity and stereochemical implications of the enzymes. Using this approach, we were able to identify nontrivial structural elements substituting the recognized phosphinic peptidomimetic scaffold of metallo-aminopeptidase inhibitors. Keywords: organophosphorus compounds; peptide analogs; enzyme inhibitors; ligand–enzyme interactions; molecular modeling and docking 1. Introduction Phosphinic dipeptides are generally acknowledged as transition-state analog inhibitors of metallo-aminopeptidases [1–3], a large and homogenous class of exopeptidases that specifically cleave the N-terminal amino acid residues of polypeptides and proteins and contain metal ion(s) in their active sites. Being ubiquitously distributed in subcellular organelles, cytoplasm, and cellular membranes throughout all of the biological kingdoms, these enzymes perform critical roles in physiological processes and diseases. In humans, they are responsible for processing peptides and proteins involved in, for example, angiogenesis, antigen presentation, cancer and metastasis, blood pressure regulation, and bacterial and protozoal infections [3–5]. The inhibitory potential of phosphinic dipeptides originates from two fundamental structural features that determine the competitive mechanism of binding, and govern the potency and specificity of the ligands. First, the presence of the central phosphinic functional group ensures steric and electronic resemblance to the scissile amide bond of hydrolyzed substrates in the tetrahedral transition state. This also involves the ability to coordinate with catalytic metal ion(s). Second, the optimized structures of the P1 and P10 side chains guarantee complementarity to the corresponding S1 and S10 binding pockets (encryptions according to the nomenclature proposed by Schechter and Berger to define the N-terminal, non-primed and C-terminal, primed residues of the scissile amide bond in peptide substrates, and the corresponding binding pockets [6]). Mammalian, protozoan, and bacterial enzymes of the M01 and M17 families have probably been the most comprehensively studied metallo-aminopeptidases with phosphinic dipeptides [2,5]. Biomolecules 2020, 10, 659; doi:10.3390/biom10040659 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, x 2 of 26 Biomolecules 2020, 10, 659 2 of 25 Mammalian, protozoan, and bacterial enzymes of the M01 and M17 families have probably been the most comprehensively studied metallo-aminopeptidases with phosphinic dipeptides [2,5]. Alanine aminopeptidaseaminopeptidase (aminopeptidase (aminopeptidase N, APN, N, M01.001,APN, M01.001, EC 3.4.11.2) EC and 3.4.11.2) leucine aminopeptidase and leucine (LAP,aminopeptidase M17.001, EC(LAP, 3.4.11.1) M17.001, can EC be 3.4.11.1) considered can be as considered the prototypical as the representatives.prototypical representatives. Importantly, Importantly,the greatest results the greatest of inhibition results of were inhibition achieved we forre achieved compounds for compounds comprising sidecomprising chains originatingside chains originatingfrom nonproteinogenic from nonproteinogenic amino acids. amino Such acids. fragments Such of fragments optimal ligandsof optimal are ligands not trivial are and not cantrivial hardly and canbe predicted hardly be by predicted directly translatingby directly thetranslating results of the studies results on of peptidyl studies substrates.on peptidyl These substrates. residues These can residuesbe conveniently can be conveniently designed with designed the aid ofwith molecular the aid of modeling molecular methods; modeling however, methods; this however, process maythis processdemand may a few demand iterations a few of iterations design, synthesis, of design, andsynthesis, evaluation. and evaluation. For example, For example, it took us it ten took years us ten to yearsinhibit (toK i)inhibit mammalian (Ki) APNmammalian and LAP byAPN phosphinic and LAP dipeptides by phosphinic at a low nanomolar dipeptides/subnanomolar at a low nanomolar/subnanomollevel [7,8]. ar level [7,8]. The synthesis of each individuallyindividually modifiedmodified metallo-aminopeptidasemetallo-aminopeptidase inhibitor requires the development of multistep synthetic pathways with orthogonal protective strategies, leading to fundamental building blocks. In In the the most most typical typical procedure, procedure, P1-specific P1-specific N-protected N-protected aminoalkyl-H- aminoalkyl-H- phosphinic acidacid and and P1 0-specificP1′-specificα-substituted α-substituted acrylate acrylate are such are components such components (Scheme 1)[(Scheme1]. Aldehydes, 1) [1]. whoseAldehydes, structures whose encode structures the P1 encode and P1 0 theresidues, P1 and are P1 primary′ residues, substrates are primary for the preparation substrates offor these the preparationsynthons. Thus, of these the proper synthons. and reliableThus, the selection proper of and the reliable fundamental selection starting of the materials fundamental is of invaluable starting materialssignificance is of as invaluable it results in significance either fruitful as orit results vain e ffinorts. either fruitful or vain efforts. Scheme 1. Retrosynthetic analysis of phosphinic dipeptidedipeptide and dehydrodipeptidedehydrodipeptide analogs. The P1 phenylethyl residueresidue (homophenylalanine) (homophenylalanine) is conservedis conserved as canonical. as canonical. The pathwaysThe pathways involve involve P10-specific P1′- specificelectrophiles electrophiles that can that be obtained can be obtained from aldehydes. from aldehydes. In the current work, we address the design issue to a virtual screening approach. In In particular, particular, the P1P10′ residuesresidues of of active active and and specific specific phosphinic phosphinic dipeptide dipeptide inhibitors inhibitors of metallo-aminopeptidases of metallo-aminopeptidases were weretargeted. targeted. A total A oftotal 900 of commercially 900 commercially available available aldehydes aldehydes were selected were selected as potential as potential precursors precursors of these ofP1 0theseresidues, P1′ residues, while the while P1 fragment the P1 frag (mimickingment (mimicking homophenylalanine) homophenylalanine) remained remained conserved. conserved. Docking Dockinganalysis wasanalysis performed was performed with 52 crystal with structures 52 crystal of 27structures enzymes. of In addition,27 enzymes. P10-dehydrodipeptide In addition, P1′- dehydrodipeptideanalogs were included analogs in the were study, included as the aldehydes in the stud mayy, as also the be aldehydes the substrates may ofalso the be P1 the0-unsaturated substrates ofcounterparts. the P1′-unsaturated One of the counterparts. methods for theirOne of preparation the methods involves for their substitution preparation of P1-specific involves substitution N-protected ofaminoalkyl-H-phosphinic P1-specific N-protected acids aminoalkyl-H-phosphinic with Morita–Baylis–Hillman acids with acetates Morita–Baylis–Hillman (Scheme1)[ 9]. The latteracetates are (Schemeobtained 1) by [9]. acetylation The latter ofare allyl obtained alcohols, by acetylation which, in turn, of allyl are alcohols, synthesized which, from in turn, aldehydes are synthesized and alkyl fromacrylates. aldehydes Similar toand the alkyl saturated acrylates. compounds, Simila phosphinicr to the dehydrodipeptidessaturated compounds, were reportedphosphinic as dehydrodipeptidesinhibitors of metallo-aminopeptidases, were reported as inhibitors although toof a metallo-aminopeptidases, lesser extent [10]. although to a lesser extentAs [10]. the main outcome of this study, we defined a set of novel nonproteinogenic P10 substituents that areAs potentialthe main structuraloutcome of fragments this study, of active we defined pseudodipeptide a set of novel inhibitors nonproteinogenic of metallo-aminopeptidases. P1′ substituents Thethat detailsare potential of interactions structural with thefragments target proteins of ac weretive analyzedpseudodipeptide at the molecular inhibitors level of by metallo- docking aminopeptidases.of the individual stereoisomersThe details of interactions in the enzyme with active the target sites. proteins Stereochemical were analyzed considerations

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