molecules

Review Human Copper-Containing Amine Oxidases in Design and Development

Serhii Vakal 1, Sirpa Jalkanen 2, Käthe M. Dahlström 1 and Tiina A. Salminen 1,* 1 Structural Bioinformatics Laboratory, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Tykistökatu 6A, FI-20520 Turku, Finland; serhii.vakal@abo.fi (S.V.); Kathe.Dahlstrom@abo.fi (K.M.D.) 2 MediCity Research Laboratory, University of Turku, Tykistökatu 6A, FI-20520 Turku, Finland; sirpa.jalkanen@utu.fi * Correspondence: tiina.salminen@abo.fi; Tel.: +358-40-515-1201

 Academic Editor: Aimin Liu  Received: 18 February 2020; Accepted: 10 March 2020; Published: 12 March 2020

Abstract: Two members of the copper-containing family are physiologically important : (1) (hDAO; AOC1) with a preference for diamines is involved in degradation of and (2) Vascular adhesion -1 (hVAP-1; AOC3) with a preference for monoamines is a multifunctional cell-surface receptor and an . hVAP-1-targeted inhibitors are designed to treat inflammatory diseases and cancer, whereas the off-target binding of the designed inhibitors to hDAO might result in adverse drug reactions. The X-ray structures for both human are solved and provide the basis for computer-aided inhibitor design, which has been reported by several research groups. Although the putative off-target effect of hDAO is less studied, computational methods could be easily utilized to avoid the binding of VAP-1-targeted inhibitors to hDAO. The choice of the model organism for preclinical testing of hVAP-1 inhibitors is not either trivial due to species-specific binding properties of designed inhibitors and different repertoire of copper-containing amine oxidase family members in mammalian species. Thus, the facts that should be considered in hVAP-1-targeted inhibitor design are discussed in light of the applied structural bioinformatics and structural biology approaches.

Keywords: copper-containing amine oxidases; vascular adhesion protein-1; diamine oxidase; inhibitor design; protein-inhibitor interactions; computer-aided drug design

1. Introduction In humans, the copper-containing amine oxidase (CAO) family consists of three proteins [1]. Diamine oxidase (hDAO; AOC1) prefers diamines and is involved in the degradation of histamine [2], while retina-specific amine oxidase (hRAO; AOC2) is a with enzymatic activity in retina [3], and vascular adhesion protein-1 (hVAP-1; AOC3) is a multifunctional cell-surface receptor and an enzyme with preference for monoamines [4]. Both hDAO and hVAP-1 are physiologically essential proteins, but little is known about the physiological function of hRAO. In the CAO-catalyzed reaction, primary amines are converted to the corresponding accompanied by the production of and (for a review, see [5]). The structural characterization of human CAOs has revealed a dimeric protein with a deeply buried active site characterized by highly conserved residues and several common features involved in the catalytic reaction, including a topaquinone (TPQ) and a conserved aspartate residue, as well as three conserved histidine residues that bind the copper ion crucial for the TPQ biogenesis (hVAP-1: [6–10]; hDAO: [11–13] (Figure1A,B). Three arms that protrude from one monomer to the other one stabilize the tight dimer of human CAOs, where each monomer consists of three domains, namely, D2, D3, and D4 (Figure1A). The D4 forms

Molecules 2020, 25, 1293; doi:10.3390/molecules25061293 www.mdpi.com/journal/molecules Molecules 2020, 25, 1293 2 of 29 Molecules 2020, 25, x FOR PEER REVIEW 2 of 28 thenamely deeply, D2, buried D3, and catalytic D4 (Figure site, and 1A). Arm The I D4 of monomerforms the Bdeeply (pink) buried contributes catalytic to ligand site, and binding Arm intoI of monomermonomer AB ( (cyan)pink) contributes (Figure1B). to ligand binding into monomer A (cyan) (Figure 1B).

FigureFigure 1.1. Structural features of human copper-containingcopper-containing amineamine oxidaseoxidase (CAO)(CAO) dimer.dimer. ((AA)) Side view β B ofof thethe hVAP-1hVAP-1 dimer dimer (PDB (PDB code code 4BTY). 4BTY) Three. Three long long-hairpin β-hairpin arms arms stabilize stabilize the tightthe tight dimer; dimer ( ) top; (B view) top of hVAP-1 shows the D2, D3, and D4 domains in each monomer. In the deeply buried active site, the view of hVAP-1 shows the D2, D3, and D4 domains in each monomer. In the deeply buried active copper ion (orange sphere) is coordinated by the three conserved histidine residues (H520, H522, and site, the copper ion (orange sphere) is coordinated by the three conserved histidine residues (H520, H684 in hVAP-1). TPQ (TPQ471) is shown in off-copper, productive conformation, and the catalytic H522, and H684 in hVAP-1). TPQ (TPQ471) is shown in off-copper, productive conformation, and aspartate (Asp386) resides next to the quinone cofactor. Arm I (pink) extending from D4 of monomer B the catalytic aspartate (Asp386) resides next to the quinone cofactor. Arm I (pink) extending from D4 (pink) forms one wall of the active site channel. The black arrow depicts the direction from the catalytic of monomer B (pink) forms one wall of the active site channel. The black arrow depicts the direction site via the active site channel formed by the D3 and D4 domains to the surface of the protein. from the catalytic site via the active site channel formed by the D3 and D4 domains to the surface of Inthe additionprotein. to its enzymatic activity, hVAP-1 is an adhesion protein involved in leukocyte trafficking, whose roles in leukocyte adhesion and inflammatory conditions are well characterized In addition to its enzymatic activity, hVAP-1 is an adhesion protein involved in leukocyte (for review see [14–17]). In adhesion, Sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9), a trafficking, whose roles in leukocyte adhesion and inflammatory conditions are well characterized leukocyte surface receptor [18], forms multivalent interactions with hVAP-1 on the endothelial cell (for review see [14–17]). In adhesion, Sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9), a surface [19]. Typically, healthy humans have a low level of hVAP-1 activity, but the levels are elevated leukocyte surface receptor [18], forms multivalent interactions with hVAP-1 on the endothelial cell upon inflammation when hVAP-1 is translocated onto the luminal surface of the endothelium [20–22]. surface [19]. Typically, healthy humans have a low level of hVAP-1 activity, but the levels are Therefore, molecules that bind to hVAP-1 can be used as tracers in positron emission tomography elevated upon when hVAP-1 is translocated onto the luminal surface of the (PET) to detect the site of inflammation in vivo [18]. Additionally, the anti-inflammatory potential endothelium [20–22]. Therefore, molecules that bind to hVAP-1 can be used as tracers in positron of VAP-1 inhibitors is shown in several inflammation models (reviewed in [17]), which have proven emission tomography (PET) to detect the site of inflammation in vivo [18]. Additionally, the that the enzymatic activity of hVAP-1 is crucial for leukocyte adhesion and especially required for anti-inflammatory potential of VAP-1 inhibitors is shown in several inflammation models (reviewed the hVAP-1-dependent extravasation in vivo [23]. Moreover, anti-hVAP-1 antibodies, which do not in [17]), which have proven that the enzymatic activity of hVAP-1 is crucial for leukocyte adhesion affect the enzymatic activity of hVAP-1, independently inhibit hVAP-1-mediated adhesion. Thus, both and especially required for the hVAP-1-dependent extravasation in vivo [23]. Moreover, anti-hVAP-1 antibodies and small molecular inhibitors could be used as anti-inflammatory to treat acute and antibodies, which do not affect the enzymatic activity of hVAP-1, independently inhibit hVAP-1-mediated adhesion. Thus, both antibodies and small molecular inhibitors could be used as anti-inflammatory drugs to treat acute and chronic inflammatory conditions. There is also

Molecules 2020, 25, 1293 3 of 29 chronic inflammatory conditions. There is also substantial evidence that hVAP-1 inhibitors could be utilized in preventing tumor progression and metastatic spread of cancer (reviewed in [14,17]). hDAO is the main enzyme in the extracellular degradation of histamine [24]. It is expressed in the , placenta, intestine, thymus, and seminal vesicles [25], and its functions are related to cell proliferation, inflammation, allergic response, and ischemia [11]. Decreased hDAO levels have been linked to histamine intolerance [26], and a decreased hDAO activity coupled to a high presence of histamine increases the probability for anaphylactic shock-like symptoms [27]. Normally, the plasma concentration of hDAO is elevated during pregnancy, and low levels of hDAO increase the risk of premature termination [28]. It is known that some pharmaceuticals, which are not targeted towards hDAO, inhibit its normal function (off-target effect) and thus cause harmful side effects (adverse drug reactions; ADRs) [11,27]. Consequently, it is a crucial drug safety issue to identify substances, which potently inhibit hDAO activity, to avoid their usage in the treatment of patients with hDAO deficiencies and conditions like varieties of urticaria, asthma, atopic dermatitis, mastocytosis, mastcell activation syndromes, and likely, pregnant women. This review concentrates on the structural chemistry of hVAP-1 inhibitors whose binding site has been either predicted by computational studies or deciphered by X-ray crystallography. The current knowledge of the off-target effect of hDAO inhibition is also summarized and discussed in light of the hVAP-1-targeted inhibitor design. Furthermore, we highlight important facts and considerations that need to be taken into account in the hVAP-1 inhibitor design process to obtain potent and selective drug molecules.

2. Catalytic Mechanism of Copper Amine Oxidases VAP-1 is a primary amine oxidase and, thus, catalyzes the oxidative deamination of primary amines, both exogenous (allylamine, benzylamine) and endogenous (methylamine and aminoacetone). According to modern concepts [29–31], the reaction proceeds through a ping-pong mechanism. In the reductive half-reaction, TPQ is reduced by the substrate to generate the product (Figure2A); then, in the oxidative half-reaction, the reduced TPQ is reoxidized by molecular oxygen, which is accompanied by a release of hydrogen peroxide and ammonia [4,32] (Figure2B). The overall reaction balance can be written as R-CH -NH + + H O + O R-COH + H O + NH +. 2 3 2 2 → 2 2 4 The reductive half-reaction comprises three steps (Figure2A); first, the oxidized TPQ reacts with a protonated primary amine to form a so-called “substrate Schiff base” (quinone imine), and then, this adduct is converted to a “product Schiff base” (quinolaldimine), which is facilitated by a conserved Asp-386 catalyzing proton abstraction [33]. In the final step, the aldehyde product (R-COH) is hydrolytically released generating the reduced TPQ (aminoquinol). TPQ coordinates the Cu(II) ions; two states named “off”-copper and “on”-copper have been characterized and shown to exist in equilibrium in solution [34]. In the “off”-copper conformation, oxidized TPQ does not interact with Cu(II), which is coordinated by three histidine residues and two water molecules, while in the “on”-copper state, the waters are displaced, and TPQ interacts with the copper. The oxidative half-reaction proceeds through four steps via one of two possible ways (Figure2B). In both scenarios, the intermediate molecules are the same, but the electron transfer pathway differs. Currently, it is not clear whether molecular oxygen reacts directly with reduced TPQ, TPQ semiquinone, or Cu(I) [31] as there is evidence for each option. In the first scenario, oxygen interacts with Cu(I) – bound to TPQ-semiquinone and then is reduced to a reactive O2• via inner-sphere electron transfer (Figure2B, upper scheme) [ 35]. Alternatively, according to Mure et al. [36], oxygen binds to hydrophobic residues near the reduced TPQ, which reduces O2 to O2•− via outer-sphere electron transfer. Then, in both scenarios, Cu(II)-hydroperoxide and TPQ-iminoquinone are generated [37]. At the final step, iminoquinone is hydrolyzed, and ammonia is released with the concomitant production of hydrogen peroxide and oxidized TPQ-Cu(II) regeneration. Molecules 2020, 25, 1293 4 of 29

Recent kinetic and computational studies suggest that a redox-active metal in copper amine oxidasesMolecules 20 is20 needed, 25, x FOR to PEER catalyze REVIEW the reduction of O2 to H2O2 [38]. Cu(I) reacts with O2 by inner-sphere4 of 28 electron transfer to eliminate charge accumulation due to O2•−, thus lowering the free energy barrier byby minimizing minimizing the the outer-sphere outer-sphere reorganization reorganization energy energy [39[39].]. Copper Copper also also facilitates facilitates long-range long-range proton-coupledproton-coupled electronelectron transfertransfer toto bindbind oxygenoxygen viavia thethe H-bondH-bond networknetwork [[3838].]. However,However, itit shouldshould bebe notednoted thatthat mostmost ofof thethe datadata onon catalyticcatalytic detailsdetails havehave beenbeen acquiredacquired usingusing non-mammaliannon-mammalian bacterial, plant,plant, andand fungalfungal amineamine oxidases,oxidases, primarilyprimarily EscherichiaEscherichia colicoli,, ArthrobacterArthrobacter globiformisglobiformis,, andand HansenulaHansenula polymorphapolymorpha orthologs.orthologs.

FigureFigure 2.2. ProposedProposed AOC AOC catalytic catalytic mechanism. mechanism. (A) Mechanism Mechanism of of reductive reductive half-reaction; half-reaction; ((BB)) twotwo proposedproposed mechanismsmechanisms ofof oxidativeoxidative half-reaction;half-reaction; inner-sphereinner-sphere electronelectron transfertransfer pathwaypathway isis shownshown above,above, whilewhile thethe outer-sphereouter-sphere isis below.below. ProteinProtein representedrepresented asas aa blueblue rectangle.rectangle. BasedBased onon thethe schemesschemes presentedpresented inin [[3131].]. 3. Medical Relevance of Targeting hVAP-1 3. Medical Relevance of Targeting hVAP-1 3.1. Basis for Clinical Targeting of VAP-1 3.1. Basis for Clinical Targeting of VAP-1 Due to the multifunctional nature of VAP-1 and its involvement in inflammation via adhesive leukocyte–endothelialDue to the multifunctional cell interactions nature and of production VAP-1 and of its end-products, involvement which in inflammation modulate other via adhesionadhesive andleukocyte signaling–endothelial molecules cell triggering interactions inflammation, and production VAP-1 inhibition of end- providesproducts, us which with a modulate novel approach other toadhesion overcome and several signaling diseases molecules having triggering inflammatory inflammation, components. VAP Moreover,-1 inhibition the enzymatic provides activity us with of a VAP-1novel modifies approach its to substrate overcome toan several aldehyde diseases that is having able to promote inflammatory the formation components of advanced. Moreover, glycation the end-productsenzymatic activity damaging of VAP vasculature,-1 modifies for example, its substrate in . to an Indeed, aldehyde a multitude that is able of preclinical to promote studies the usingformation mice, of rats, advanced and rabbits glycation have shown end-products beneficial damagingeffects of targeting vasculature VAP-1, for in several example disease, in diabetes. models. TheyIndeed, include a multitude autoimmune of preclinical and other studies inflammations, using mice, viral rats and, and bacterial rabbits infections, have shown ischemia-reperfusion beneficial effects injuries,of targeting fibrosis, VAP cancer,-1 in several and metabolic disease models diseases. They (reviewed include in autoimmune [17]). These studies,and other together , with the findingsviral and that bacterial hVAP-1 infections, is translocated ischemia to the-reperfusion endothelial injuries, cell surface fibrosis, from cancer intracellular, and metabolic storage granulesdiseases at(reviewed sites ofinflammation in [17]). These and studies that increased, together concentrations with the findings of hVAP-1 that hVAP are found-1 is intranslocated several diseases, to the formendothelial the basis cell for surface clinical targetingfrom intracellular of VAP-1 (reviewed storage granules in [17]). Moreover,at sites of easy inflammation accessibility ofand VAP-1 that onincreased inflamed concentrations endothelium for of potential hVAP-1 are imaging found agents in several makes diseasesVAP-1 an, form optimal the target basis to for search clinical for inflammatorytargeting of fociVAP that-1 (reviewed is often challenging in [17]). inMoreover, clinics. easy accessibility of VAP-1 on inflamed endothelium for potential imaging agents makes VAP-1 an optimal target to search for inflammatory foci that is often challenging in clinics.

3.2. Clinical Trials

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3.2. Clinical Trials After the discovery of leukocyte ligands for VAP-1 [18,40], a VAP-1 binding peptide of Siglec-9 has been developed as a novel imaging agent. This peptide binds to VAP-1-positive vessels in rheumatoid synovium [41], and the peptide conjugated with 68Ga-Dota has just recently successfully passed the phase I clinical trial [42]. It is going to further clinical trials meant to test this peptide as a diagnostic andMolecules follow-up 2020, 25, x FOR tool PEER for REVIEW arthritic lesions in PET imaging. 5 of 28 Molecules 2020, 25, x FOR PEER REVIEW 5 of 28 Several companies are also developing therapeutics to block the function of hVAP-1. They After the discovery of leukocyte ligands for VAP-1 [18,40], a VAP-1 binding peptide of Siglec-9 includeAfter both the discovery antibodies of leukocyte and small ligands molecular for VAP-1 inhibitors[18,40], a VAP (Table-1 binding1). Currently,peptide of Siglec active-9 clinical trials or has been developed as a novel imaging agent. This peptide binds to VAP-1-positive vessels in has been developed as a novel imaging agent. This peptide binds to VAP-1-positive vessels in completedrheumatoid trials synovium with [41 accessible], and the peptide information conjugated about with their 68Ga-Dota outcome has just are recently discussed successfully below. BioTie Therapies rheumatoid synovium [41], and the peptide conjugated with 68Ga-Dota has just recently successfully (currentlypassed the Acorda)phase I clinical developed trial [42]. a It fully is going human to further anti-hVAP-1 clinical trials antibody meant to test Timolumab this peptide (BTT1023).as a It has been passed the phase I clinical trial [42]. It is going to further clinical trials meant to test this peptide as a diagnostic and follow-up tool for arthritic lesions in PET imaging. well-tolerateddiagnostic and follow and shown-up tool for effi arthriticcacy both lesions in earlyin PET clinicalimaging. trials for rheumatoid arthritis and psoriasis. In Several companies are also developing therapeutics to block the function of hVAP-1. They contrast,Several a phase companies 2 proof-of-concept are also developing trial thera forpeutics 19 patients to block su theffering function from of hVAPprimary-1. They sclerosing cholangitis include both antibodies and small molecular inhibitors (Table 1). Currently, active clinical trials or include both antibodies and small molecular inhibitors (Table 1). Currently, active clinical trials or didcompleted not meet trials the with pre-defined accessible e informationfficacy criteria about in their the outcome interim are analysis, discussed and below. the trialBioTie was terminated [43]. completed trials with accessible information about their outcome are discussed below. BioTie TherapiesAstellas’ (currently ASP8232 Acorda) VAP-1 developed inhibitor a fully was testedhuman in anti placebo-controlled-hVAP-1 antibody Timolumab phase II trials for diabetic Therapies (currently Acorda) developed a fully human anti-hVAP-1 antibody Timolumab (BTT1023). It has been well-tolerated and shown efficacy both in early clinical trials for rheumatoid macular(BTT1023). edema It has been and well diabetic-tolerated nephropathy and shown efficacy [44,45 both]. In in macular early clinical edema, trials for this rheumatoid inhibitor was not effective, arthritis and psoriasis. In contrast, a phase 2 proof-of-concept trial for 19 patients suffering from althougharthritis and it e psoriasis.fficiently In inhibited contrast, a VAP-1 phase 2 enzymatic proof-of-concept activity trial in for plasma. 19 patients How suffering well the from orally administered primary sclerosing cholangitis did not meet the pre-defined efficacy criteria in the interim analysis, primary sclerosing cholangitis did not meet the pre-defined efficacy criteria in the interim analysis, inhibitorand the trial reached was terminated the vitreous [43]. fluid was not tested and the authors speculate that the route of delivery and the trial was terminated [43]. shouldAstellas’ be reassessed ASP8232 VAP in the-1 inhibitor future trials.was tested In contrast,in placebo- 12-weekcontrolled dailyphase treatmentII trials for diabetic with orally administered Astellas’ ASP8232 VAP-1 inhibitor was tested in placebo-controlled phase II trials for diabetic macular edema and diabetic nephropathy [44,45]. In macular edema, this inhibitor was not effective, ASP8232macular edema significantly and diabetic reduced nephropathy albuminuria [44,45]. In macular of diabetic edema patients., this inhibitor The was results not effective of this, trial suggest that although it efficiently inhibited VAP-1 enzymatic activity in plasma. How well the orally diabeticalthough kidney it efficiently disease inhibited can be VAP delayed-1 enzymatic by inhibiting activity inVAP-1 plasma. activity. How well In both the of orally these trials, ASP8232 administered inhibitor reached the vitreous fluid was not tested and the authors speculate that the administered inhibitor reached the vitreous fluid was not tested and the authors speculate that the wasroute safe of delivery and well-tolerated. should be reassessed in the future trials. In contrast, 12-week daily treatment with route of delivery should be reassessed in the future trials. In contrast, 12-week daily treatment with orallyPharmaxis administered developed ASP8232 significantly BXS4728A reduced to inhibit albuminuria VAP-1 of diabetic activity patients and. The performed results of a phase I clinical orally administered ASP8232 significantly reduced albuminuria of diabetic patients. The results of this trial suggest that diabetic kidney disease can be delayed by inhibiting VAP-1 activity. In both of trial.this trial Moreover, suggest that this diabetic compound kidney disease turned can outbe delayed to be by safe inhibiting and well-tolerated. VAP-1 activity. In both It is of orally bioavailable these trials, ASP8232 was safe and well-tolerated. andthese has trials, long-lasting ASP8232 wasinhibitory safe and well e-toleratedffects.. Boehringer-Ingelheim acquired this compound and tested Pharmaxis developed BXS4728A to inhibit VAP-1 activity and performed a phase I clinical trial. Pharmaxis developed BXS4728A to inhibit VAP-1 activity and performed a phase I clinical trial. itMoreover, in a phase this compound II trial for turned non-alcoholic out to be safe steatohepatitisand well-tolerated. (NASH)It is orally bioavailable that is a growingand has health problem Moreover, this compound turned out to be safe and well-tolerated. It is orally bioavailable and has connectedlong-lasting to inhibitory obesity effects. and type Boehringer 2 diabetes-Ingelheim and acquired can result this in compound permanent and livertested damage. it in a The study met long-lasting inhibitory effects. Boehringer-Ingelheim acquired this compound and tested it in a phase II trial for non-alcoholic steatohepatitis (NASH) that is a growing health problem connected to itsphase pre-specified II trial for non endpoints-alcoholic steatohepatitis including a (NASH) decrease that inis a NASH growing biomarkers health problem but connected due to to potentially harmful obesity and type 2 diabetes and can result in permanent liver damage. The study met its interactionsobesity and withtype 2 a diabetesdrug (not and defined can result in thein permanent press release) liver useddamage. by The NASH study patients, met its the company will pre-specified endpoints including a decrease in NASH biomarkers but due to potentially harmful pre-specified endpoints including a decrease in NASH biomarkers but due to potentially harmful notinteractions continue with to a develop drug (notthe defined inhibitor in the press to this release) group used of by patients. NASH patients, However, the company they continuewill to run phase interactions with a drug (not defined in the press release) used by NASH patients, the company will IInot trials continue for diabeticto develop retinopathy, the inhibitor to butthis group in this of multi-centerpatients. However, double-blind they continue studyto run phase involving 100 patients not continue to develop the inhibitor to this group of patients. However, they continue to run phase II trials for diabetic retinopathy, but in this multi-center double-blind study involving 100 patients withII trial moderatelys for diabetic retinopathy, severe non-proliferative but in this multi-center diabetic double retinopathy,-blind study involving the diabetic 100 patients macular edema is not with moderately severe non-proliferative diabetic retinopathy, the diabetic macular edema is not targetedwith moderately [46]. Currently, severe non also-proliferative Terns Pharmaceuticals diabetic retinopathy, runs the phase diabetic I trial macular for NASH edema is with not its VAP-1 inhibitor, targeted [46]. Currently, also Terns Pharmaceuticals runs phase I trial for NASH with its VAP-1 targeted [46]. Currently, also Terns Pharmaceuticals runs phase I trial for NASH with its VAP-1 TERN-201inhibitor, TERN (https:-201//www.ternspharma.com (https://www.ternspharma.com/pipeline)/pipeline).. Besides Besides these, these, several several other companies have inhibitor, TERN-201 (https://www.ternspharma.com/pipeline). Besides these, several other VAP-1-targetedcompanies have VAP programs-1-targeted in programs their pipeline in their forpipeline pulmonary for pulmonary diseases, diseases, pain, pain, uveitis, uveitis, multiple sclerosis, companies have VAP-1-targeted programs in their pipeline for pulmonary diseases, pain, uveitis, multiple sclerosis, inflammatory bowel diseases, Alzheimer’s disease, and Parkinson’s disease at inflammatorymultiple sclerosis, bowel inflammatory diseases, bowel Alzheimer’s diseases, Alzheimer disease,’s and disease Parkinson’s, and Parkinson’s disease disease at preclinical at or inactive preclinical or inactive state. Which one of these will end up in clinical trials remains to be seen. state.preclinical Which or inactive one of state. these Which will one end of upthese in will clinical end up trials in clinical remains trials remains to be seen. to be seen. Table 1. List of hVAP-1 inhibitors in ongoing or completed clinical trials as of 15 January 2020. TableTable 1. 1.ListList of hVAP of hVAP-1-1 inhibitors inhibitors in ongoing in or ongoing completed or clinical completed trials as clinical of 15 January trials 2020. as of 15 January 2020. Type of Name and Structure Type of Company Current Status Ref. Name and Structure Inhibition Company Current Status Ref. Name and Structure TypeInhibition of Inhibition Company Current Status Ref. FDA Approved in FDA Approved in Hydralazine 15.01.1953 FDAfor the Approved in 15.01.1953 Hydralazine Novartis 15.01.1953 for the Novartis treatment of for the treatment of Mechanism- InternationalNovartis treatment of Mechanism- International hypertension; laterhypertension; [47,48] later Mechanism-basedbased AG,International hypertension; AG, later [47,48] [47,48] based AG, discontinued duediscontinued to the due to the SwitzerlandSwitzerland discontinued due to the Switzerland development of newerdevelopment of newer development of newer medications. medications. medications. Discontinued; not LJP1207 Discontinued; notDiscontinued; not LJP1207LJP1207 appropriate for drug La Jolla La Jollaappropriate for drugappropriate for drug Mechanism- La Jolla development due to its Mechanism-basedMechanism- PharmaceuticalPharmaceutical development duedevelopment to its [49–53 due] to its [49–53] based Pharmaceutical potentially toxic [49–53] based Company,Company, USA USApotentially toxic potentially toxic Company, USA allylhydrazine allylhydrazine structureallylhydrazine structure structure ASP8232 R Tech Ueno Discontinued on Phase2 ASP8232 Unknown R Tech Ueno Discontinued on Phase2 [44,45] Undisclosed Structure Unknown Ltd., Japan; trials (NCT02302079, [44,45] Undisclosed Structure Ltd., Japan; trials (NCT02302079,

Molecules 2020, 25, 1293 6 of 29 Molecules 2020, 25, x FOR PEER REVIEW 6 of 28 Molecules 2020, 25, x FOR PEER REVIEW 6 of 28

Astellas NCT02218099) due to Astellas NCT02218099) due to TablePharma 1. Cont. Europe strategic prioritization Molecules 2020, 25, x FOR PEER REVIEW Pharma Europe strategic prioritization 6 of 28 BV, The Molecules 2020, 25, x FOR PEER REVIEW BV, The 6 of 28 Name and Structure Type of InhibitionNetherlands Company Current Status Ref. NetherlandsAstellas NCT02218099) due to PXS--4159A (R10)(R10) PXS-4159A (R10) PharmaAstellas EuropeR Tech UenostrategicNCT02218099) Ltd., prioritization Discontinueddue to on Phase2 ASP8232 PharmaBV, TheEuropeJapan; Astellasstrategic prioritizationtrials (NCT02302079, MechanismUnknown- Pharmaxis In preclinical toxicology [44,45] Undisclosed Structure Mechanism- NetherlandsPharmaxisBV, PharmaThe EuropeIn preclinical BV, toxicologyNCT02218099) [54] due to based Ltd., Australia evaluation [54] PXS-4159A (R10) based LtdNetherlands., AustraliaThe Netherlands evaluation strategic prioritization PXS-4159APXS-4159A (R10) (R10) Mechanism- Pharmaxis In preclinical toxicology

PXS 4728A (BI 1467335) [54] PXS 4728A (BI 1467335) Mechanismbased - LtdPharmaxis., AustraliaPharmaxis In Completedpreclinical Ltd.,evaluation toxicologyPhaseIn preclinicalII toxicology Mechanism-based Completed Phase II [54] [54] based Ltd., AustraliaAustralia clinicalevaluation trials for the evaluation clinical trials for the Mechanism- Pharmaxis treatment of Mechanism- Pharmaxis treatment of [55–57] based Ltd., Australia non-alcoholic [55–57] PXS 4728A (BI 1467335) based Ltd., Australia non-alcoholic Completedsteatohepatitis Phase II PXS 4728A (BI 1467335) steatohepatitis PXS 4728A (BI 1467335) clinicalCompleted(NCT03166735) trials Phase for the II (NCT03166735) Mechanism- Pharmaxis clinicaltreatment trialsCompleted for of the Phase II clinical PXS-4681A [55–57] PXS-4681A Mechanismbased - LtdPharmaxis., AustraliaPharmaxis Ltd.,nontreatment-alcoholic trialsof for the treatment of Mechanism-based [55–57] [55–57] based Ltd., AustraliaAustralia steatohepatitisnon-alcoholicnon-alcoholic steatohepatitis (NCT03166735)steatohepatitis (NCT03166735) Mechanism- Pharmaxis Mechanism- Pharmaxis (NCT03166735)Discontinued [58,59] PXS-4681A based Ltd., Australia Discontinued [58,59] based Ltd., Australia PXS-4681APXS-4681A

Mechanism- Pharmaxis Pharmaxis Ltd.,Discontinued [58,59] LJP1586 Mechanism-basedMechanismbased - LtdPharmaxis., Australia Discontinued [58,59] LJP1586 AustraliaDiscontinued [58,59] based LtdLa., Australia Jolla Mechanism- La Jolla [22,60– Mechanism- Pharmaceutical Discontinued [22,60– based Pharmaceutical Discontinued 64] based 64] Company, USA Company, USA LJP1586LJP1586

LJP1586 EliLa Lilly Jolla and La Jolla Mechanism- Eli Lilly and Phase I clinical trials for [22,60– Mechanism-basedPharmaceuticalCompany;La JollaPharmaceutical PhaseDiscontinued I clinical trials forDiscontinued [22,60–64] TERN-201 Mechanismbased - Company; the treatment of [22,6064] – TERN-201 Mechanism- Company,PharmaceuticalTernsCompany, USA USAtheDiscontinued treatment of [65] Undisclosed Structure based Terns non-alcoholic [6465]] Undisclosed Structure based Company,Pharmaceutical USA non-alcoholic PharmaceuticalEli Lilly and steatohepatitis s Eli LillyPhase andsteatohepatitis I clinicalPhase trials for I clinical trials for the TERN-201 EliCompany; Lillys and Mechanism-basedEliCambridge LillyCompany; and Terns treatment of non-alcoholic [65] UndisclosedTERN-201 Structure Mechanism- Cambridge Phasethe I treatmentclinical trials of for Company;Terns Completed Phase II [65] UndisclosedTERN- 201Structure Mechanismbased - Biotechnology,Pharmaceuticals Completedthenon treatment-alcoholic Phase of II steatohepatitis TERN-201 Mechanism- PharmaceuticalBiotechnology,Terns clinicalthe treatment trials for of the [65] PRX167700 TernsUK; clinical trials for the [65] UndisclosedPRX167700 Structure based UK; Cambridgesteatohepatitisnon-alcoholicCompleted Phase II clinical Unknown Pharmaceuticals treatmenttreatment ofof Undisclosed Structure Unknown Proximagen, steatohepatitistreatment of UndisclosedPRX167700 Structure Proximagen,sBiotechnology, steatohepatitisosteoarthritis UK; trials for the treatment of Unknown CambridgeUK; Roche,s osteoarthritis Undisclosed Structure UK; Roche,Proximagen, Completed(NCT01945346) UK; Phase II osteoarthritis Biotechnology,Cambridge (NCT01945346) Switzerland clinicalCompleted trials Phase for the II PRX167700 Biotechnology,SwitzerlandUK;Roche, Switzerland (NCT01945346) SzV--1287 Unknown clinicaltreatment trials for of the UndisclosedPRX167700SzV-1287SzV-1287 Structure Proximagen,UK; In preclinical studies; Unknown In preclinicalosteoarthritistreatment studies; of Undisclosed Structure Proximagen,UK; Roche, patented as a mean to patented(NCT01945346)osteoarthritis as a meanIn to preclinical studies; SemmelweisSwitzerlandUK; Roche, treat hyperalgesia and Mechanism- SemmelweisSemmelweis treat(NCT01945346) hyperalgesiapatented and as a mean to treat SzV-1287 Mechanism- SwitzerlandUniversity, allodynia in traumatic [66–69] Mechanism-basedbased University,University, allodyniaIn preclinical in traumatic hyperalgesiastudies; [66 and–69] allodynia [66–69] SzV-1287 based Hungary neuropathy or SzV-1287 Hungary Hungaryneuropathyin or traumatic neuropathy or patentedIn preclinicalneurogenic as a meanstudies; to neurogenicneurogenic inflammation Semmelweis treatpatentedinflammation hyperalgesia as a mean and to Mechanism- inflammation SemmelweisUniversity, treatallodynia hyperalgesia in traumatic and [66–69] Mechanismbased - U-V002 (R7) University,Hungary allodynianeuropathy in traumatic or [66–69] U-V002U-V002 (R7) (R7) based Hungary neuropathyneurogenic or inflammationneurogenic inflammation Mechanism- R Tech Ueno U-V002 (R7) Mechanism- R Tech UenoR Tech Ueno Ltd.,Unknown [70–73] Mechanism-basedbased Ltd., Japan; Unknown Unknown[70–73] [70–73] U-V002 (R7) based Ltd., Japan; Japan;

Mechanism- R Tech Ueno Unknown [70–73] Mechanismbased - RLtd Tech., Japan; Ueno Mechanism- R Tech Ueno Unknown [70–73] BTT-2027 Unknown [70–73] BTT-2027BTT-2027 based Ltd., Japan; Biotie Mechanism- BiotieBiotie Therapies Mechanism-basedMechanism- Therapies Discontinued Discontinued[20] [20] based Therapies Discontinued [20] based Corp. Corp. BTT-2027 Corp. Molecules 2020, 25, x FOR PEER REVIEW 7 of 28 BTT-2027 BTT-2027 Biotie BTT2052 Mechanism- Biotie Discontinued [74] BTT2052BTT2052 Mechanism- TherapiesBiotie Discontinued [7420] Mechanismbased - Therapies TherapiesCorp. Discontinued [20] based Corp.Biotie Therapies Mechanism-basedbased Corp. Discontinued [74] Corp. Corp. BTT2052 Mechanism- Biotie Discontinued [74]

BTT2052 Mechanism- Biotie Discontinued [74]

4. Structural Biology and Computer-Aided Drug Design in hVAP-1-Targeted Inhibitor Design As hVAP-1 is a suitable drug design target, several potential hVAP-1 inhibitors have been published including , thiazoles or other nitrogen-containing heterocyclic cores, allylamines and propargylamines, amino acid derivatives, benzamides, oxime-based inhibitors, aminoglycoside antibiotics, vitamin B1 derivatives, and peptides (Reviewed, e.g., in [32,75,76]). Nevertheless, most of the current hVAP-1 inhibitors are mechanism-based and bind irreversibly to the TPQ cofactor in a similar way as 2-hydrazinopyridine (2HP; PDB code 2C11) [7] (Figure 3). The irreversible binding mode is an undesirable feature for human drugs, since only new protein synthesis restores the function [77]. Thus, the design of reversible inhibitors specific for hVAP-1 would be highly beneficial. So far, pyridazinones (Figure 3; PDB codes 4BTY, 4BTX, and 4BTW; [10]), to our knowledge, are the only published inhibitors designed to have a reversible binding mode to hVAP-1. Figure 3 simultaneously shows both mechanism-based and reversible binding sites and modes via the overlaid 2HP and pyridazinone complex structures. It serves as a binding-site map for further discussion on the binding pose of the reviewed inhibitors for which the structural data is not publicly available (Table 1 and Table 2). Computer-aided drug design has been reported for several mechanism-based inhibitors (Table 2), and the results have provided foundations for some of the inhibitors that have entered clinical trials and/or have been extensively used in preclinical studies (Table 1).

Figure 3. Binding site map for the reviewed hVAP-1 inhibitors. The binding mode of mechanism-based inhibitors is depicted using the 2HP–hVAP-1 complex (PDB code 2C11; (A) mechanism-based; boxed in blue) as a representative structure and the binding mode of reversible inhibitors is illustrated using the overlaid pyridazinone–hVAP-1 complex (PDB code 4BTY; (B) reversible; boxed in red). Circled numbering 1–7 with color-coding pinpoint the common interaction sites in the catalytic site (blue box) and the active site channel (red box) of hVAP-1. The same coding scheme is used in Table 2.

Molecules 2020, 25, x FOR PEER REVIEW 7 of 28

based Therapies Corp.

Molecules 2020, 25, 1293 7 of 29

4. Structural Biology and Computer-Aided Drug Design in hVAP-1-Targeted Inhibitor Design 4. Structural Biology and Computer-Aided Drug Design in hVAP-1-Targeted Inhibitor Design As hVAP-1 is a suitable drug design target, several potential hVAP-1 inhibitors have been publishedAs hVAP-1 including is a suitable hydrazines, drug design thiazoles target, or several other potential nitrogen-containing hVAP-1 inhibitors heterocyclic have been cores, publishedallylamines including and propa hydrazines,rgylamines, thiazoles amino or other acid nitrogen-containing derivatives, benzamides, heterocyclic oxime cores,-based allylamines inhibitors, andaminoglycoside propargylamines, antibiotics, amino acid vitamin derivatives, B1 derivatives benzamides,, and oxime-basedpeptides (Reviewed inhibitors,, e.g. aminoglycoside, in [32,75,76]). antibiotics,Nevertheless, vitamin most B1 of derivatives,the current andhVAP peptides-1 inhibitors (Reviewed, are mechanism e.g., in [32-based,75,76 and]). Nevertheless, bind irreversibly most to ofthe the TPQ current cofactor hVAP-1 in a inhibitors similar way are as mechanism-based 2-hydrazinopyridine and bind (2HP; irreversibly PDB code to2C11) the TPQ[7] (Figure cofactor 3). in The a similarirreversible way as binding 2-hydrazinopyridine mode is an (2HP; undesirable PDB code feature 2C11) [ for7] (Figure human3). drugs The irreversible, since only binding new mode protein issynthesis an undesirable restores feature the function for human [77 drugs,]. Thus, since the only design new of protein reversible synthesis inhibitors restores specific the function for hVAP [77].-1 Thus,would the be designhighly ofbeneficial. reversible So far, inhibitors pyridazinones specific ( forFigure hVAP-1 3; PDB would codes be 4BTY highly, 4BTX beneficial., and 4BTW So; [ far,10]), pyridazinonesto our knowledge, (Figure are3; the PDB only codes published 4BTY, 4BTX, inhibitors and 4BTW;designed [ 10 to]), have to our a reversible knowledge, bind areing the mode only to publishedhVAP-1. inhibitorsFigure 3 simultaneously designed to have shows a reversible both mechanism binding mode-based to hVAP-1. and reversible Figure3 binding simultaneously sites and showsmodes both via the mechanism-based overlaid 2HP and and pyridazinone reversible bindingcomplex sites structures and modes. It serves via as the a binding overlaid-site 2HP map and for pyridazinonefurther discussion complex on structures.the binding It pose serves of asthe a binding-sitereviewed inhib mapitors for for further which discussion the structural on the data binding is not posepublicly of the available reviewed (Table inhibitors 1 and for Table which 2).the Computer structural-aided data drug is not design publicly has available been reported (Tables for1 and several2). Computer-aidedmechanism-based drug inhibitors design has (Table been 2 reported), and the for results several have mechanism-based provided foundations inhibitors for (Table some2), of and the theinhibitors results have that providedhave entered foundations clinical fortrials some and/or of the have inhibitors been extensively that have entered used in clinical preclinical trials andstudies/or have(Table been 1).extensively used in preclinical studies (Table1).

FigureFigure 3. Binding3. Binding site map site for map the reviewed for the hVAP-1 reviewed inhibitors. hVAP- The1 inhibitors. binding mode The of mechanism-basedbinding mode of inhibitorsmechanism is depicted-based inhibitors using the 2HP–hVAP-1is depicted using complex the (PDB 2HP– codehVAP 2C11;-1 complex (A) mechanism-based; (PDB code 2C11; boxed (A) inmechanism blue) as a representative-based; boxed structurein blue) as and a therepresentative binding mode structure of reversible and the inhibitors binding is mode illustrated of reversible using theinhibitors overlaid is pyridazinone–hVAP-1 illustrated using the complex overlaid (PDB pyridazinone code 4BTY;–hVAP (B) reversible;-1 complex boxed (PDB in code red). 4BTY; Circled (B) numberingreversible; 1–7 boxed with in color-coding red). Circled pinpoint numbering the common1–7 with interactioncolor-coding sites pinpoint in the catalyticthe common site (blueinteraction box) andsites the in active the catalytic site channel site (blue (red box) ofand hVAP-1. the active The site same channel coding (red scheme box) of is usedhVAP in-1. Table The2 same. coding scheme is used in Table 2.

Molecules 2020,, 25,, x FOR PEER REVIEW 8 of 28 Molecules 2020, 25, x FOR PEER REVIEW 8 of 28 Molecules 2020, 25, x FOR PEER REVIEW 8 of 28 MoleculesMolecules2020, 25 ,20 129320, 25, x FOR PEER REVIEW 8 of8 28 of 29 Table 2. List of computational studies modeling the interactionsinteractions between between various various inhibitors inhibitors and and Table 2. List of computational studies modeling the interactions between various inhibitors and hVAPTable-1 . 2.. List of computational studies modeling the interactions between various inhibitors and hVAPTable- 1 2.. List of computational studies modeling the interactions between various inhibitors and hVAP-1. Table 2.hVAPList of-1. computational studies modeling the interactions betweenUsed various inhibitors and hVAP-1. Used Docking Inhibitor Type of Inhibitor,, IC50IC50 VAPUsed-1 Docking Ref. Inhibitor Type of Inhibitor, IC50 VAP-1 DockingTool Ref. Inhibitor TypeType of of Inhibitor Inhibitor,, IC50 UsedStructureVAPUsed VAP-1-1 DockingTool Ref. Inhibitor Structure DockingTool Ref. Inhibitor TypeHydrazine of IC50Inhibitor Alcohols, IC50 StructureStructureVAP-1 Tool Ref. Alcohols Tool R1:: Compound 2a,,b Hydrazine Alcohols Structure R1: Compound 2a,b Hydrazine Alcohols R1: Compound 2a,b Hydrazine Alcohols Mechanism-based, R1R1: Compound: Compound 2a,,b Mechanism-based, 2C11 GOLD 3.1.1 [78] Mechanism0.04–0.15 µM-based, 2C11 GOLD 3.1.1 [78] Mechanism-based,Mechanism0.04–0.15 -µMbased, 2C11 GOLDGOLD 3.1.1 [78] 0.04–0.15 µM 2C112C11 GOLD 3.1.1 [[7878] ] 0.04–0.150.04–0.15 µµMM 3.1.1 Interacting residues:: Covalent bond between hydrazine nitrogen and TPQ; H-bonds between hydrazine nitrogen and thethe Interacting residues: Covalent bond between hydrazine nitrogen and TPQ; H-bonds between hydrazine nitrogen and the Interactinghydroxyl residues group: Covalent of R1 withbond TPQ between and D386;hydrazine hydrophobic nitrogen interactionsand TPQ; H -withbonds Y384, between F389, hydrazine Y394, L468, nitrogen L469. and the hydroxyl group of R1 with TPQ and D386; hydrophobic interactions with Y384, F389, Y394, L468, L469. InteractingInteracting residueshydroxyl :residues Covalent group: Covalent bond of R1 betweenbond with betweenTPQ hydrazine and hydrazine D386; nitrogenhydrophobic nitrogen and and interactions TPQ; TPQ; H-bonds H- bondswith betweenY384, between F389, hydrazine Y394, L468, nitrogen nitrogen L469. and and the the

hydroxylR2:: Compound group 8of R1 with TPQ and D386; hydrophobic interactions with Y384, F389, Y394, L468, L469. hydroxyl groupR2: Compound of R1 with 8 TPQ and D386; hydrophobic interactions with Y384, F389, Y394, L468, L469. R2R:2 Compound: Compound 88 R2: Compound 8

Mechanism-based, Mechanism-based, 2C11 GOLD 3.1.1 [78] Mechanism-based,Mechanism1.2 µM - based, 2C11 GOLDGOLD 3.1.1 [78] Mechanism1.2 µM-based, 2C112C11 GOLD 3.1.1 [[7878] ] 1.21.2µ µMM 2C11 GOLD3.1.1 3.1.1 [78] 1.2 µM

Interacting residues:: Covalent bond between hydrazine nitrogen and TPQ; H-bonds between nitrogen and TPQ and D386, and Interacting residues: Covalent bond between hydrazine nitrogen and TPQ; H-bonds between nitrogen and TPQ and D386, and Interacting residuesbetween: Covalent thethe bond hydroxyl between group hydrazine and D386; nitrogen hydrophobic and TPQ; interactions H-bonds withbetween Y384, nitrogen Y394, F389. and TPQ and D386, and Interacting residues: Covalentbetween bond the hydroxyl between group hydrazine and D386; nitrogen hydrophobic and TPQ; interactions H-bonds with between Y384, nitrogen Y394, F389. and TPQ and D386, Interacting residuesbetween: Covalent the bond hydroxyl between group hydrazine and D386; nitrogen hydrophobic and TPQ; interactions H-bonds between with Y384, nitrogen Y394, andF389. TPQ and D386, and and between the hydroxyl group and D386; hydrophobic interactions with Y384, Y394, F389. R3:: Compoundbetween 12 the hydroxyl group and D386; hydrophobic interactions with Y384, Y394, F389. R3R:3: Compound Compound 1212 R3: Compound 12 R3: Compound 12

Mechanism-based, Mechanism-based,Mechanism-based, 2C11 GOLDGOLD 3.1.1 [78] Mechanism0.26 µM- based, 2C112C11 GOLD 3.1.1 [78[78] ] Mechanism0.260.26 µµMM-based, 2C11 GOLD3.1.1 3.1.1 [78] 0.26 µM 2C11 GOLD 3.1.1 [78] 0.26 µM

Interacting residues:: Covalent bond between hydrazine nitrogen and TPQ; H-bonds between thethe hydroxyl group and D386; Interacting residues: Covalent bond between hydrazine nitrogen and TPQ; H-bonds between the hydroxyl group and D386; InteractingInteracting residues residues: Covalent: Covalent bond bond between betweenhydrophobic hydrazine hydrazine interactions nitrogennitrogen with and and Y384, TPQ; TPQ; Y394, H H-bonds-bonds F389. between between the hydroxyl the hydroxyl group group and D386; and hydrophobic interactions with Y384, Y394, F389. Interacting residues: CovalentD386; bond hydrophobicbetweenhydrophobic hydrazine interactions interactions nitrogen with withand Y384,TPQ; Y384, HY394, Y394,-bonds F389. F389.between the hydroxyl group and D386;

R4R4::: Compound 11a11a–d– d hydrophobic interactions with Y384, Y394, F389. R4: Compound 11a–d R4: Compound 11a–d R4: Compound 11a–d Mechanism-based,Mechanism-based, GOLD Mechanism-based, 2C112C11 GOLD 3.1.1 [78[78] ] Mechanism0.28–1.180.28–1.18 µM-µbased,M 2C11 GOLD3.1.1 3.1.1 [78] Mechanism0.28–1.18 -µMbased, 2C11 GOLD 3.1.1 [78] 0.28–1.18 µM 2C11 GOLD 3.1.1 [78] 0.28–1.18 µM

Interacting residues:: Covalent bond between hydrazine nitrogen and TPQ; 11a,b form H-bond with TPQ, while 11c,d with InteractingInteracting residues residues: Covalent: Covalent bond bond between between hydrazine hydrazine nitrogen nitrogen and and TPQ; TPQ; 11a,b 11a,b form form H-bond H-bond with TPQ, TPQ, while while 11c,d 11c,d with with D386;Interacting hydrophobic residues: interactionsCovalent bond with between A370, Y384, hydrazine F389, nitrogenY394, L468, and L469; TPQ; 11c,d 11a,b form form π H–π-bond interaction with TPQ, with while Y372 11c,dor Y384. with D386; hydrophobicD386; hydrophobic interactions interactions with A370,with A370, Y384, Y384, F389, F389, Y394, Y394, L468, L468, L469; L469; 11c,d 11c,d form form ππ––ππ interaction with with Y372 Y372 or orY384. Y384. InteractingD386; hydrophobic residues: C interactionsovalent bond with between A370, hydrazineY384, F389, nitrogen Y394, L468, and L469;TPQ; 11a,b11c,d form Hπ–-bondπ interaction with TPQ, with while Y372 11c,d or Y384. with

D386; hydrophobic interactions with A370, Y384, F389,ThiazolesThiazoles Y394, L468, L469; 11c,d form π–π interaction with Y372 or Y384. Thiazoles R5:: Compound 10 Thiazoles R5R:5: Compound Compound 1010 R5: Compound 10 Thiazoles R5: Compound 10 Mechanism-based,Mechanism-based, Mechanism-based, 2C112C11 GOLD GOLD 5.0 5.0[79 [79] ] Mechanism0.23 µMµM- based, 2C11 GOLD 5.0 [79] Mechanism0.23 µM-based, 2C11 GOLD 5.0 [79] 0.23 µM 2C11 GOLD 5.0 [79] 0.23 µM

InteractingInteracting residues residues: Covalent:: Covalent bond bond between between guanidine group and and TPQ; TPQ; H- H-bondbond between between guanidine guanidine group group and D386; and S D386;–O Interacting residues: Covalent bond between guanidine group and TPQ; H-bond between guanidine group and D386; S–O S–O interactioninteractioninteractionInteractingof ofof residues sulfursulfursulfur : atom atom Covalent inin in thethe thebond thiazolethiazole thiazole between ringring ring guanidinewithwith with T210T210 T210 backbonebackbonegroup backbone and carbonylcarbonyl TPQ; carbonyl H- oxygen;oxygen;bond between oxygen; protonproton guanidine– proton–π interactioninteractionπ groupinteraction ofof andamideamide D386; moietymoiety of amideS–O Moleculesinteraction 2020 of, 2 sulfur5, x FOR atom PEER in the REVIEW thiazole ring with T210 backbone carbonyl oxygen; proton–π interaction of amide moiety9 of 28 interactionInteracting ofresidues sulfur: Catomovalent in the bond thiazole between ring guanidine withmoiety T210with group withbackbone Y176. Y176.and carbonylTPQ; H-bond oxygen; between proton guanidine–π interaction group of and amide D386; moiety S–O with Y176. interactionR6: of Compound sulfur atom 35ain the thiazole ring with T210with backbone Y176. carbonyl oxygen; proton–π interaction of amide moiety

with Y176.

Mechanism-based, R6:: Compound 35a Mechanism-based, 2C11 GOLD 5.0 [80 ] R6: Compound 35a Mechanism34 nM -based, 2C11 GOLD 5.0 [80] R6: Compound 35a 34 nM 2C11 GOLD 5.0 [ 80 ] Mechanism-based,Mechanism34 nM- based, R6: Compound 35a 2C112C11 GOLD GOLD 5.0 5.0 [ [ 8080 ] ] 3434 nM nM

Interacting residues: Covalent bond between guanidine group and TPQ; π–proton interactions of sulfonyl moiety with L447, amide nitrogen with Y176, and guanidine nitrogen with Y384.

R7: Compound 35c

Mechanism-based, 2C11 GOLD 5.0 [80] 20 nM

Interacting residues: Covalent bond between guanidine group and TPQ; H-bonds between sulfonyl moiety with L447 and D446, π–proton interactions of sulfonyl moiety with L447, amide nitrogen with Y176, and guanidine nitrogen with Y384.

Indanols BTT2052

Mechanism-based, Manual 1US1 [74] 0.06 µM docking

Interacting residues: Covalent bond between hydrazine nitrogen and TPQ; H-bond between the hydroxyl group and D386; hydrophobic interactions with M211, Y384, F389, L469.

1H-imidazol-2-amines R8: Compound 19

Mechanism-based, 2C11 GOLD 5.1 [81] 32 µM

Interacting residues: Covalent bond between the amine group and TPQ; H-bond between NH of the imidazole ring with N470; π–π interaction between imidazole ring and Y384; π–proton interaction of benzene ring with L469.

R9: Compound 37b

Mechanism-based, 2C11 GOLD 5.1 [81] 19 nM

Interacting residues: Covalent bond between the amine group and TPQ; π–proton interactions of thiazole group with T212 and L447; S-O interaction of thiazole sulfur with T210 backbone carbonyl oxygen; proton–π interaction between amide moiety and Y176; CH–O interaction of acetyl moiety and D180.

Allylamines Mechanism-based, MOE R10: Compound 28 ? [54] 10 nM 2011.10

MoleculesMolecules 20 202020, 2, 52,5 x, xFOR FOR PEER PEER REVIEW REVIEW 9 9of of 28 28 Molecules 2020, 25, x FOR PEER REVIEW 9 of 28 Molecules 2020, 25, x FOR PEER REVIEW 9 of 28

Molecules 2020, 25, 1293 9 of 29

Table 2. Cont.

InteractingInteracting residues residues: C: Covalent ovalent bond bond between between guanidine guanidine group group and and TPQ; TPQ; π –πp–rotonproton interactions interactions of of sulfonyl sulfonyl moiety moiety with with L447, L447, Type of Inhibitor, Used VAP-1 Docking Interacting residuesInhibitor: Covalent amidebondamide between nitrogen nitrogen guanidine with with Y176, Y176, group and and guanidine and guanidine TPQ; nitrogenπ nitrogen–proton with interactionswith Y384. Y384. of sulfonyl moiety with Ref.L447, Interacting residues: Covalent bond between guanidine group andIC50 TPQ; π–proton interactionsStructure of sulfonyl moietyTool with L447, amide nitrogen with Y176, and guanidine nitrogen with Y384. amide nitrogen with Y176, and guanidine nitrogenπ with Y384. Interacting residuesRR7:7 Compound:: Compound Covalent 35c bond35c between guanidine group and TPQ; –proton interactions of sulfonyl moiety with R7: CompoundL447, 35c amide nitrogen with Y176, and guanidine nitrogen with Y384. RR77: Compound: Compound 35c 35c

Mechanism-based, Mechanism-based, 2C11 GOLD 5.0 [80] Mechanism20 nM -based, 2C11 GOLD 5.0 [80] 20 nM 2C11 GOLD 5.0 [80] MechanismMechanism-based,20 -nMbased, 2C112C11 GOLD GOLD 5.0 5.0[80] [80] 2020 nM nM

InteractingInteracting residues residues: C: Covalentovalent bond bond between between guanidine guanidine group group and and TPQ; TPQ; H H-bonds-bonds between between sulfonyl sulfonyl moiety moiety with with L447 L447 and and D446,D446,Interacting π –πp–rotonproton residues interactions interactions: Covalent of of bondsulfonyl sulfonyl between moiety moiety guanidine with with L447, L447, group amide amide and nitrogen nitrogenTPQ; H with- bondswith Y176, Y176, between and and guanidine sulfonylguanidine moietynitrogen nitrogen with with with L447 Y384. Y384. and Interacting residues: Covalent bond between guanidine group and TPQ; H-bonds between sulfonyl moiety with L447 and InteractingD446, residues π–proton: Covalent interactions bond of between sulfonyl guanidinemoiety with group L447, andamide TPQ; nitrogen H-bonds with Y176, between and sulfonylguanidine moiety nitrogen with with L447 Y384. and D446, π–proton interactions of sulfonyl moiety with L447, amide nitrogen with Y176, and guanidine nitrogen with Y384. D446, π–proton interactions of sulfonyl moiety with L447, amide nitrogen with Y176, and guanidine nitrogen with Y384.

IndanolsIndanols Indanols BTTBTT20522052 Indanols Indanols BTT2052BTT2052 BTT2052 MechanismMechanism-based,-based, ManualManual Mechanism-based, 1US11US1 Manual [74[74] ] Mechanism0.060.06 µM µM- based, 1US1 dockingdockingManual [74] Mechanism-based,µ 1US1 Manual [74] 0.060.06 µMM 1US1 dockingdocking [74] 0.06 µM docking

InteractingInteracting residues residues: C: Covalentovalent bond bond between between hydrazine hydrazine nitrogen nitrogen and and TPQ; TPQ; H H-bond-bond between between the the hydroxyl hydroxyl group group and and D386; D386; Interacting residues: Covalent bond between hydrazine nitrogen and TPQ; H-bond between the hydroxyl group and Interacting residues: Covalent bondhydrophobichydrophobic between hydrazine interactions interactions nitrogen with with M211, andM211, TPQ; Y384, Y384, H F389,- bondF389, L469. betweenL469. the hydroxyl group and D386; Interacting residues: CovalentD386; bond hydrophobic between hydrazine interactions nitrogen with and TPQ; M211, H- Y384,bond between F389, L469. the hydroxyl group and D386; hydrophobic interactions with M211, Y384, F389, L469. hydrophobic interactions with M211, Y384, F389, L469. 1H1-imidazol-2-aminesH1H-imidazol-imidazol- 2--2amines-amines

RR8:8 Compound: Compound 19 19 1H-imidazol-2-amines R8: Compound 19 1H-imidazol-2-amines R8: Compound 19 R8: Compound 19 MechanismMechanism-based,-based, Mechanism-based, 2C112C11 GOLDGOLD 5.1 5.1 [81[81] ] Mechanism3232 µM µM - based, 2C11 GOLD 5.1 [81] Mechanism32 µ-based,M 2C11 GOLD 5.1 [81] 32 µM 2C11 GOLD 5.1 [81] 32 µM Interacting residues: Covalent bond between the amine group and TPQ; H-bond between NH of the imidazole ring with N470; InteractingInteracting residues residues: Covalent: Covalent bond bond between between the the amine amine group and and TPQ; TPQ; H H-bond-bond between between NH NHof the of imidazole the imidazole ring with ring N470; with Interacting residuesππ–π–π interaction interaction: Covalent between betweenbond between imidazole imidazole the ringamine ring and and group Y384; Y384; and π π– pTPQ;–rotonproton H interaction- bondinteraction between of of benzene benzene NH of ringthe ring imidazole with with L469. L469. ring with N470; InteractingN470; residuesπ–π interaction: Covalent betweenbond between imidazole the amine ring group and Y384;and TPQ;π–proton H-bond interactionbetween NH of of benzene the imidazole ring ring with with L469. N470; R9: πCompou–π interactionnd 37b between imidazole ring and Y384; π–proton interaction of benzene ring with L469. R9π–:π Compound interaction between37b imidazole ring and Y384; π–proton interaction of benzene ring with L469. R9: Compound 37b R9: Compound 37b R9: Compound 37b MechanismMechanism-based,Mechanism-based,-based, 2C112C112C11 GOLDGOLD GOLD 5.1 5.1 5.1[81[81 []81 ] ] Mechanism191919 nM nM - based, Mechanism-based, 2C11 GOLD 5.1 [81] 19 nM 2C11 GOLD 5.1 [81] 19 nM

InteractingInteractingInteracting residues residues residues: Covalent: : C Covalentovalent bond bond bond between between between thethe the amineamine amine group group group and andand TPQ; TPQ; TPQ; π π–πp––protonrotonproton interactions interactions interactions of of thiazole thiazole of thiazole group group groupwith with T212 T212 with T212 andInteractingandand L447; L447; L447; S-O Sresidues -SO-O interaction interaction: Covalent of of thiazole thiazolebond thiazole between sulfur sulfur sulfur with withthe with T210amine T210 T210 backbone groupbackbone backbone and carbonyl carbonyl TPQ; carbonyl π oxygen;– poxygen;roton oxygen; protoninteractions proton–π– proton–π interaction interaction of thiazoleπ interaction between between group withamide amide between T212 Interacting residues: Covalent bond between the amine group and TPQ; π–proton interactions of thiazole group with T212 and L447; S-O interactionamide moiety ofmoietymoiety thiazole and and sulfur Y176; Y176; withCH CH–OCH– OT210–O interaction interaction interaction backbone of ofcarbonyl acetyl of acetyl acetyl moiety moiety oxygen; moiety and and proton D180.and D180.– D180.π interaction between amide and L447; S-O interaction of thiazole sulfur with T210 backbone carbonyl oxygen; proton–π interaction between amide moiety and Y176; CH–O interaction of acetyl moiety and D180. Molecules 2020, 25, x FOR PEERmoiety REVIEW and Y176; CH–OAllylamines interaction of acetyl moiety and D180. 10 of 28 AllylaminesAllylamines

R10: Compound 28 MechanismAllylamines-based, MOE R10: Compound 28 Mechanism-based, ? MOE [54] R10: Compound 28 Allylamines ? [54] Mechanism1010 nM nM - based, 2011.102011.10MOE R10: Compound 28 Mechanism-based, ? MOE [54] R10: Compound 28 10 nM ? 2011.10 [54] Mechanism-based,10 nM 2011.10MOE ? [54] 10 nM 2011.10

Interacting residues: Covalent bond between the amine group and TPQ; Hydrophobic interactions with F173 and Y394. Interacting residues: Covalent bond between the amine group and TPQ; Hydrophobic interactions with F173 and Y394.

Phenyl-piperidinyl amine R11: ELP12

Reversible, AutoDock 2C10 [82] Ki(EI)111 µM/Ki(ESI) = 163 µM 4.2

Interacting residues: Salt bridges between amino groups of R11 with the carboxylates of D180 and D446 and H-bonds with the side-chain of Y448 and with the backbone carbonyl of T424; an aromatic ring is stacked to the ring of Y176; hydrophobic interactions with F173, Y394, P397, I425, and L447; H-bond with Y394.

Glycine amides R12: Compound 4a

Mechanism-based, 2C11 GOLD 5.2 [83] 0.80 µM

Interacting residues: Covalent bond between the primary amine group and TPQ; CH–p interaction with L447, CH–O interaction with the L468 backbone carbonyl oxygen, arene--H interaction with L447.

R13: Compound 4g

Mechanism-based, 2C11 GOLD 5.2 [83] 0.34 µM

Interacting residues: Covalent bond between the primary amine group and TPQ; CH–O interaction with the L468 backbone carbonyl oxygen, arene–H interaction with L447.

R14: Compound 17h

Mechanism-based, 2C11 GOLD 5.2 [84] 25 nM

Interacting residues: Covalent bond between primary amine group and TPQ; Three CH–p interactions: central phenyl ring with L469, pyrimidine ring with L447, and the hydrogen in the piperazine with Y394; two CH–O interactions: the oxygen in the glycine amide moiety with L468, and the hydrogen of the carbonyl group in the glycine amide moiety with the carbonyl oxygen of the L468 backbone; chloro group formed a halogen–O interaction with the carbonyl oxygen of the D446 backbone.

Molecules 2020, 25, x FOR PEER REVIEW 10 of 28 Molecules 2020, 25, x FOR PEER REVIEW 10 of 28 Molecules 2020, 25, x FOR PEER REVIEW 10 of 28 Molecules 2020, 25, x FOR PEER REVIEW 10 of 28

Molecules 2020, 25, 1293 10 of 29

Table 2. Cont. Interacting residues: Covalent bond between the amine group and TPQ; Hydrophobic interactions with F173 and Y394. Interacting residues: Covalent bond between the amine group and TPQ; Hydrophobic interactions with F173 and Y394. Interacting residues: Covalent bond between the amineType group of Inhibitor, and TPQ; HydrophobicUsed VAP-1 interactions withDocking F173 and Y394. InteractingInhibitor residues: Covalent bond betweenPhenyl the amine-piperidinyl group and amine TPQ; Hydrophobic interactions with F173 and Y394.Ref. IC50 Structure Tool R11: ELP12 Phenyl-piperidinyl amine Phenyl-piperidinylPhenyl-piperidinyl amine amine R11: ELP12 Phenyl-piperidinyl amine R11: ELP12 R11R11: ELP12: ELP12

Reversible, AutoDock 2C10 [82] Ki(EI)111Reversible, µM/Ki(ESI) = 163 µM AutoDock4.2 Reversible,Reversible, 2C10 AutoDock [82] Ki(EI)111 µMReversible,/Ki(ESI) = 163 µM 2C10 AutoDock4.2AutoDock [82] Ki(EI)Ki111(EI) 111µM/µKiM(ESI)/Ki = (ESI)163 µM 2C10 4.2 [[82] Ki(EI)111 µM/Ki(ESI) = 163 µM 4.24.2 = 163 µM

Interacting residues: Salt bridges between amino groups of R11 with the carboxylates of D180 and D446 and H-bonds with the Interactingside-chain residues: of Y448 Salt and bridges with thebetween backbone amino carbonyl groups ofof T424;R11 with an aromatic the carboxylates ring is stacked of D180 to andthe ringD446 of and Y176; H- bondshydrophobic with the Interacting residues: Salt bridges between amino groups of R11 with the carboxylates of D180 and D446 and H-bonds with the InteractingsideInteracting-chain residues of residues: Y448: Salt and S bridgesalt with bridgesinteractions the between backbonebetween with amino amino carbonyl F173, groups groups Y394, of T424; P397, of R11 an I425, aromaticwithwith and the the L447; carboxylatesring carboxylates His- bondstacked withof toD180 of theY394. D180 andring andD446of Y176; D446and hydrophobic H and-bonds H-bonds with the side-chain of Y448 and with the backbone carbonyl of T424; an aromatic ring is stacked to the ring of Y176; hydrophobic with theside side-chain-chain of ofY448 Y448 andinteractions and with with the backbone the with backbone F173, carbonyl Y394, carbonyl P397,of T424; I425, of an T424;and aromatic L447; an aromatic ringH-bond is stacked with ring Y394. to is stackedthe ring of to Y176; the ring hydrophobic of Y176; interactions with F173, Y394, P397, I425, and L447; H-bond with Y394. hydrophobic interactionsinteractions with with F173, F173, Y394,Glycine Y394, P397, P397, amides I425, I425, and andL447; L447; H-bond H-bond with Y394. with Y394.

R12: Compound 4a Glycine amides GlycineGlycine amides amides R12: Compound 4a Glycine amides R12: Compound 4a R12R:12 Compound: Compound4a 4a Mechanism-based, 2C11 GOLD 5.2 [83] Mechanism0.80 µM-based, Mechanism-based,Mechanism-based, 2C11 GOLD 5.2 [83] Mechanism0.80 µM -based, 2C112C11 GOLD GOLD 5.2 5.2 [83 [83] ] 0.800.80 µµMM 2C11 GOLD 5.2 [83] 0.80 µM Interacting residues: Covalent bond between the primary amine group and TPQ; CH–p interaction with L447, CH–O Interacting residues:interaction Covalent with bond the between L468 backbone the primary carbonyl amine oxygen, group areneand TPQ;--H interaction CH–p interaction with L447. with L447, CH–O InteractingInteracting residues: residues: Covalent Covalent bond betweenbond between the primarythe primary amine amine group group and and TPQ; TPQ; CH–pCH–p interaction interaction with with L447, L447, CH CH–O–O InteractingR13: Compound residues:interaction C 4govalent with thebond L468 between backbone the primarycarbonyl amine oxygen, group arene and--H TPQ; interaction CH–p withinteraction L447. with L447, CH–O interactioninteraction with the with L468 the L468 backbone backbone carbonyl carbonyl oxygen, oxygen, arene–H arene--H interaction interaction with with L447. L447. R13: Compoundinteraction 4g with the L468 backbone carbonyl oxygen, arene--H interaction with L447. R13: Compound 4g

R13: Compound 4g R13: Compound 4g Mechanism-based, Mechanism-based, 2C11 GOLD 5.2 [83] Mechanism0.34 µM-based, 2C11 GOLD 5.2 [83] Mechanism0.34 µM-based, 2C11 GOLD 5.2 [83] Mechanism0.34 µM -based, 2C11 GOLD 5.2 [83] 0.34 µM 2C11 GOLD 5.2 [83] 0.34 µM

Interacting residues: Covalent bond between the primary amine group and TPQ; CH–O interaction with the L468 backbone InteractingInteracting residues residues:: CovalentCovalent bond bond between betweencarbonyl the theoxygen, primary primary arene amine amine–H group interaction group and TPQ; andwith CH TPQ;L447.–O CH–Ointeraction interaction with the L468 with backbone the L468 Interacting residues: Covalent bond between the primary amine group and TPQ; CH–O interaction with the L468 backbone Interacting residues: Covalentbackbone bondcarbonyl between carbonyl oxygen, the oxygen, primary arene arene–H amine–H interaction group interaction and with TPQ; L447. with CH – L447.O interaction with the L468 backbone R14: Compound 17h carbonyl oxygen, arene–H interaction with L447. R14: Compound 17h carbonyl oxygen, arene–H interaction with L447.

R14: Compound 17h R14: Compound 17h R14: Compound 17h Mechanism-based,Mechanism-based, 2C112C11 GOLD GOLD 5.2 5.2[84 [84] ] Mechanism2525 nM nM-based, Mechanism-based, 2C11 GOLD 5.2 [84] Mechanism25 nM -based, 2C11 GOLD 5.2 [84] 25 nM 2C11 GOLD 5.2 [84] 25 nM

InteractingInteracting residues residues:: Covalent Covalent bond bond between between primary primary amineamine group group and and TPQ; TPQ; Three Three CH CH–p–p interactions: interactions: central central phenyl phenylring withInteracting L469, pyrimidineresidues: Covalent ring with bond L447, between and the primary hydrogen amine in thegroup piperazine and TPQ; with Three Y394; CH two–p interactions: CH–O interactions: central phenylthe oxygen ring in ring withInteracting L469, pyrimidine residues: Covalent ring with bond L447, between and primary the hydrogen amine group in the and piperazine TPQ; Three with CH Y394;–p interactions: two CH–O central interactions: phenyl ring the withthe glycine InteractingL469, pyrimidine amide residues: moiety ring Covalent with with L468, L447, bond and and between the the hydrogen hydrogen primary of inamine the the carbonyl piperazinegroup and group withTPQ; in Y394; Threethe glycine two CH CH–p amide interactions:–O interactions: moiety centralwith the the phenyloxygen carbonyl ringin oxygenwith in L469, the glycine pyrimidine amide ring moiety with L447, with and L468, the hydrogen and the hydrogen in the piperazine of the carbonylwith Y394; grouptwo CH in–O the interactions: glycine amide the oxygen moiety in theoxygen withglycine ofL469, the amide pyrimidineL468 moiety backbone; withring chlorowithL468, L447, andgroup theand formed hydrogen the hydrogen a halogen of the in carbonyl– theO interaction piperazine group with inwith the the Y394; glycine carbonyl two amide CH oxygen– Omoi interactions: etyof the with D446 the the backbone.carbonyl oxygen in with thethe carbonyl glycine amide oxygen moiety of the with L468 L468, backbone; and the hydrogen chloro group of the formed carbonyl a group halogen–O in the glycine interaction amide with moi theety with carbonyl the carbonyl oxygen oxygenthe glycine of the L468 amide backbone; moiety with chloro L468, group and formed the hydrogen a halogen of –theO interactioncarbonyl group with inthe the carbonyl glycine oxygen amide moiof theety D446 with backbone.the carbonyl oxygen of the L468 backbone; chloro group formedof the a halogen D446 backbone.–O interaction with the carbonyl oxygen of the D446 backbone. oxygen of the L468 backbone; chloro group formed a halogen–O interaction with the carbonyl oxygen of the D446 backbone.

4.1. X-Ray Structures of hVAP-1Complexes for Docking Studies and Inspiration for Drug Design Most of the docking studies (Table2, Table S1) have been conducted using the 2-hydrazinopyridine (2HP) complex of hVAP-1 as the target protein (PDB code 2C11) [7] and, thus, the focus is on the special features of this structure and its comparison to other solved structures. Moreover, the crystal structures of hVAP-1 in complex with imidazole (PDB codes 2Y73 and 2Y74; [9]) are presented as Molecules 2020, 25, x FOR PEER REVIEW 11 of 28 Molecules 2020, 25, x FOR PEER REVIEW 11 of 28 Molecules 2020, 25, x FOR PEER REVIEW 11 of 28 Molecules 2020, 25, x FOR PEER REVIEW 11 of 28 4.1.4.1. X -XR-ayRay Structures Structures of ofhVAP hVAP-1 -C1omplexes Complexes for for Docking Docking Studies Studies and and Inspiration Inspiration for for Drug Drug Design Design 4.1. X-Ray Structures of hVAP-1 Complexes for Docking Studies and Inspiration for Drug Design 4.1. X-Ray Structures of hVAP-1 Complexes for Docking Studies and Inspiration for Drug Design MostMost of of the the docking docking studies studies ( Table (Table 2, 2, Table Table S1 S1) ) have have been been conducted conducted using using the the 2-hydrazinopyridineMost of the docking (2HP) complexstudies of (Table hVAP - 21, as Table the target S1) protein have been(PDB conductedcode 2C11) [7] using and , thethus, 2-hydrazinopyridineMost of the docking(2HP) complex studies of hVAP (Table- 1 2as, the Table target S1 )protein have (PDB been code conducted 2C11) [7] usingand, thus, the Molecules 20202-hydrazinopyridinethe, 25 focus, 1293 is on the (2HP)special complex features of of hVAP this structure-1 as the targetand its protein comparison (PDB codeto other 2C11) solved [7] and structures, thus, 11. of 29 2the-hydrazinopyridine focus is on the special (2HP) features complex of of this hVAP structure-1 as the and target its comparisonprotein (PDB to code other 2C11) solved [7] structures and, thus,. theMoreover, focus is on the the crystal special structures features of of hVAP this structure-1 in complex and itswith comparison imidazole to (PDB other codes solved 2Y73 structures and 2Y74;. theMoreover, focus is the on crystalthe special structures features of hVAPof this- 1structure in complex and with its comparison imidazole (PDB to other codes solved 2Y73 structuresand 2Y74;. Moreover,[9]) are presentedthe crystal as structures they unintentionally of hVAP-1 in have complex provided with imidazole valuable data(PDB on codes the 2Y73 inhibito andr binding2Y74; Moreover,[9]) are presented the crystal as structures they unintentionally of hVAP-1 havein complex provided with valuable imidazole data (PDB on codesthe inhibito 2Y73 andr binding 2Y74; they unintentionally[9]properties) are presented of have hVAP as provided- they1. All unintentionally publicly valuable available data have X onprovided-ray the structures inhibitor valuable of binding hVAP data -on1 and propertiesthe hDAO inhibito arer of binding listed hVAP-1. in All [9]properties) are presented of hVAP as-1. they All publiclyunintentionally available have X-ray provided structures valuable of hVAP data-1 on and the hDAO inhibito arer listed binding in publiclyproperties availableTable 3. of X-ray hVAP structures-1. All publicly of hVAP-1 available and X-ray hDAO structures are listed of hVAP in Table-1 and3 . hDAO are listed in propertiesTable 3. of hVAP-1. All publicly available X-ray structures of hVAP-1 and hDAO are listed in Table 3. Table 3. Table 3. A comprehensive list of experimentally solved structures of hVAP-1 and diamine oxidase Table 3.TableA comprehensive 3. A comprehensive list oflist experimentallyof experimentally solvedsolved structures structures of ofhVAP hVAP-1-1 and anddiamine diamine oxidase oxidase Table(DAO) 3. Adeposited comprehensive into the list Protein of experimentally Data Bank as ofsolved 15 January structures 2020 of. hVAP-1 and diamine oxidase (DAO) depositedTable(DAO) 3. deposited A intocomprehensive the into Protein the Protein list Data of Dataexperimentally Bank Bank as as of of 15 solved15 JanuaryJanuary structures 20 2020.20. of hVAP-1 and diamine oxidase (DAO) deposited into the as of 15 January 2020. (DAO) deposited into the Protein Data Bank as of 15 January 2020. InhibitionInhibition Resolution,Resolution, ExpressionExpression DateDate Inhibition PDBPDB ID ID Resolution, Expression Date LigandsLigands InhibitionTInhibitionype,Type, Ref.Ref. PDB IDResolution, Resolution,Å Å Expression ExpressionSystemSystem DateDepositedDDateeposited Ligands InhibitionType, Ref. PDB IDPDB ID Resolution,Å ExpressionSystem DepositedDate LigandsLigands IC50Type,IC50Type,/Ki / Ki Ref. Ref. PDB ID Å Å SystemSystem DepositedDeposited Ligands IC50Type,/Ki Ref. Å System DepositedVascularVascular Adhesion Adhesion Protein Protein 1 1 IC50IC50/Ki /Ki Vascular Adhesion Protein 1 IC50/Ki Cricetulus Vascular Adhesion Protein 1 Cricetulus Vascular Adhesion Protein 1 Vascular Adhesion Protein 1 1 1 1PU41PU4 3.203.20 Cricetulusgriseusgriseus 24.06.0324.06.03 — — n.a.n.a.1 [8][8] 1PU4 3.20 Cricetulusgriseus 24.06.03 — n.a. [8] 1PU4 3.20 Cricetulus(CHOgriseus(CHO cells) cells) 24.06.03 — n.a.1 [8] 1PU4 3.20 (CHOgriseus cells) 24.06.03 — n.a.1 1 [8] 1PU4 3.20 griseus(CHOCricetulusCricetulus cells) 24.06.03 — n.a. [8] (CHOCricetulus cells) 1US11US1 2.902.90 (CHOCricetulus cells)griseusgriseus 17.11.0317.11.03 — — n.a.n.a. [8][8] 1US1 2.90 Cricetulusgriseus 17.11.03 — n.a. [8] 1US1 2.90 Cricetulus(CHOgriseus(CHO cells) cells) 17.11.03 — n.a. [8] 1US1 2.90 (CHOgriseus cells) 17.11.03 — n.a. [8] 1US1 2.90 griseusH(CHOomoHomo sapiens cells) sapiens 17.11.03 — n.a. [8] H(CHOomo sapiens cells) 2C102C10 2.502.50 (CHOH cells)omo(HEK293(HEK293 sapiens 09.09.0509.09.05 — — n.a.n.a. [7][7] 2C10 2.50 Homo(HEK293 sapiens 09.09.05 — n.a. [7] 2C10 2.50 Homo sapiens(HEK293cells)cells) 09.09.05 — n.a. [7] 2C10 2.50 (HEK293cells) 09.09.05 — n.a. [7] 2C10 2.50 (HEK293cells) 09.09.052-Hydrazinopyridine2-Hydrazinopyridine — n.a. [7] H. sapiens 2-Hydrazinopyridine Mechanism cells)H.cells) sapiens 2-Hydrazinopyridine Mechanism 2C11 2.90 H. (HEK293sapiens 09.09.05 Mechanism- based, [7] 2C11 2.90 (HEK293 09.09.05 2-Hydrazinopyridine2-Hydrazinopyridine - based, [7] H. sapiensH. sapiens Mechanism2 2C11 2.90 (HEK293cells) 09.09.05 - based,n.d.2 [7] cells) Mechanism-n.d.2 2C112C11 2.90 2.90 (HEK293(HEK293 09.09.0509.09.05 - based,2 [7] [7] cells) based,n.d. n.d.2 cells)cells) ImidazoleImidazole n.d.2 H. sapiens Imidazole Mechanism H. sapiens Imidazole Mechanism 2Y73 2.60 (human 28.01.11 Imidazole - based, [9] 2Y73 2.60 H. sapiensH(human. sapiens 28.01.11 Imidazole Mechanism- based, [9] 2Y73 2.60 H(human. sapiensserum) 28.01.11 Mechanism- Mechanism-based,n.d.2 2 [9] 2Y73 2.60 (humanserum) 28.01.11 n.d.2 2 [9] 2Y73 2.60 (human 28.01.11 - based,2 n.d. [9] serum)serum) n.d. serum) ImidazoleImidazole n.d.2 H. sapiens Imidazole Mechanism H. sapiensH. sapiens ImidazoleImidazole Mechanism 2Y742Y74 2.952.95 H(human. sapiens(human 28.01.1128.01.11 Imidazole Mechanism- Mechanism-based,- based, [9][9] 2Y742Y74 2.95 2.95 (humanH(human. sapiens 28.01.1128.01.11 Mechanism- based, [9] [9] 2Y74 2.95 (humanserum) 28.01.11 - based,n.d.2 2 2[9] serum) based,n.d.2 n.d. 2Y74 2.95 serum)(human 28.01.11 - based,2 [9] serum) n.d. serum)Cricetulus n.d.2 CricetulusCricetulus 3ALA 2.90 Cricetulusgriseus 29.07.10 — n.a. [85] 3ALA3ALA 2.90 2.90 griseusgriseus 29.07.1029.07.10 — n.a. n.a.[85] [85] 3ALA 2.90 Cricetulusgriseus(CHO cells) 29.07.10 — n.a. [85] (CHO(CHO cells) cells) 3ALA 2.90 (CHOgriseus cells) 29.07.10 —R15: n.a. [85] R15R15:: (CHO cells) 5-(cyclohexylamino)R15: -2-phenyl-6-(1H-1,2 5-(cyclohexylamino)-2-phenyl-6-(15-(cyclohexylamino)-2-phenyl-6-(1HH-1,2-1,2, 5-(cyclohexylamino),4-triazol-5-yl)R15:-3(2-2 -phenylH)-pyridazinone-6-(1H-1,2 4-triazol-5-yl)-3(2,4-triazol-5-yl)-3(2HH)-pyridazinone)-pyridazinone 5-(cyclohexylamino),4-triazol-5-yl)-3(2-H2-)phenyl-pyridazinone-6-(1H-1,2 H. sapiens ,4-triazol-5-yl)-3(2H)-pyridazinone Reversible H. sapiensH. sapiens ReversibleReversible 4BTW 2.80 H. sapiens(human 19.06.13 ReversibleIC50 = 71 [10] 4BTW4BTW 2.80 2.80 (human(human 19.06.1319.06.13 IC50IC50 = 71 = [10] [10] 4BTW 2.80 H(human. sapiensserum) 19.06.13 ReversibleIC50 =nM 71 [10] serum)serum) nM71 nM 4BTW 2.80 (humanserum) 19.06.13 IC50nM = 71 [10] serum) nM

R16R16: R16:: 5-isopropylaminoR16:-2 -phenyl-6-(1 H-1,2,4- 5-isopropylamino-2-phenyl-6-(15-isopropylamino-2-phenyl-6-(1H-1,2,4H-1,2,- 5-isopropylaminotriazol-5-yl)R16:--23(2-phenylH )-pyridazinone-6-(1H-1,2,4 - 4-triazol-5-yl)-3(2triazol-5-yl)-3(2HH)-)-pyridazinonepyridazinone 5-isopropylaminotriazol-5-yl)-3(2-2H-phenyl)-pyridazinone-6-(1H-1,2,4 - H. sapiensH. sapiens triazol-5-yl)-3(2H)-pyridazinone ReversibleReversible H. sapiens Reversible 4BTX4BTX 2.78 2.78 (humanH. sapiens(human 19.06.1319.06.13 ReversibleIC50IC50 = 290 = [10[] 10] 4BTX 2.78 (human 19.06.13 IC50 = 290 [10] 4BTX 2.78 serum)H(human. sapiensserum) 19.06.13 ReversibleIC50 290=nM 290 nM [10] serum) nM 4BTX 2.78 (humanserum) 19.06.13 IC50nM = 290 [10] serum) nM Molecules 2020, 25, x FOR PEER REVIEW 12 of 28

5-[4-(4-methylpiperazin-1-yl)phenylami 4BTY4BTY 3.103.10 H. Hsapiens. sapiens 19.06.1319.06.13 R17: 5-[4-(4-methylpiperazin-1-yl)R17:R17: ReversibleReversible [10[]10 ] 4BTY 3.10 H(human. sapiens 19.06.13 no]-2-(4-chlorophenyl)-6-R17: (1H-1,2,4-triaz ReversibleIC50 = 20 [10] 4BTY 3.10 H. sapiens 19.06.13 phenylamino]-2-(4-chlorophenyl)-6-R17: Reversible [10] serum) nM 4BTY 3.10 H. sapiens 19.06.13 (1H-1,2,4-triazol-5-yl)-3(2ol-5-yl)-3(2R17:H)-pyridazinone H)-pyridazinone Reversible [10]

H. sapiens Reversible 4BTY 3.10 (human 19.06.13 IC50 = [10] serum) 20 nM

Diamine Oxidase Drosophila 3HI7 1.80 melanogaster 19.05.09 — n.a. [11] (S2 cells) Berenil (4-[(2E)-3-(4-carbamimidoylphenyl)triaz -2-en-1-yl]benzene-1-carboximidamide) Drosophila Reversible 3HIG 2.09 melanogaster 19.05.09 [11] Ki = 13 nM (S2 cells)

Pentamidine (1,5-bis(4-amidinophenoxy)pentane) Drosophila Reversible 3HII 2.15 melanogaster 20.05.09 [11] Ki = 290 nM (S2 cells)

Drosophila 3K5T 2.11 melanogaster 08.10.09 — n.a. [13] (S2 cells) Aminoguanidine Drosophila Mechanism 3MPH 2.05 melanogaster 27.04.10 - based, [12] (S2 cells) Ki = 140 nM

1 Not applicable due to the absence of inhibitors in the crystallographic unit. 2 Inhibition constants are not known.

4.1.1. Irreversible Complex of 2HP with hVAP-1 The X-ray structure for the extracellular part (residues 29-763) of hVAP-1 in complex with 2HP was solved at 2.9 Å resolution by Jakobsson et al. in 2005 (PDB code 2C11; [7]). The crystals were obtained by soaking the crystals of the holoenzyme (PDB code 2C10) with 5 mM CuCl2 and 8 mM 2HP. Due to the addition of CuCl2, additional Cu2+ ions were detected, and one of them interacts with the residues in the Arg726-Gly725-Asp728 hairpin loop, changing its conformation, which likely causes the lack of 34 C-terminal residues (729-763). The resulting complex structure also lacks large portions of the N-terminal residues 29-57. Despite these non-natural features, the 2HP adduct in the complex structure exists primarily as a and shows how 2HP interacts with the catalytic site (Figure 4A). The TPQ is in off-copper conformation, where 2HP may react with C5 of TPQ. The catalytic Asp386 forms hydrogen bonds with the N2 and N3 nitrogens of 2HP, which stacks with Tyr384 and Phe389. Furthermore, Leu468 and Leu469 form hydrophobic interactions with 2HP. Since the computationally designed ligands have been planned to interact covalently with TPQ, the 2HP adduct structure has been most frequently used as a target in docking studies (Table 2).

4.1.2. Diverse Binding Modes Between Imidazole and hVAP-1 Two structures in complex with imidazoles were obtained when soluble hVAP-1 was extracted from human serum and crystallized in imidazole containing buffer [9]. Both of them similarly interact with two imidazoles that inhibit the enzymatic activity; the first one by binding to TPQ and Molecules 2020, 25, x FOR PEER REVIEW 12 of 28 Molecules 2020,, 25,, x FOR PEER REVIEW 12 of 28 5-[4-(4-methylpiperazin-1-yl)phenylami 5-[4-(4-methylpiperazin-1-yl)phenylami (human(human no]-2-(4-chlorophenyl)-6-(1H-1,2,4-triaz IC50IC50 = =20 20 (human no]-2-(4-chlorophenyl)-6-(1H-1,2,4-triaz IC50 = 20 Molecules 2020 25 serum)serum) ol-5-yl)-3(2H)-pyridazinone nMnM , , 1293 serum) ol-5-yl)-3(2H)-pyridazinone nM 12 of 29

Table 3. Cont.

Inhibition Resolution, Expression Date PDB ID Ligands Type, Ref. Å System Deposited IC50/Ki Diamine Oxidase DiamineDiamine Oxidase Oxidase Drosophila DrosophilaDrosophila 3HI7 1.80 melanogaster 19.05.09 — n.a. [11] 3HI73HI7 1.80 1.80 melanogastermelanogaster 19.05.0919.05.09 — — n.a. n.a.[11] [11] (S2(S2 cells) cells) (S2 cells)(S2 cells) BerenilBerenil BerenilBerenil (4(4-[(2-[(2E)E-)3--3(4-(4-carbamimidoylphenyl)triaz-carbamimidoylphenyl)triaz (4-[(2(4-[(2EE)-3-(4-carbamimidoylphenyl)triaz-)-3-(4-carbamimidoylphenyl)triaz -2--2en-en-1--1yl]benzene-yl]benzene-1--1carboximidamide)-carboximidamide) Drosophila 2-en-1-yl]benzene-1-carboximidamide)-2-en-1-yl]benzene-1-carboximidamide) DrosophilaDrosophila Reversible 3HIG 2.09 melanogaster 19.05.09 ReversibleReversible [11] 3HIG3HIG 2.09 2.09 melanogastermelanogaster 19.05.0919.05.09 Ki = 13 nM [11] [11] (S2 cells) Ki = Ki13 nM= 13 nM (S2 cells)(S2 cells)

Pentamidine PentamidinePentamidine (1,5-bis(4-amidinophenoxy)pentane) Drosophila (1,5-(1,5-bis(4(4-amidinophenoxy)pentane)-amidinophenoxy)pentane) DrosophilaDrosophila Reversible 3HII 2.15 melanogaster 20.05.09 ReversibleReversible [11] 3HII3HII 2.15 2.15 melanogastermelanogaster 20.05.0920.05.09 Ki = 290 nM [11] [11] (S2 cells) Ki = Ki290= nM290 nM (S2 cells)(S2 cells)

Drosophila DrosophilaDrosophila 3K5T 2.11 melanogaster 08.10.09 — n.a. [13] 3K5T3K5T 2.11 2.11 melanogastermelanogaster 08.10.0908.10.09 — — n.a. n.a.[13] [13] (S2 cells) (S2 cells)(S2 cells) Aminoguanidine AminoguanidineAminoguanidine DrosophilaDrosophila MechanismMechanism- Drosophila Mechanism 3MPH 2.05 melanogastermelanogaster 27.04.10 - based, [12] 3MPH3MPH 2.05 2.05 melanogaster 27.04.1027.04.10 - based,based, [12] [12] (S2 cells) Ki = 140 nM (S2 cells)(S2 cells) Ki = Ki140= nM140 nM

1 1 2 2 Not applicable1 N Notot applicable applicable due to thedue due absence to to the the absence absence of inhibitors of of inhibitors inhibitors in the in crystallographic in the the crystallographic crystallographic unit. unit unitInhibition.. 2. Inhibition Inhibition constants constants constants are are not are known. notnot known. known. 4.1.1. Irreversible Complex of 2HP with hVAP-1 4.1.1.4.1.1. Irreversible Irreversible C Complexomplex of of 2HP 2HP with with hVAP hVAP-1- 1 The X-ray structure for the extracellular part (residues 29-763) of hVAP-1 in complex with 2HP TheThe X X-ray-ray structure structure for for the the extracellular extracellular part part (residues (residues 29 29-763)-763) of of hVAP hVAP-1- 1in in complex complex with with 2HP 2HP was solvedwaswas solved atsolved 2.9 at Åat 2.9 resolution2.9 Å Å resolution resolution by by Jakobssonby Jakobsson Jakobsson etet et al.al. al. in inin 2005 20052005 (PDB (PDB (PDB code code code 2C11; 2C11; 2C11; [7] [7]).). The [ 7The]). crystals crystals The crystals were were were obtained by soaking the crystals of the holoenzyme (PDB code 2C10) with 5 mM CuCl2 and 8 mM obtainedobtained by soaking by soaking the crystalsthe crystals of of the the holoenzyme holoenzyme (PDB (PDB code code 2C10) 2C10) with with 5 mM 5 CuCl mM2CuCl and 82 mMand 8 mM 2+ 2HP.2HP. Due Due to to the the addition addition of of CuCl CuCl2,2 ,additional additional Cu Cu2+2+ ions ions were were detected detected, ,and and one one of of them them interacts interacts 2HP. Due2HP. to Due the to addition the addition of CuCl of CuCl2, additional2, additional CuCu ionsions were were detected detected,, and one and of one them of interacts them interacts with the residues in the Arg726-Gly725-Asp728 hairpin loop, changing its conformation, which with thewith residues the residues in the in Arg726-Gly725-Asp728 the Arg726-Gly725-Asp728 hairpin hairpin loop, loop changing,, changing its its conformation,conformation, which which likely likelylikely causes causes the the lack lack of of 34 34 C C-terminal-terminal residues residues ( 729(729-763-763).) .The The resulting resulting complex complex structure structure also also lacks lacks causes thelikely lack causes of 34theC-terminal lack of 34 C-terminal residues residues (729-763). (729-763 The). The resulting resulting complex complex structure structure also also lacks lacks large largelarge portions portions of of the the N N-terminal-terminal residues residues 29 29-57.-57. Despite Despite these these non non-natural-natural features, features, the the 2HP 2HP adduct adduct portions of the N-terminal residues 29-57. Despite these non-natural features, the 2HP adduct in the inin the the complex complex structure structure ex existsists primarily primarily as as a ahyd hydrazonerazone and and shows shows how how 2HP 2HP interact interacts swith with the the complexcatalyticcatalytic structure site site ( existsFigure(Figure 4 primarily A4A)..) The. The TPQ TPQ as is ais in hydrazonein off off-copper-copper conformation and conformation shows,,how where, where 2HP 2HP 2HP interacts may may react react withwith with C5 the C5 of catalyticof site (FigureTPQ.TPQ.4 TheA). The Thecatalytic catalytic TPQ Asp Asp is386 in386 o form formff-coppers shydrogen hydrogen conformation, bonds bonds with with the where the N2 N2 and 2HP and N3 N3 may nitrogens nitrogens react withof of 2HP, 2HP, C5 which ofwhich TPQ. The catalyticstacksstacks Asp386 with with forms Tyr384 Tyr384 hydrogen and and Phe389. Phe389. bonds Furthermore Furthermore with,,the ,Leu Leu N2468468 and and Leu469 N3 Leu469 nitrogens form form hydrophobic hydrophobic of 2HP, which interactions interactions stacks with with 2HP. Since the computationally designed ligands have been planned to interact covalently with Tyr384 andwith Phe389.2HP. Since Furthermore, the computationally Leu468 designed and Leu469 ligands have form been hydrophobic planned to interact interactions covalently with with 2HP. Since TPQTPQ, ,the the 2HP 2HP adduct adduct structure structure has has been been most most freque frequentlyntly used used as as a atarget target in in docking docking studies studies ( Table(Table the computationallyTPQ, the 2HP adduct designed structure ligands has been have most been freque plannedntly used to as interact a target covalently in docking studies with TPQ,(Table the 2HP 2).2). adduct structure has been most frequently used as a target in docking studies (Table2). 4.1.2.4.1.2. Diverse Diverse Binding Binding Modes Modes Between Between Imidazole Imidazole and and hVAP hVAP-1- 1 TwoTwo structures structures in in complex complex with with imidazoles imidazoles were were obtained obtained when when soluble soluble hVAP hVAP-1- 1was was extracted extracted fromfrom human human serum serum and and crystallized crystallized in in imidazole imidazole containing containing buffer buffer [9] [9].. . B B Bothoth of of them them similarly similarly interactinteract with with two two imidazoles imidazoles tha that tinhibit inhibit the the enzymatic enzymatic activity; activity; the the first first one one by by binding binding to to TPQ TPQ and and Molecules 2020, 25, x FOR PEER REVIEW 13 of 28 the second one by blocking access to the active site. Both structures were crystallized under similar conditions but, interestingly, have the TPQ cofactor in different conformations. In one of them, TPQ is in the off-copper conformation, and the imidazole is covalently bound to TPQ (Figure 4B; PDB code 2Y74), whereas in the other one TPQ is in the on-copper conformation and the imidazole binds non-covalently to it (Figure 4C; PDB code 2Y73). Compared to the 2HP complex (Figure 4A), the first imidazole makes only one hydrogen bond with Asp386 and stacks between Tyr384 and Leu468 in both complexes (Figure 4B,C). Despite the different conformation of TPQ, the binding site of the non-covalently bound imidazole totally overlaps with the covalently bound imidazole and both N1 and N3 form hydrogen bonds to O2 of TPQ and Asp386, respectively (Figure 4C). The second imidazole (yellow; Figure 4D) stacks with Tyr176, makes hydrophobic interactions with Leu469, Phe368, and Leu447 of Arm I from the other monomer, and its N3 forms a hydrogen bond with the hydroxyl group of Tyr394. In both the off-copper (Figure 4D) and on-copper (Figure 4E,F) structures, the second imidazole has the same binding site but the hydrogen bonding network with Thr212 is different in monomer B of the on-copper complex (Figure 4F). In monomer A of both complexes and monomer B of the off-copper complex, the N1 nitrogen makes a water-mediated hydrogen bond with the main chain nitrogen of Thr212 (Figure 4D,E). In chain B of the on-copper complex, the side chain of Thr212 has a different conformation, and its hydroxyl group forms an additional hydrogen bond with the water molecule that interacts with N1 of the second imidazole (magenta, Figure 4F). Thus, the binding mode of the second imidazole differs in the monomers of hVAP-1 when the first Moleculesimidazole2020 is, 25 bound, 1293 non-covalently to on-copper TPQ (Figure 4D,F). The closest distance between13 of the 29 two imidazoles is 7.5 Å .

FigureFigure 4.4. TheThe bindingbinding modesmodes ofof 2HP2HP andand imidazolesimidazoles toto hVAP-1.hVAP-1. Chain A ofof thethe hVAP-1hVAP-1 dimerdimer isis shownshown inin pinkpink andand chainchain BB inin cyan;cyan; ((AA)) thethe covalentcovalent adductadduct ofof 2HP2HP andand TPQTPQ (grey;(grey; PDBPDB codecode 2C11);2C11); ((BB)) thethe covalentcovalent adductadduct ofof imidazoleimidazole andand TPQTPQ (grey;(grey; PDBPDB codecode 2Y74);2Y74); ((CC)) thethe non-covalentnon-covalent complexcomplex ofof imidazoleimidazole (PDB(PDB codecode 2Y73);2Y73); ((DD)) thethe bindingbinding sitesite ofof thethe secondsecond imidazoleimidazole (yellow)(yellow) inin thethe covalentcovalent imidazoleimidazole complex complex (PDB (PDB code code 2Y74); 2Y74); (E ()E the) the binding binding site site of of the the second second imidazole imidazole (yellow) (yellow) in chainin chain A ofA the non-covalent imidazole complex (PDB code 2Y73); (F) the different binding mode of the second imidazole in chain B of the non-covalent imidazole complex (PDB code 2Y73).

4.1.2. Diverse Binding Modes Between Imidazole and hVAP-1 Two structures in complex with imidazoles were obtained when soluble hVAP-1 was extracted from human serum and crystallized in imidazole containing buffer [9]. Both of them similarly interact with two imidazoles that inhibit the enzymatic activity; the first one by binding to TPQ and the second one by blocking access to the active site. Both structures were crystallized under similar conditions but, interestingly, have the TPQ cofactor in different conformations. In one of them, TPQ is in the off-copper conformation, and the imidazole is covalently bound to TPQ (Figure4B; PDB code 2Y74), whereas in the other one TPQ is in the on-copper conformation and the imidazole binds non-covalently to it (Figure4C; PDB code 2Y73). Compared to the 2HP complex (Figure4A), the first imidazole makes only one hydrogen bond with Asp386 and stacks between Tyr384 and Leu468 in both complexes (Figure4B,C). Despite the di fferent conformation of TPQ, the binding site of the non-covalently bound imidazole totally overlaps with the covalently bound imidazole and both N1 and N3 form hydrogen bonds to O2 of TPQ and Asp386, respectively (Figure4C). The second imidazole (yellow; Figure4D) stacks with Tyr176, makes hydrophobic interactions with Leu469, Phe368, and Leu447 of Arm I from the other monomer, and its N3 forms a hydrogen bond with the hydroxyl group of Tyr394. In both the off-copper (Figure4D) and on-copper (Figure4E,F) structures, the second imidazole has the same binding site but the hydrogen bonding network with Thr212 is different in monomer B of the on-copper complex (Figure4F). In monomer A of both complexes and monomer B of the o ff-copper Molecules 2020, 25, 1293 14 of 29 complex, the N1 nitrogen makes a water-mediated hydrogen bond with the main chain nitrogen of Thr212 (Figure4D,E). In chain B of the on-copper complex, the side chain of Thr212 has a di fferent conformation, and its hydroxyl group forms an additional hydrogen bond with the water molecule that interacts with N1 of the second imidazole (magenta, Figure4F). Thus, the binding mode of the second imidazole differs in the monomers of hVAP-1 when the first imidazole is bound non-covalently to on-copper TPQ (Figure4D,F). The closest distance between the two imidazoles is 7.5 Å.

4.2. Mechanism-Based Inhibitors

4.2.1. Hydrazines Nurminen et al. (2011) [78] used the 2HP–hVAP-1 complex as a target for covalent docking of modified hydrazines (Table3). The stacking interactions with Tyr384 and Phe389 formed by compound R1 are equal to those formed between 2HP and hVAP-1 (Figure4A), but the additional hydroxyl group of R1 forms a hydrogen bond with the catalytic Asp386. The addition of a larger hydrophobic group to the C2 position of the hydrazines increased the selectivity of the inhibitors towards hVAP-1 over monoamine oxidase (MAO). Compounds R2 and R3 differ by the N-methyl group of R3, which changed the binding mode in such a way that the methyl group points towards Leu469. Therefore, the hydrogen bond between the same nitrogen and Asp386 that occurs in R2 is lost in R3, and this allows better hydrophobic interactions for the C2 substituent in R3. Thus, computational modeling explained the better binding affinity of R3 compared to R2. The docking studies combined with molecular dynamics simulations revealed that the flexibility of Met211, which is part of the hydrophobic pocket formed by Tyr384, Phe389, Tyr394, Leu468, and Leu469 (Figure4A), allows accommodation of larger hydrophobic groups and even aromatic ring (compounds 11a–d) to the C2 position. La Jolla Pharmaceutical has been developing another hydrazine compound, namely LJP1207, with phenylpropenyl moiety attached to the hydrazine group. Initially, it showed promising results in a series of animal models, including autoimmune encephalomyelitis [52], chronic inflammation modeling ulcerative colitis [53], transient forebrain ischemia [86], and liver cancer [87]. However, due to its potentially toxic allylhydrazine structure, further development of LJP1207 was discontinued. In general, the safety issues related to the development of hydrazine derivatives have inspired hVAP-1 targeted inhibitor development towards other types of inhibitors [32].

4.2.2. Thiazoles Inoue et al. [79] identified a thiazole hit compound with an IC50 value of 3.5 µM for hVAP-1 inhibition using high-throughput screening (HTS) and structure-activity relationship (SAR) studies. The developed thiazole compounds have a guanidine moiety that mimics a substrate Schiff base intermediate complex with TPQ. In accordance, the best compound R5 of the study had unsubstituted quinidine and 0.23 µM hVAP-1 inhibition. When R5 was covalently docked to the N1 nitrogen of the 2HP adduct (PDB code 2C11), the guanidine moiety established a tight hydrogen-bonding network with Asp386 providing a structural explanation for its importance as an unsubstituted functional group (Table3). The sulfur atom in the thiazole ring formed a hydrogen bond with the backbone nitrogen of Thr210 and the amido group a proton–π interaction with Tyr176. Compound R5 was further analyzed by an in vivo test, and it was proved to be an inhibitor of macular edema [81]. It also exhibited improved binding to rat VAP-1 (rVAP-1) and more than 435-fold selectivity towards VAP-1 over DAO and MAO-B. The fact that compound R5 had significantly lower VAP-1 inhibitory activity in humans than in rats encouraged Inoue et al. [80] to develop the compound further. They used molecular docking together with pharmacophore modeling and combined features of R5 and another compound from HTS, which did not have a guanidinyl group. Series of thiazole and pyrazole compounds were subjected to SAR studies where thiazole was found to have a better VAP-1 inhibitory potency over pyrazole. The results were also analyzed in light of the sequence comparison of rat and human VAP-1. As a result, compound R6 was first identified but it established weak inhibitory activity Molecules 2020, 25, 1293 15 of 29 for rVAP-1. Based on the sequence analysis and docking results, the methanesulfonylphenyl moiety of R6 locates near Leu447, and the corresponding Phe447 in the rVAP-1 causes a steric hindrance for binding. Thus, they designed compound R7 (U-V002) by changing the phenyl group in R6 to benzyl group, which increased inhibitory activity for rVAP-1 50-fold and hVAP-1 about 2-fold [80]. U-V002, developed by R Tech Ueno Ltd.® showed promising inhibiting potency towards both human and rat VAP-1 with IC50 7 nM and 8 nM, respectively, with low effect on MAO-A and MAO-B (IC50 > 10 µM ≈ for both) [70]. U-V002 also showed promising anti-inflammatory and anti-neovascularization effects in animal models [71,73] and, furthermore, it was evaluated preclinically for the treatment of diabetic retinopathy [72]; unfortunately, current development status of the compound is unknown.

4.2.3. Indanols Biotie Therapies Corporation developed a series of hydrazine indanols of which BTT2052 was a potent inhibitor of hVAP-1, and its binding mode was computationally studied by manual docking to TPQ [74]. It binds like other hydrazines (Figure3A) and, similarly to the hydroxyl group of the C2 substituted hydrazines (4.2.1), the hydroxyl group of indanol forms a hydrogen bond with Asp386, while the rings form hydrophobic interactions with Met211, Tyr384, Phe389, and Leu469. Despite BTT2052 being a potent inhibitor of hVAP-1 and not inhibiting MAO, it was an effective inhibitor of hDAO and hRAO [74]. This is understandable in light of our current knowledge of human CAO active site channels [1], showing that the residues in the catalytic site are highly conserved and residues in the active site channel contribute to the subtype specificity. As a small inhibitor, BTT2052 does not interact with these residues in the active site channel. The same considerations are valid in the case of a highly similar molecular entity, BTT-2027, which was shown to have high selectivity to hVAP-1 over twenty test targets with Ki 54 nM [20]. Although no modeling studies have been published, it can be ≈ hypothesized to bind tightly to hDAO and hRAO in the same orientation as BTT2052 does.

4.2.4. H-Imidazol-2-Amines Inoue et al. [81] continued their earlier work with thiazole compounds (4.2.2) intending to design compounds that bind better to hVAP-1, as the developed compound R5 [79] was a much better inhibitor of rat than hVAP-1. They first used the thiazole pharmacophore of compound R5 (named 2 in [81]) and optimized the phenylguanidine biostere, 1H-benzimidazol-2-amine (compound R8) to design compound R9 by synthesis and SAR of 1H-imidazol-2-amines compounds. The inhibitor VAP-1 activity of R9 was 19 nM towards human and 5.1 nM towards rat enzyme [81]. In their study, docking analysis of compound R8 suggested that its benzene ring is not optimally positioned in the active site of hVAP-1. Based on these results, they added a thiazole substituent in the 4-position of the benzene ring to create compound R9. The docking studies of R9 indicated that the stronger interactions formed by the aminothiazole moiety are responsible for its increased inhibitory activity compare to compound R5 (Table2). Compound R9 was highly selective towards VAP-1 over other CAOs and MAOs, and it was orally active in a pathological rat model of macular edema [81]. Later, R9 proved not to be suitable for further clinical development due to its insufficient pharmacokinetic properties in rats [83].

4.2.5. Allylamines Pharmaxis Ltd.® published their first paper on allylamine inhibitors of hVAP-1 in 2012 [54]. Their project was inspired by the irreversible haloallylamine inhibitor of MAO-B, , the inhibition mode of which is hypothesized to rely on a covalently attached reactive intermediate that is alkylated to release fluorine, thus leading to irreversible inhibition [54]. In their study, modeling and SAR were systematically used to design a highly potent compound R10 (PXS-4159) with promising pharmacokinetic profile and good in vitro and in vivo inhibitory activity. It showed a high preference towards hVAP-1 as compared to MAO-A, MAO-B, and hDAO, which is evident from the IC50 values of 10 nM vs. >30, 1.64, and >30 µM, respectively [54]. Modeling showed a crucial role for the hydrophobic interactions between the cyclohexyl moiety and Phe173-Tyr394 for the stabilization of Molecules 2020, 25, 1293 16 of 29

R10 in the binding pocket. To the best of our knowledge, PXS-4159 is still subjected to the preclinical tests. After that, Pharmaxis reported several other haloallyamines (Table1). In PXS-4728A (later renamed to BI 1467335), the cyclohexyl group was changed to the other lipophilic (isobutyl) moiety. In 2019, BI 1,467,335 completed the Phase II clinical trials for the treatment of non-alcoholic fatty liver disease; however, no results have been released yet. Another haloallylamine, PXS-4681A, which is no longer under development, has a highly polar sulfonamide group instead of the lipophilic cyclohexyl moiety, which can establish multiple hydrogen bonds with the same Tyr394 residue. Moreover, La Jolla Pharmaceutical Company® has been evaluating haloallylamine LJP1586, which has high potency against both human and rat VAP-1 with IC50 values between 4 and 43 nM [22]. It showed promising results in animal models of subarachnoid hemorrhage-associated cerebrovascular dilating dysfunction [63], intracerebral hemorrhagic stroke [61], multiple sclerosis [60], and temporary middle cerebral artery occlusion [64]. However, no data on the further clinical development of the compound are available.

4.2.6. Glycine Amides Yamaki et al. (2017) [83] reported the development of their first VAP-1-targeted glycine amide derivatives as their previous work on thiazole substituted 1H-imidazol-2-amine (compound 37b in Table2) was not suitable for further clinical development. The first glycine amide sca ffold with micromolar hVAP-1 inhibitor activity was identified by screening their in-house compound library, and its structural optimization was conducted using an extensive synthesis of glycine amide derivatives combined with SAR. Thereafter, they found that compound R12, a tertiary amide derivative of the initial compound, had increased stability but much weaker in vitro potency to human and rat VAP-1 [83]. At this point, molecular docking of R12 with hVAP-1 was used to model the Schiff base intermediate with TPQ. Based on the molecular docking results, substituents to the 4-position of the most solvent-exposed phenyl ring were designed and synthesized. Based on the parallel SAR and artificial membrane permeability assay, compound R13 (Table2) showed the best properties and was selected for further studies. Next, the binding mode of R13 was predicted using docking analysis and its binding pose shown to be similar to that of R12. The morpholine group at the 4-position of the phenyl ring protrudes from the active site cavity to the solvent between Arm I from the other monomer and Phe173 of D3 and the stacking interaction of the phenyl ring with Leu447 of Arm I is preserved like in R12 [83]. Further studies of compound R13 (Compound 1 in [84]) revealed that it had species-specific binding properties and was a better inhibitor of rat than human VAP-1. Similarly to the thiazole design, the sequence comparison of human and rat VAP-1 suggested that Leu447/Phe and Phe173/Thr differences are responsible for the weaker binding to hVAP-1. As the 4-position of the phenyl ring had been proven optimal in the previous study [83], replacement of the phenyl ring by various heteroaromatic rings was tested [84]. Several rounds of synthesis and SAR revealed compound R14 with a nanomolar binding activity to human and rat VAP-1 and reduced CYP inhibition. The docking study predicted good stacking interactions between the first phenyl ring and Leu469, pyrimidine and Leu447 of ArmI, and piperazine and Tyr394. Furthermore, the chlorine of the surface phenyl group interacts with Asp446. Ex vivo and in vivo studies suggest that R14 could be suitable for treating diabetic nephropathy [84].

4.3. Reversible Inhibitors

4.3.1. Pyridazinones The hVAP-1–imidazole complex structures (PDB core 2Y73 and 2Y74) revealed a novel, secondary binding site for imidazole in the active site channel, which inspired the design of reversibly binding pyridazinone inhibitors [10]. Three of the designed inhibitors were studied in more detail, and the X-ray structures in complex with hVAP-1 were solved for R15 (IC50 70 µM; PDB code 4BTX), R16 (IC50 290 µM; PDB code 4BTW), and R17 (IC50 20 µM; PDB code 4BTY) (Table3). The binding mode of the pyridazinones is similar and the 2.8 Å X-ray structure of the R15–hVAP-1 complex is shown as an Molecules 2020, 25, 1293 17 of 29 example of the binding mode (Figure5). For all the pyridazinones, the pyridazine ring stacks between Tyr176Molecules and2020, Leu44725, x FOR fromPEER REVIEW Arm I of the other monomer and makes hydrophobic interactions17 withof 28 Phe227. In addition, the carbonyl group of pyridazinone forms a hydrogen bond with the hydroxyl groupinhibitors ofTyr394 stacks with (Figures the5 aromaticA and4B). rings The of phenyl Phe389 group and Tyr394 in the inhibitorsand forms stacks hydrophobic with the interactions aromatic ringswith Leu468 of Phe389 and and Leu469 Tyr394 (Figure and forms 5A). hydrophobicThe hydroxyl interactions group of Tyr448 with Leu468 originating and Leu469 from A (Figurerm I of5 A).the Theother hydroxyl monomer group, the of carbonylTyr448 originating group of from Asp180 Arm, Iand of the the other hydroxyl monomer, group the carbonyl of Thr210 group form of Asp180,hydrogen and-bond theing hydroxyl networks group that ofinterac Thr210t with form the hydrogen-bonding triazole nitrogen of networks the inhibitors that interact. In the A with chains the, triazolethe methyl nitrogen group of of the Thr212 inhibitors. makes In the hydrophobic A chains, the interactions methyl group with ofthe Thr212 phenyl makes ring hydrophobic(Figure 5A), interactionswhereas, in all with the the B chains phenyl, the ring hydroxyl (Figure 5groupA), whereas, of Thr212 in allforms the a B water chains,-mediated the hydroxyl hydrogen group bond of Thr212with the forms triazole a water-mediated nitrogen (Figure hydrogen 5B). The variable bond with part the of triazolethe pyridazinones nitrogen (Figure binds5 nearB). The the variableopening partof the of theactive pyridazinones site and form bindss hydrophobic near the opening interactions of the active with site Leu177 andforms and Phe173 hydrophobic (Figure interactions 5B). The withbinding Leu177 sites and of pyridazinones Phe173 (Figure and5B). the The second binding imidazole sites of pyridazinones overlap, and andalso, thethe second binding imidazole mode in overlap,chain B of and the also, on-copper the binding TPQ modecomplex in chain(PDB Bcode of the 2Y73) on-copper shares TPQsome complex similarities: (PDB Tyr394 code 2Y73) and Thr212 shares someare involved similarities: in the Tyr394 hydrogen and Thr212 bonding are, involved and Tyr176 in the π hydrogen-stacks with bonding, them and (Figure Tyr176 5C).π -stacks Upon withpyridazinone them (Figure binding,5C). Uponthe side pyridazinone chain of Phe173 binding, changes the side conformation chain of Phe173 to accommodate changes conformation the bulky toligand accommodate, and Leu177 the moves bulky slightly ligand, (Figure and Leu177 5C). The moves TPQ, slightly however, (Figure is in5 aC). different The TPQ, conformation however, isin inthe a non diff-erentcovalent conformation complexes in of the pyridazinone non-covalent (off complexes-copper TPQ) of pyridazinone and imidazinone (off-copper (on copper TPQ) TPQ) and imidazinone(Figure 5C). (on copper TPQ) (Figure5C).

Figure 5.5. Pyridazinone binding binding to to hVAP hVAP-1;-1; ( A)) b bindinginding mode of of pyridazinone R15 to to chain A; ( B) bindingbinding mode ofof pyridazinonepyridazinone R15R15 totochain chain B. B. Leu447 Leu447 from from the the Arm Arm I ofI of chain chain A A (cyan, (cyan, behind behind R15) R15 is) notis not labeled; labeled (C; ()C comparison) comparison of of pyridazinone pyridazinone R15 R15 (pink) (pink) and and the the second second imidazole imidazole (orange) (orange) binding binding to chainto chain B ofB hVAP-1of hVAP (grey).-1 (grey) The. The point point of viewof view is rotatedis rotated to showto show the the hydrogen hydrogen bonding bonding interactions interactions of R15of R15 (black (black dashed dashed lines) lines) and and imidazole imidazole (orange). (orange). Labels Labels for Tyr176 for Tyr176 (pink), (pink), Leu447, Leu447 and Tyr448, and Tyr448 (cyan) stacking(cyan) stacking with the with inhibitors the inhibitors are not are shown not shown for clarity. for clarity.

A m modelingodeling study by Blig Bligt-Lindt-Lindénén et al. [ 88] revealed the differences differences in the secondary imidazole binding site organization in human and monkey VAP VAP-1-1 vs.vs. rat and murine VAP-1.VAP-1. The width of the entry channelchannel inin ratrat andand murine murine VAP-1 VAP- was1 was limited limited by by Phe447, Phe447, which which is not is not the the case case for thefor humanthe human and monkeyand monkey enzyme enzyme with awith smaller a smaller leucine residue.leucine residue Besides,. rodentsBesides, have rodents larger have residues larger than residues primates than in positionsprimates 177in positions (Gln vs. Leu) 177 (Gln and 180 vs. (Gln Leu) vs. and Asp), 180 which(Gln vs. makes Asp), the which channel makes even the narrower. channel There even arenarrower. also additional There are di alsofferences additional in the compositiondifferences in of the residues composition lining theof residues active site lining channel the thatactive might site achannelffect ligand that binding. might affect Phe173 ligand and Leu177 binding. in hVAP-1Phe173 are and changed Leu177 to in polar hVAP residues-1 are in changed rodent enzymes: to polar Thr173residues in in mVAP-1, rodent Asp173 enzymes: in rVAP-1, Thr173 and in mVAP Gln177-1, in Asp173 both rodent in rVAP VAP-1s.-1, and Rats Gln177 also have in larger both rodent Thr in positionVAP-1s. 761Rats as also compared have larger to Ser761 Thr in in position hVAP-1 and761 as Ala761 compared in mVAP-1. to Ser761 All in these hVAP residues-1 and are Ala761 located in atmVAP the entrance-1. All these of theresidues active are site located channel at andthe entrance might also of the affect active ligand site recognition.channel and Thismight finding also affect can explainligand recognition. the high specificity This finding of compounds can explainR15 the, R16high, andspecificitR17 toy of hVAP-1 compounds as compared R15, R16 to, theand murine R17 to VAP-1hVAP-1 (mVAP-1) as compared [10]. to For the example, murine methylpiperazineVAP-1 (mVAP-1) [ moiety10]. For of example,R17 is in methylpiperazine close contact with moiety residues of R17 is in close contact with residues 761 and 177, its phenyl ring interacts with residues 173, 177 and 447 and its imidazole ring with residue 180, which differs between human and rodent VAP-1. The largest and most lipophilic compound R17 shows the best binding profile, which is in agreement

Molecules 2020, 25, 1293 18 of 29

761 and 177, its phenyl ring interacts with residues 173, 177 and 447 and its imidazole ring with residue 180, which differs between human and rodent VAP-1. The largest and most lipophilic compound R17 shows the best binding profile, which is in agreement with the hypothesis that primate VAP-1 prefers bulkier and more hydrophobic ligands than rodent VAP-1 [88].

4.3.2. Phenyl-Piperidinyl Amine Before the publication of pyridazinone complexes, Emanuela et al. (2012) reported docking and inhibitor activity studies of R11 as a plausible skeleton for the design of reversibly binding inhibitors of hVAP-1 [82] (Table2). Based on the docking results, R11 (about 100 µM inhibitor) was predicted to have two different binding modes. In one of them, the inhibitor aligns with the opening of the active site cavity, reaching towards Asp180. In the other binding mode, one end of R11 similarly aligns with the opening, but the other one intrudes to the active site and stacks between Phe389 and Leu469.

5. hDAO as an Off-Target

5.1. Biological Function DAO, with distinct specificity for diamine substrates, was originally described as a histaminase in 1929 [89]. hDAO is the main enzyme catabolizing ingested histamine [26,90]. Unlike VAP-1, DAO is a soluble protein, which is released from kidney and intestinal epithelial cells and requires an external stimulus [91]. Pathologies that limit DAO secretion from basolateral vesicles, e.g., inflammation and degenerative intestinal disorders, lead to hDAO deficiency [26,92,93]. Furthermore, low DAO levels are known to be caused by genetic mutations. The preferred substrates for DAO are histamine (Km = 2.8 0.07 µM), 1-methylhistamine (Km = 3.4 0.3 µM), and putrescine (Km = 20 1 µM) [25]. ± ± ± Of the DAO substrates, histamine regulates several physiological processes (e.g., gastric acid secretion, central nervous system functioning, hypersensitivity reactions, bronchial asthma, tumorigenesis, and multiple elements of immune regulation). hDAO has a central role in histamine intolerance, which is caused by histamine-rich food, alcohol, or drugs that release histamine or inhibit hDAO and, thus, leads to an excess of histamine and symptoms similar to an allergic reaction in the patients [26]. hDAO deficiency has been described in patients with atopic eczema, chronic urticaria, chronic abdominal pain or inflammatory bowel disease, migraines, and asthma [94–99]. Histamine also induces contractions and spontaneous abortions when injected into pregnant animals [28,100–102]. The enzymatic activity of DAO increases several 100-fold during pregnancy, and low DAO activity in the first trimester has been associated with a 16-fold increased risk of fetal death [28,103]. Women suffering from early-onset preeclampsia (delivery before week 34 associated with a 10-fold higher risk of mortality [104]) were shown to have significantly lower hDAO levels in the first trimester of pregnancy compared to healthy controls, which is believed to be damaging for the vascular remodeling modulating the uteroplacental blood flow [105]. Based on a mice study, DAO regulates the homeostatic histamine and putrescine levels, which is important in embryo implantation [106].

5.2. Off-Target Function Despite strictly controlled development processes, any drug bears a risk of unintended and potentially harmful responses, i.e., adverse drug reactions (ADRs) [107]. Commonly, ADRs are caused by the drug molecule interacting with targets beyond the one(s) it was designed for, so-called off-targets [108]. DAO is an important off-target due to its role as the frontline enzyme for clearing extra-cellular histamine [11,26]. A drug molecule with an off-target activity that causes inhibition of hDAO can cause a rise in histamine levels and related ADRs resembling allergic reactions (e.g., headaches, gastrointestinal disorders, skin reactions, flushing, sneezing, asthma-like wheezing, muscle aches, hypotension, and arrhythmias) and, at its worst, anaphylactic shock. Inhibition of pig and sheep DAO with the potent and irreversible inhibitor aminoguanidine, followed by oral challenge with reasonable levels of histamine, significantly induced anaphylactic shock-like symptoms and increased Molecules 2020, 25, 1293 19 of 29

Molecules 2020, 25, x FOR PEER REVIEW 19 of 28 death rates compared to control animals [27,109]. Relevant to humans, high levels of histamine are foundalcohol, in some and common can also food be induced (e.g., cheese, by drugs sausages, releasing yogurts, histamine etc.), in or alcohol, blocking and DAO can also[26,110 be] induced. Inhibition by drugsof DAO releasing can also histamine increase or the blocking risk of DAOdeveloping [26,110 intestinal]. Inhibition carcinoma, of DAO canindicating also increase that prolonged the risk of use developingof drugs intestinal inhibiting carcinoma, DAO may indicating elevate that the prolonged cancer risk use [111,112 of drugs]. The inhibiting patients DAO of mayatopic elevate eczema, thechronic cancer risk urticaria, [111,112 chronic]. The abdominal patients of pain atopic or eczema, inflammatory chronic bowel urticaria, disease, chronic migraines, abdominal and pain asthma or inflammatory[94–99] that have bowel hDAO disease, deficiency migraines,, as well and asthmaas pregnant [94– 99women] that have, form hDAO high-risk deficiency, groups asfor well severe as pregnantADRs if women, treated form with high-risk drugs groupscausing for histamine severe ADRs relea ifse treated or drugs with drugs having causing an off histamine-target and releaseactivity or drugs-blocking having effect an oonff-target hDAO. and Currently, activity-blocking aminoguanidine, effect on hDAO.berenil, Currently, pentamidine, aminoguanidine, and metformin berenil,have pentamidine, been indicated and to metforminhave off-target, have activity been indicated-blocking to effects have o onff-target, hDAO, activity-blocking but many more edrugsffects are on hDAO,expected but to manyshow moresimila drugsr behavior, are expected thereby topotentially show similar causing behavior, severe thereby ADRs [13, potentially27,113]. causing severe ADRs [13,27,113]. 5.3. Unintended Drug–hDAO Interactions at the Atomic Level 5.3. Unintended Drug–hDAO Interactions at the Atomic Level Berenil and pentamidine are diamine derivatives, which are drugs for treating trypanosomiasis andBerenil pneumocystis and pentamidine pneumonia are diamine infections, derivatives, interact with which the are minor drugs groove for treating of DNA trypanosomiasis [114,115]. Both of andthe pneumocystism have amidophenyl pneumonia moieties infections, at the interact ends with but thevary minor in the groove length of and DNA che [114mical,115 nature]. Both o off the themcentral have amidophenyl linkers. The moieties X-ray structures at the ends of but hDAO vary in the complex length with and chemical berenil nature(PDB code of the 3HIG) central and linkers.pentamide The X-ray (PDB structures code 3HII) of hDAO revealed in complex the binding with berenil mode (PDBof the code inhibitors 3HIG) [11] and pentamide(Table 3, Figure (PDB 6). codeAminoguanidine 3HII) revealed theis a binding potent inhibitor mode of of the DAO inhibitors with reported [11] (Table IC503, Figure values6 ).of Aminoguanidine≈ 30 μM [116] and is ≈ 150 a potentnM [12] inhibitor. It is an of expe DAOrimental with reported therapeutic, IC50 which values prev of ents30 µ advancedM[116] and glycation150 nMend [ 12product]. It is (AGE) an ≈ ≈ experimentalformation therapeutic,[117,118]. Several which clinical prevents trials advanced conducted glycation on amino end productguanidine (AGE) indicate formationd that [ 117it would,118]. be Severalbeneficial clinical for trials treating conducted patient ons with aminoguanidine diabetic nephropathy indicated but that also it would caused be many beneficial adverse for reactions treating in patientshigh with doses diabetic, e.g., nephropathy flu-like symptoms, but also caused abnormal many adversekidney reactions function in tests, high doses, and effects e.g., flu-like on the symptoms,gastrointestinal abnormal tract kidney [117 function–119]. tests, In clinical and eff ects therapy, on the the gastrointestinal peak plasma tract concentration [117–119]. In of clinicalaminoguanidine therapy, the peak is ≈ 50 plasma μM [ concentration117], which is of much aminoguanidine higher than is the 50IC50µM[ of117 hDAO], which and is enough much to ≈ higherinhibit than the the IC50majority of hDAO of peripheral and enough hVAP to inhibit-1 (IC50 the majority≈ 30 μM) of as peripheral well [12 hVAP-1]. Aminoguanidine (IC50 30 µM) may, ≈ as wellhowever, [12]. Aminoguanidine be used in small may, doses however, for treating be used diabetic in small nephropathy doses for treating as well diabetic as other nephropathy AGE-related as welldisorders as other 118] AGE-related. In the X-ray disorders structure 118]. complexes, In the X-ray pentamidine structure complexes, and berenil pentamidine do not interact and covalently berenil do notwith interact the TPQ covalently cofactor ( withFigure the 6 TPQA, B) cofactorbut amin (Figureoguanidine6A,B) form but aminoguanidines a covalent adduct forms with a covalentTPQ (Figure adduct6C). with Its guanidiTPQ (Figurenium6 C). group Its guanidinium makes polar group interactions makes polar with interactions Asp373 and with stacks Asp373 between and stacks Val458, betweenTyr371 Val458,, and Trp376 Tyr371, (Figure and Trp376 6C). Metformin, (Figure6C). a Metformin, drug used afor drug the usedtreatment for the of treatmenttype 2 diabetes, of type causes 2 diabetes,gastrointestinal causes gastrointestinal side effects sidefor 30 eff%ects–50% for 30%–50%of patients, of patients,and 5% has and s 5%uch has severe such severesymptoms symptoms that they thatdiscontinue they discontinue the usage the usageof metformin of metformin [113]. [ 113Based]. Based on the on molecular the molecular structure structure of metformin of metformin (Figure (Figure6C),6 itC), also it also interacts interacts covalently covalently with with TPQ TPQ but butlikel likelyy forms forms even even more more interactions. interactions.

Figure 6. hDAO interactions with off-targets. (A) Pentamidine; (B) Berenil; (C) Aminoguanidine. Figure 6. hDAO interactions with off-targets. (A) Pentamidine; (B) Berenil; (C) Aminoguanidine. Phe435 of Arm I from the other monomer is shown in orange. Phe435 of Arm I from the other monomer is shown in orange. The binding modes of pentamidine (Figure6A) and berenil (Figure6B) resemble each other but The binding modes of pentamidine (Figure 6A) and berenil (Figure 6B) resemble each other but also have apparent differences. In these complex structures, TPQ is in on-copper conformation and also have apparent differences. In these complex structures, TPQ is in on-copper conformation and does not make direct contact with the inhibitors. In both of the inhibitors, the buried amidinium does not make direct contact with the inhibitors. In both of the inhibitors, the buried amidinium group interacts with the catalytic Asp373, and the connected phenyl ring stacks with Tyr371 and group interacts with the catalytic Asp373, and the connected phenyl ring stacks with Tyr371 and Trp376. Pentamidine binds in a U-shaped form and the other amidinium group forms hydrogen bonds with Ser380 and the main chain of Glu377and Trp376. Its central linker region does not make

Molecules 2020, 25, 1293 20 of 29

Trp376. Pentamidine binds in a U-shaped form and the other amidinium group forms hydrogen bonds with Ser380 and the main chain of Glu377and Trp376. Its central linker region does not make any significant interactions. Berenil extends straight out from the active site, and the central triazine linker is positioned between Tyr459 and Asp186, and Asp186 makes electrostatic interactions with the nitrogens in the triazine moiety. The second phenyl ring stacks between Phe148 and Phe435 of ArmI. The second amidinium group is exposed to solvent near the surface opening of the active site channel.

6. Special Considerations in hVAP-1-Targeted Drug Design

6.1. Species-Specific Features in VAP-1Ligand Recognition Due to the fact that rodents are routinely used for estimating inhibitor potency, it is crucial to elucidate the difference in ligand binding among human, mouse, and rat VAP-1. Sequence analysis shows a high degree of similarity between hVAP-1 and rodent VAP-1s: hVAP-1 is 80% identical with rVAP-1 and 83% identical with mVAP-1 [120]. Nevertheless, species differences in ligand binding to VAP-1 were documented using SSAO-activity in plasma, umbilical artery, or homogenized tissues more than two decades ago [121,122]. In contrast to the very first modeling study by Marti et al. [123], who failed to find the sequence basis for the observed species-specific enzymatic properties of VAP-1, Bligt-Lindén et al. [88] showed that hVAP-1 has broader and more hydrophobic active site channel than rodent VAP-1; thus, hVAP-1 should prefer larger and more hydrophobic inhibitors. Indeed, there are no major differences in the active site near TPQ, while the active site channel differs between human and rodent VAP-1 [88]. The different cavity architecture is caused by sequence variations in positions 173, 177, 180, and 761 from one chain and 447 from the other one, which are responsible for the species-specific differences in inhibitor binding (Figure7A) [ 88]. Highly species-specific pyridazinone inhibitors R15, R16, and R17 with a preference for hVAP-1 were recently structurally studied [10]. Comparison of the X-ray structures of hVAP-1 complexes and modeling of the inhibitor complexes for rodent and monkey VAP-1 suggested that the same residues are responsible for the species-specific inhibitor binding properties of pyridazinones (Figure7A). Since these residues are scattered all over the active site channel, it is a challenging task to design an equally potent inhibitor towards primate andMolecules rodent 2020 VAP-1, 25, x FOR [10 PEER]. REVIEW 21 of 28

Figure 7.7.( A()A Species-specific) Species-specific residues residues in the in VAP-1 the activeVAP- site1 active channel. site Residues channel in. rat Residues (wheat) /mouse in rat (yellow)(wheat)/mouse VAP-1 are(yellow) listed belowVAP-1 the are residue listed labels below of the hVAP-1 residue (cyan labels/pink). of ThehVAP catalytic-1 (cyan/pink) center around. The TPQcatalytic is totally center conserved, around TPQ and is the totally residue conserved differences, and concentrate the residue on differences the Arm I concentrate and the first on helix the of Arm D3; (IB and) key the residue first helix differences of D3; between(B) key residue hVAP-1 differences and hDAO (greenbetween/orange). hVAP- The1 and Aminoguanidine–hDAO hDAO (green/orange). complexThe Aminoguanidine (green; PDB code–hDAO 3MPH) complex superimposed (green; PDB with hVAP-1 code 3MPH) structure superimposed (PDB code 2C10). with Binding hVAP-1 sitesstructure depicted (PDB similarly code 2C10). as in Binding Figure3 sites. depicted similarly as in Figure 3.

6.2. TheModeling Risk forstudy Interaction by Inoue with etOther al. [ 81CAO] suggested Sub-Family that Members differences in residues 173 (Phe in hVAP-1 vs. Thr in rVAP-1) and 447 (Leu vs. Phe) could be responsible for a species-specific inhibitor potency of R6 In addition to species-specific binding properties (Table S2), differences in the expression profile of the CAO sub-families should be considered when model organisms are selected for in vivo studies [1]. As an example, the pyridazinone inhibitors had a similar in vitro potency against VAP-1 from human and cynomolgus monkey [10], but cynomolgus monkey is not a good model organism for in vivo studies since it also has soluble plasma amine oxidase (SAO; AOC4), the closest CAO family member to VAP-1 [1]. In fact, of all studied primates, only human and gorilla lacked SAO [1]. Of rodents, mice had the same CAO sub-families (VAP-1, DAO, and RAO) as humans, whereas rats have only VAP-1 and DAO [1]. Thus, the best preclinical model organism for hVAP-1-targeted ligand design would be mouse [1] and, naturally, transgenic mouse overexpressing human VAP-1 on endothelium is an even better alternative for in vivo studies to avoid both sub-family cross-reactions and species-specific binding properties [17]. Of the CAO sub-families expressed in human, RAO is also a monoamine oxidase, but it has a different substrate preference profile, and its enzymatic activity has been detected only in the eye [3]. DAO, in turn, is a known off-target and interfering with its normal function as histaminase with drug molecules results in ADRs [11,113,117–119]. At the moment, to the best of our knowledge, modeling studies on VAP-1-targeting inhibitors that could result in off-target interactions with DAO have not been published. The X-ray structure of hDAO has been solved in complex with the experimental drug aminoguanidine, which inhibits both DAO and VAP-1. Aminoguanidine inhibits DAO significantly better than VAP-1, but its clinically used concentration also inhibits hVAP-1 [116]. Comparison of the hDAO-aminoguanidine complex with hVAP-1 structure shows that of the interacting residues only Tyr371 is conserved while the Trp376/Phe389, Val458/Leu468, and Tyr459/Leu469 replacements likely contribute to its weaker binding into hVAP-1 (Figure 7B). The position corresponding to Asp186 in hDAO (Figure 7A) is highly conserved within each CAO subfamily (Thr212 in VAP-1, His in RAO and Asn in SAO) and, thus, suggested to be important for the substrate selectivity of CAOs [11]. The corresponding Thr212, together with Thr210 and Tyr394 in hVAP-1 (Phe184 and Val381 in hDAO), is involved in imidazole and pyridazinone binding. In general, the residues in the active site channel of hVAP-1 are not conserved in hDAO, and furthermore, the tip of Arm I has a totally different conformation in these two CAOs. However, it is advisable to test the binding of designed hVAP-1 inhibitors for their effect on hDAO as the catalytic site is highly conserved and small adjustments and rearrangements of side chain conformations in the active site channel might allow their binding to hDAO as well.

Molecules 2020, 25, 1293 21 of 29

(Figure7A). Based on their hypothesis, the highly hydrophilic Asp173 in mVAP-1 can be responsible for a high preference for hVAP-1 over mVAP-1 [88]. Foot et al. [54] modeled the binding orientation of compound R10 that is structurally close to PXS-4728A and revealed the essential role of hydrophobic interactions with Phe173 and Tyr394. Based on their analysis, the species-specificity in position 173 (hydrophobic Phe in hVAP-1, polar Thr in rVAP-1, and negatively charged Asp in mVAP-1; Figure7A) is a probable reason for a slightly higher potency of PXS-4728A towards hVAP-1 as compared to rodent VAP-1. Kubota et al. [120] recently performed extensive biochemical comparison of inhibitor potency toward human, mouse and rat VAP-1 and revealed apparent species-specific differences: was 10 and 3 times more potent toward hVAP-1 (85.9 µM) as compared to rVAP-1 (993 µM) and mVAP-1 (295 µM), respectively; in contrast, hydralazine more efficiently inhibited rodent VAP-1 than hVAP-1 (1.2 µM for rVAP-1 and 3.1 µM for mVAP-1 vs. 7.8 µM for hVAP-1). Another small inhibitor LJP-1207 showed less species-specificity: it was 2 and 2.5 times more potent towards hVAP-1 than to rVAP-1 and mVAP-1, respectively (252 nM vs. 120 nM and 102 nM). The slightly bulkier inhibitor PXS-4728A showed a weak preference for hVAP-1, while large compound R7 developed by Astellas showed a slight preference for hVAP-1 (23 nM) over the rat ortholog (50 nM) but much weaker binding to the mouse ortholog (414 nM). As a summary of the previous studies on species-specific binding properties of hVAP-1 inhibitors, it is clear that computational methods should be used to predict the species-specific binding properties in hVAP-1-targeted ligand design. In general, the likelihood of species-specificity increases with enlarged ligand size and is most prominent for those ligands that interact with the active site channel farther away from the catalytic site [1].

6.2. The Risk for Interaction with Other CAO Sub-Family Members In addition to species-specific binding properties (Table S2), differences in the expression profile of the CAO sub-families should be considered when model organisms are selected for in vivo studies [1]. As an example, the pyridazinone inhibitors had a similar in vitro potency against VAP-1 from human and cynomolgus monkey [10], but cynomolgus monkey is not a good model organism for in vivo studies since it also has soluble plasma amine oxidase (SAO; AOC4), the closest CAO family member to VAP-1 [1]. In fact, of all studied primates, only human and gorilla lacked SAO [1]. Of rodents, mice had the same CAO sub-families (VAP-1, DAO, and RAO) as humans, whereas rats have only VAP-1 and DAO [1]. Thus, the best preclinical model organism for hVAP-1-targeted ligand design would be mouse [1] and, naturally, transgenic mouse overexpressing human VAP-1 on endothelium is an even better alternative for in vivo studies to avoid both sub-family cross-reactions and species-specific binding properties [17]. Of the CAO sub-families expressed in human, RAO is also a monoamine oxidase, but it has a different substrate preference profile, and its enzymatic activity has been detected only in the eye [3]. DAO, in turn, is a known off-target and interfering with its normal function as histaminase with drug molecules results in ADRs [11,113,117–119]. At the moment, to the best of our knowledge, modeling studies on VAP-1-targeting inhibitors that could result in off-target interactions with DAO have not been published. The X-ray structure of hDAO has been solved in complex with the experimental drug aminoguanidine, which inhibits both DAO and VAP-1. Aminoguanidine inhibits DAO significantly better than VAP-1, but its clinically used concentration also inhibits hVAP-1 [116]. Comparison of the hDAO-aminoguanidine complex with hVAP-1 structure shows that of the interacting residues only Tyr371 is conserved while the Trp376/Phe389, Val458/Leu468, and Tyr459/Leu469 replacements likely contribute to its weaker binding into hVAP-1 (Figure7B). The position corresponding to Asp186 in hDAO (Figure7A) is highly conserved within each CAO subfamily (Thr212 in VAP-1, His in RAO and Asn in SAO) and, thus, suggested to be important for the substrate selectivity of CAOs [11]. The corresponding Thr212, together with Thr210 and Tyr394 in hVAP-1 (Phe184 and Val381 in hDAO), is involved in imidazole and pyridazinone binding. In general, the residues in the active site channel of hVAP-1 are not conserved in hDAO, and furthermore, the tip of Arm I has a totally different conformation in these two CAOs. However, it is advisable to test the binding of designed hVAP-1 Molecules 2020, 25, 1293 22 of 29 inhibitors for their effect on hDAO as the catalytic site is highly conserved and small adjustments and rearrangements of side chain conformations in the active site channel might allow their binding to hDAO as well.

Supplementary Materials: The following supplements are available online, Table S1: List of traditional and IUPAC names of the compounds referred to in the text; Table S2: List of amino acids relevant to inhibitor binding that differ among VAP-1 orthologs and copper-containing amine oxidase sub-families. Author Contributions: Conceptualization, T.A.S. and S.J.; writing—original draft preparation, S.V., K.M.D., S.J., T.A.S.; writing—review and editing, S.V., K.M.D., S.J., T.A.S.; visualization, T.A.S.; supervision, T.A.S.; project administration, S.V., T.A.S.; funding acquisition, S.J., T.A.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Sigrid Juselius Foundation (T.A.S.), the Magnus Ehrnrooth foundation (T.A.S), the Tor, Joe, and Pentti Borgs Foundation (T.A.S.), Medicinska Understödsföreningen Liv och hälsa (T.A.S.), and the Academy of Finland (No. 288534 T.A.S.). Acknowledgments: We thank the bioinformatics (J.V. Lehtonen), translational activities, and structural biology infrastructure support from Biocenter Finland and Instruct-FI and CSC IT Center for Science for computational infrastructure support at the Structural Bioinformatics Laboratory, Åbo Akademi University. Conflicts of Interest: S.J. owns stocks of Faron Ventures and Faron Pharmaceuticals. The other authors declare no conflict of interest.

Abbreviations

2HP 2-hydrazinopyridine; ADRs adverse drug reactions; AOC1 hDAO human diamine oxidase; AOC2 hRAO retina-specific amine oxidase; AOC3 hVAP-1 vascular adhesion protein-1; CAO copper-containing amine oxidase; DAO diamine oxidase; HTS high-throughput screening; MAO monoamine oxidase; mVAP-1 murine ortholog of vascular adhesion protein-1; PDB Protein Data Bank; PET positron emission tomography; rVAP-1 rat ortholog of vascular adhesion protein-1; TPQ topaquinone.

References

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