Structure-Based Macrocyclization of Substrate Analogue NS2B-NS3 Protease Inhibitors of Zika,West Nile and Dengue viruses

A series of cyclic active-site-directed inhibitors of the NS2B-NS3 proteases from Zika (ZIKV), West Nile (WNV), and dengue-4 (DENV4) viruses has been designed. The most potent compounds contain a reversely incorporated d -lysine residue in the P1 position. Its side chain is connected to the P2 backbone, its α-amino group is converted into a guanidine to interact with the conserved Asp129 side chain in the S1 pocket, and its C terminus is connected to the P3 residue via different linker segments. The most potent compounds inhibit the ZIKV protease with K_i values <5 nM. Crystal structures of seven ZIKV protease inhibitor complexes were determined to support the inhibitor design. All the cyclic compounds possess high selectivity against trypsin-like serine proteases and furin-like proprotein convertases. Both WNV and DENV4 proteases are inhibited less efficiently. Nonetheless, similar structure-activity relationships were observed for these enzymes, thus suggesting their potential application as pan-flaviviral protease inhibitors.     

Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

A series of new substrate analogue inhibitors of the WNV NS2B–NS3 protease containing decarboxylated arginine mimetics at the P1 position was developed. Among the various analogues, trans‐(4‐guanidino)cyclohexylmethylamide (GCMA) was identified as the most suitable P1 residue. In combination with dichloro‐substituted phenylacetyl groups at the P4 position, three inhibitors with inhibition constants of <0.2 μM were obtained. These GCMA inhibitors have a better selectivity profile than the previously described agmatine analogues, and possess negligible affinity for the trypsin‐like serine proteases thrombin, factor Xa, and matriptase. A crystal structure in complex with the WNV protease was determined for one of the most potent inhibitors, 3,4‐dichlorophenylacetyl‐Lys‐Lys‐GCMA (K_i=0.13 μM ). The inhibitor adopts a horseshoe‐like conformation, most likely due to a hydrophobic contact between the P4 phenyl ring and the P1 cyclohexyl group, which is further stabilized by an intramolecular hydrogen bond between the P1 guanidino group and the P4 carbonyl oxygen atom. These inhibitors are stable, readily accessible, and have a noncovalent binding mode. Therefore, they may serve as suitable lead structures for further development.     

Natural and engineered plasmin inhibitors: applications and design strategies

The serine protease plasmin is ubiquitously expressed throughout the human body in the form of the zymogen plasminogen. Conversion to active plasmin occurs through enzymatic cleavage by plasminogen activators. The plasminogen activator/plasmin system has a well-established function in the removal of intravascular fibrin deposition through fibrinolysis and the inhibition of plasmin activity; this has found widespread clinical use in reducing perioperative bleeding. Increasing evidence also suggests diverse, although currently less defined, roles for plasmin in a number of physiological and pathological processes relating to extracellular matrix degradation, cell migration and tissue remodelling. In particular, dysregulation of plasmin has been linked to cancer invasion/metastasis and various chronic inflammatory conditions; this has prompted efforts to develop inhibitors of this protease. Although a number of plasmin inhibitors exist, they commonly suffer from poor potency and/or specificity of inhibition that either results in reduced efficacy or prevents clinical use. Consequently, there is a need for further development of high-affinity plasmin inhibitors that maintain selectivity over other serine proteases. This review summarises clearly defined and potential applications for plasmin inhibition. The properties of naturally occurring and engineered plasmin inhibitors are discussed in the context of current knowledge regarding plasmin structure, specificity and function. This includes design strategies to obtain the potency and specificity of inhibition in addition to controlled temporal and spatial distribution tailored for the intended use.     

Crystal Structures of BapA Complexes with β‐Lactam‐Derived Inhibitors Illustrate Substrate Specificity and Enantioselectivity of β‐Aminopeptidases

β‐Aminopeptidases have exclusive biocatalytic potential because they react with peptides composed of β‐amino acids, which serve as building blocks for the design of non‐natural peptidomimetics. We have identified the β‐lactam antibiotic ampicillin and the ampicillin‐derived penicilloic acid as novel inhibitors of the β‐aminopeptidase BapA from Sphingosinicella xenopeptidilytica (K_i values of 0.69 and 0.74 mM , respectively). We report high‐resolution crystal structures of BapA in noncovalent complexes with these inhibitors and with the serine protease inhibitor 4‐(2‐aminoethyl)benzenesulfonyl fluoride. All three inhibitors showed similar binding characteristics; the aromatic moiety extended into a hydrophobic binding pocket of the active site, and the free amino group formed a salt bridge with Glu133 of BapA. The exact position of the inhibitors and structural details of the ligand binding pocket illustrate the specificity and the enantioselectivity of BapA‐catalyzed reactions with β‐peptide substrates.     

Microscale Parallel Synthesis of Acylated Aminotriazoles Enabling the Development of Factor XIIa and Thrombin Inhibitors

Herein we report a microscale parallel synthetic approach allowing for rapid access to libraries of N-acylated aminotriazoles and screening of their inhibitory activity against factor XIIa (FXIIa) and thrombin, which are targets for antithrombotic drugs. This approach, in combination with post-screening structure optimization, yielded a potent 7 nM inhibitor of FXIIa and a 25 nM thrombin inhibitor; both compounds showed no inhibition of the other tested serine proteases. Selected N-acylated aminotriazoles exhibited anticoagulant properties in vitro influencing the intrinsic blood coagulation pathway, but not extrinsic coagulation. Mechanistic studies of FXIIa inhibition suggested that synthesized N-acylated aminotriazoles are covalent inhibitors of FXIIa. These synthesized compounds may serve as a promising starting point for the development of novel antithrombotic drugs.     

Understanding binding selectivity toward trypsin and factor Xa: the role of aromatic interactions

A congeneric series of four bis-benzamidine inhibitors sharing a dianhydrosugar isosorbide scaffold in common has been studied by crystal structure analysis and enzyme kinetics with respect to their binding to trypsin and factor Xa. Within the series, aromatic interactions are an important determinant for selectivity discrimination among both serine proteases. To study the selectivity-determining features in detail, we used trypsin mutants in which the original binding site is gradually substituted to finally resemble the factor Xa binding pocket. The influence of these mutations has been analyzed on the binding of the closely related inhibitors. We present the crystal structures of the inhibitor complexes obtained by co-crystallizing an "intermediate" trypsin mutant. They could be determined to a resolution of up to 1.2 A, and we measured the inhibitory activity (K(i)) of each ligand against factor Xa, trypsin, and the various mutants. From these data we were able to derive a detailed structure-activity relationship which demonstrates the importance of aromatic interactions in protein-ligand recognition and their role in modulating enzyme selectivity. Pronounced preference is experienced to accommodate the benzamidine anchor with meta topology in the S(1) specificity pocket. One ligand possessing only para topology deviates strongly from the other members of the series and adopts a distinct binding mode addressing the S(1)' site instead of the distal S(3)/S(4) binding pocket.     

A Bisbenzamidine Phosphonate as a Janus‐faced Inhibitor for Trypsin‐like Serine Proteases

A hybrid approach was applied for the design of an inhibitor of trypsin‐like serine proteases. Compound 16 [(R,R)‐ and (R,S)‐diphenyl (4‐(1‐(4‐amidinobenzylamino)‐1‐oxo‐3‐phenylpropan‐2‐ylcarbamoyl)phenylamino)(4‐amidinophenyl)methylphosphonate hydrochloride], prepared in a convergent synthetic procedure, possesses a phosphonate warhead prone to react with the active site serine residue in a covalent, irreversible manner. Each of the two benzamidine moieties of 16 can potentially be accommodated in the S1 pocket of the target enzyme, but only the benzamidine close to the phosphonate group would then promote an irreversible interaction. The Janus‐faced inhibitor 16 was evaluated against several serine proteases and caused a pronounced inactivation of human thrombin with a second‐order rate constant (k_inac/K_i) of 59 500 M ^?1 s^?1. With human matriptase, 16 showed preference for a reversible mode of inhibition (IC₅₀=2.6 μM ) as indicated by linear progress curves and enzyme reactivation.     

Therapeutic Strategies for Disseminated Intravascular Coagulation Associated with Aortic Aneurysm

Aortic aneurysms are sometimes associated with enhanced-fibrinolytic-type disseminated intravascular coagulation (DIC). In enhanced-fibrinolytic-type DIC, both coagulation and fibrinolysis are markedly activated. Typical cases show decreased platelet counts and fibrinogen levels, increased concentrations of fibrin/fibrinogen degradation products (FDP) and D-dimer, and increased FDP/D-dimer ratios. Thrombin-antithrombin complex or prothrombin fragment 1 + 2, as markers of coagulation activation, and plasmin-α₂ plasmin inhibitor complex, a marker of fibrinolytic activation, are all markedly increased. Prolongation of prothrombin time (PT) is not so obvious, and the activated partial thromboplastin time (APTT) is rather shortened in some cases. As a result, DIC can be neither diagnosed nor excluded based on PT and APTT alone. Many of the factors involved in coagulation and fibrinolysis activation are serine proteases. Treatment of enhanced-fibrinolytic-type DIC requires consideration of how to control the function of these serine proteases. The cornerstone of DIC treatment is treatment of the underlying pathology. However, in some cases surgery is either not possible or exacerbates the DIC associated with aortic aneurysm. In such cases, pharmacotherapy becomes even more important. Unfractionated heparin, other heparins, synthetic protease inhibitors, recombinant thrombomodulin, and direct oral anticoagulants (DOACs) are agents that inhibit serine proteases, and all are effective against DIC. Inhibition of activated coagulation factors by anticoagulants is key to the treatment of DIC. Among them, DOACs can be taken orally and is useful for outpatient treatment. Combination therapy of heparin and nafamostat allows fine-adjustment of anticoagulant and antifibrinolytic effects. While warfarin is an anticoagulant, this agent is ineffective in the treatment of DIC because it inhibits the production of coagulation factors as substrates without inhibiting activated coagulation factors. In addition, monotherapy using tranexamic acid in cases of enhanced-fibrinolytic-type DIC may induce fatal thrombosis. If tranexamic acid is needed for DIC, combination with anticoagulant therapy is of critical importance.     

Screening of 5- and 6-Substituted Amiloride Libraries Identifies Dual-uPA/NHE1 Active and Single Target-Selective Inhibitors

The K⁺-sparing diuretic amiloride shows off-target anti-cancer effects in multiple rodent models. These effects arise from the inhibition of two distinct cancer targets: the trypsin-like serine protease urokinase-type plasminogen activator (uPA), a cell-surface mediator of matrix degradation and tumor cell invasiveness, and the sodium-hydrogen exchanger isoform-1 (NHE1), a central regulator of transmembrane pH that supports carcinogenic progression. In this study, we co-screened our library of 5- and 6-substituted amilorides against these two targets, aiming to identify single-target selective and dual-targeting inhibitors for use as complementary pharmacological probes. Closely related analogs substituted at the 6-position with pyrimidines were identified as dual-targeting (pyrimidine 24 uPA IC₅₀ = 175 nM, NHE1 IC₅₀ = 266 nM, uPA selectivity ratio = 1.5) and uPA-selective (methoxypyrimidine 26 uPA IC₅₀ = 86 nM, NHE1 IC₅₀ = 12,290 nM, uPA selectivity ratio = 143) inhibitors, while high NHE1 potency and selectivity was seen with 5-morpholino (29 NHE1 IC₅₀ = 129 nM, uPA IC₅₀ = 10,949 nM; NHE1 selectivity ratio = 85) and 5-(1,4-oxazepine) (30 NHE1 IC₅₀ = 85 nM, uPA IC₅₀ = 5715 nM; NHE1 selectivity ratio = 67) analogs. Together, these amilorides comprise a new toolkit of chemotype-matched, non-cytotoxic probes for dissecting the pharmacological effects of selective uPA and NHE1 inhibition versus dual-uPA/NHE1 inhibition.     

Beyond Heparinization: Design of Highly Potent Thrombin Inhibitors Suitable for Surface Coupling

During extracorporeal circulation, when blood comes in contact with artificial surfaces, patients receive a standard treatment with anticoagulants to avoid blood coagulation. Dialysis patients in particular are systemically treated with heparin up to four times a week, causing a high burden for the body. For potential anticoagulant modification of external materials, such as dialysis equipment, a series of highly potent thrombin inhibitors was developed. All inhibitors share the general formula arylsulfonyl‐P3‐Pro‐4‐amidinobenzylamide, where P3 is glycyl or a trifunctional amino acid residue in L ‐configuration. Among this series, several derivatives inhibit thrombin with K_i values of less than 1 nM . Specificity measurements revealed that this inhibitor type is highly specific for thrombin with negligible activity against related trypsin‐like serine proteases. X‐ray analysis of the most potent analogue in complex with thrombin demonstrated that the N‐terminal arylsulfonyl group occupies the aryl binding site, whereas the P3 side chain is directed into the solvent and therefore is well suited for further coupling. Based on their in vitro profile, these inhibitors are suitable candidates for the development of hemocompatible materials with anticoagulant properties.     

Computational Selectivity Assessment of Protease Inhibitors against SARS-CoV-2

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious global health threat. Since no specific therapeutics are available, researchers around the world screened compounds to inhibit various molecular targets of SARS-CoV-2 including its main protease (M^pro) essential for viral replication. Due to the high urgency of these discovery efforts, off-target binding, which is one of the major reasons for drug-induced toxicity and safety-related drug attrition, was neglected. Here, we used molecular docking, toxicity profiling, and multiple molecular dynamics (MD) protocols to assess the selectivity of 33 reported non-covalent inhibitors of SARS-CoV-2 M^pro against eight proteases and 16 anti-targets. The panel of proteases included SARS-CoV M^pro, cathepsin G, caspase-3, ubiquitin carboxy-terminal hydrolase L1 (UCHL1), thrombin, factor Xa, chymase, and prostasin. Several of the assessed compounds presented considerable off-target binding towards the panel of proteases, as well as the selected anti-targets. Our results further suggest a high risk of off-target binding to chymase and cathepsin G. Thus, in future discovery projects, experimental selectivity assessment should be directed toward these proteases. A systematic selectivity assessment of SARS-CoV-2 M^pro inhibitors, as we report it, was not previously conducted.     

Functionalization of Fluorinated Benzenesulfonamides and Their Inhibitory Properties toward Carbonic Anhydrases

Substituted tri‐ and tetrafluorobenzenesulfonamides were designed, synthesized, and evaluated as high‐affinity and isoform‐selective carbonic anhydrase (CA) inhibitors. Their binding affinities for recombinant human CA I, II, VA, VI, VII, XII, and XIII catalytic domains were determined by fluorescent thermal shift assay, isothermal titration calorimetry, and a stopped‐flow CO₂ hydration assay. Variation of the substituents at the 2‐, 3‐, and 4‐positions yielded compounds with a broad range of binding affinities and isoform selectivities. Several 2,4‐substituted‐3,5,6‐trifluorobenzenesulfonamides were effective CA XIII inhibitors with high selectivity over off‐target CA I and CA II. 3,4‐Disubstituted‐2,5,6‐trifluorobenzenesulfonamides bound CAs with higher affinity than 2,4‐disubstituted‐3,5,6‐trifluorobenzenesulfonamides. Many such fluorinated benzenesulfonamides were found to be nanomolar inhibitors of CA II, CA VII, tumor‐associated CA IX and CA XII, and CA XIII. X‐ray crystal structures of inhibitors bound in the active sites of several CA isoforms provide structure–activity relationship information for inhibitor binding affinities and selectivity.     

Effect on inhibitory activity of mutation at reaction site P4 of the Streptomyces subtilisin inhibitor, SSI

The protein Streptomyces subtilisin inhibitor, SSI, efficientlyinhibits a bacterial serine protease, subtilisin BPN'. We recentlydemonstrated that functional change in SSI was possible simplyby replacing the amino acid residue at the reactive P1 site(methionine 73) of SSI. The present paper reports the additionaleffect of replacing methionine 70 at the P4 site of SSI(Lys73)on inhibitory activity toward two types of serine proteases,trypsin (or lysyl endopeptidase) and subtilisin BPN'. Conversionof methionine 70 at the P4 site of SSI(Lys73) to glycine oralanine resulted in increased inhibitory activity toward trypsinand lysyl endopeptidase, while replacement with phenylalanineweakened the inhibitory activity toward trypsin. This suggeststhat steric hindrance at the P4 site of SSI(Lys73) is an obstaclefor its binding with trypsin. In contrast, the same P4 replacementshad hardly any effect on inhibitory activity toward subtilisinBPN'. Thus the subsite structure of subtilisin BPN' is tolerantto these replacements. This contrast in the effect of P4 substitutionmight be due to the differences in the S4 subsite structuresbetween the trypsin-like and the subtilisin-like proteases.These findings demonstrate the importance of considering structuralcomplementarity, not only at the main reactive site but alsoat subsites of a protease, when designing stronger inhibitors.     

α-Triazolylboronic Acids: A Promising Scaffold for Effective Inhibitors of KPCs

Boronic acids are known reversible covalent inhibitors of serine β-lactamases. The selectivity and high potency of specific boronates bearing an amide side chain that mimics the β-lactam's amide side chain have been advanced in several studies. Herein, we describe a new class of boronic acids in which the amide group is replaced by a bioisostere triazole. The boronic acids were obtained in a two-step synthesis that relies on the solid and versatile copper-catalyzed azide–alkyne cycloaddition (CuAAC) followed by boronate deprotection. All of the compounds show very good inhibition of the Klebsiella pneumoniae carbapenemase KPC-2, with K_i values ranging from 1 nM to 1 μM, and most of them are able to restore cefepime activity against K. pneumoniae harboring bla_KPC-2. In particular, compound 1 e , bearing a sulfonamide substituted by a thiophene ring, proved to be an excellent KPC-2 inhibitor (K_i=30 nM); it restored cefepime susceptibility in KPC-Kpn cells (MIC=0.5 μg/mL) with values similar to that of vaborbactam (K_i=20 nM, MIC in KPC-Kpn 0.5 μg/mL). Our findings suggest that α-triazolylboronates might represent an effective scaffold for the treatment of KPC-mediated infections.     

Mapping the catechol binding site in dopamine D₁ receptors: synthesis and evaluation of two parallel series of bicyclic dopamine analogues

A novel class of isochroman dopamine analogues, originally reported by Abbott Laboratories, have >100‐fold selectivity for D₁‐like over D₂‐like receptors. We synthesized a parallel series of chroman compounds and showed that repositioning the oxygen atom in the heterocyclic ring decreases potency and confers D₂‐like receptor selectivity to these compounds. In silico modeling supports the hypothesis that the altered pharmacology for the chroman series is due to potential intramolecular hydrogen bonding between the oxygen in the chroman ring and the meta‐hydroxy group of the catechol moiety. This interaction realigns the catechol hydroxy groups and disrupts key interactions between these ligands and critical serine residues in TM5 of the D₁‐like receptors. This hypothesis was tested by the synthesis and pharmacological evaluation of a parallel series of carbocyclic compounds. Our results suggest that if the potential for intramolecular hydrogen bonding is removed, D₁‐like receptor potency and selectivity are restored.     

Deciphering Specificity Determinants for FR900359‐Derived Gqα Inhibitors Based on Computational and Structure–Activity Studies

Direct targeting of intracellular Gα subunits of G protein‐coupled receptors by chemical tools is a challenging task in current pharmacological studies and in the development of novel therapeutic approaches. In this study we analyzed novel FR900359‐based analogs from natural sources, synthetic cyclic peptides, as well as all so‐far known G_qα inhibitors in a comprehensive study to devise a strategy for the elucidation of characteristics that determine interactions with and inhibition of G_q in the specific FR/YM‐binding pocket. Using 2D NMR spectroscopy and molecular docking we identified unique features in the macrocyclic structures responsible for binding to the target protein correlating with inhibitory activity. While all novel compounds were devoid of effects on G_i and G_s proteins, no inhibitor surpassed the biological activity of FR. This raises the question of whether depsipeptides such as FR already represent valuable chemical tools for specific inhibition of G_q and, at the same time, are suitable natural lead structures for the development of novel compounds to target Gα subunits other than G_q.     

Ahp Cyclodepsipeptides: The Impact of the Ahp Residue on the “Canonical Inhibition” of S1 Serine Proteases

S1 serine proteases are by far the largest and most diverse family of proteases encoded in the human genome. Although recent decades have seen an enormous increase in our knowledge, the biological functions of most of these proteases remain to be elucidated. Chemical inhibitors have proven to be versatile tools for studying the functions of proteases, but this approach is hampered by the limited availability of inhibitor scaffold structures with the potential to allow rapid discovery of selective, noncovalent small‐molecule protease inhibitors. The natural product class of Ahp cyclodepsipeptides is an unusual class of small‐molecule canonical inhibitors; the incorporation of protease cleavage sequences into their molecular scaffolds enables the design of specific small‐molecule inhibitors that simultaneously target the S and S′ subsites of the protease through noncovalent mechanisms. Their synthesis is tedious, however, so in this study we have investigated the relevance of the Ahp moiety for achieving potent inhibition. We found that although the Ahp residue plays an important role in inhibition potency, appropriate replacement with β‐hydroxy amino acids results in structurally less complex derivatives that inhibit serine proteases in the low micromolar range.     

Targeting Cystalysin,a Virulence Factor of Treponema denticola‐Supported Periodontitis

Cystalysin from Treponema denticola is a pyridoxal 5′‐phosphate dependent lyase that catalyzes the formation of pyruvate, ammonia, and sulfide from cysteine. It is a virulence factor in adult periodontitis because its reaction contributes to hemolysis, which sustains the pathogen. Therefore, it was proposed as a potential antimicrobial target. To identify specific inhibitors by structure‐based in silico methods, we first validated the crystal structure of cystalysin as a reliable starting point for the design of ligands. By using single‐crystal absorption microspectrophotometry, we found that the enzyme in the crystalline state, with respect to that in solution, exhibits: 1) the same absorption spectra for the catalytic intermediates, 2) a close pK_a value for the residue controlling the keto enamine ionization, and 3) similar reactivity with glycine, L ‐serine, L ‐methionine, and the nonspecific irreversible inhibitor aminoethoxyvinylglycine. Next, we screened in silico a library of 9357 compounds with the Fingerprints for Ligands and Proteins (FLAP) software, by using the three‐dimensional structure of cystalysin as a template. From the library, 17 compounds were selected and experimentally evaluated by enzyme assays and spectroscopic methods. Two compounds were found to competitively inhibit recombinant T. denticola cystalysin, with inhibition constant (K_i) values of 25 and 37 μM . One of them exhibited a minimum inhibitory concentration (MIC) value of 64 μg mL^?1 on Moraxella catarrhalis ATCC 23246, which proves its ability to cross bacterial membranes.     

Design and Synthesis of Tricyclic JAK3 Inhibitors with Picomolar Affinities as Novel Molecular Probes

The Janus kinase (JAK) signaling pathway is of particular importance in the pathology of inflammatory diseases and oncological disorders, and the inhibition of Janus kinase 3 (JAK3) with small molecules has proven to provide therapeutic immunosuppression. A novel class of tricyclic JAK inhibitors derived from the 3‐methyl‐1,6‐dihydrodipyrrolo[2,3‐b:2′,3′‐d]pyridine scaffold was designed based on the tofacitinib–JAK3 crystal structure by applying a rigidization approach. A convenient synthetic strategy to access the scaffold via an intramolecular Heck reaction was developed, and a small library of inhibitors was prepared and characterized using in vitro biochemical as well as cellular assays. IC₅₀ values as low as 220 pM could be achieved with selectivity for JAK3 over other JAK family members. Both activity and selectivity were confirmed in a cellular STAT phosphorylation assay, providing also first‐time data for tofacitinib. Our novel inhibitors may serve as tool compounds and useful probes to explore the role of JAK3 inhibition in pharmacodynamics studies.