Water-medicated specifically targeting the S1 pockets among serine proteases using an arginine analogue.

Because of the high similarity in structure and sequence, it is challenging to distinguish the S1 pocket among serine proteases, primarily due to the only variability at residue 190 (A190 and S190). Peptide or protein-based inhibitors typically target the negatively charged S1 pocket using lysine or arginine as the P1 residue, yet neither discriminates between the two S1 pocket variants. This study introduces two arginine analogues, L-4-guanidinophenylalanine (12) and L-3-(N-amidino-4-piperidyl)alanine (16), as novel P1 residues in peptide inhibitors. 16 notably enhances affinities across all tested proteases, whereas 12 specifically improved affinities towards proteases possessing S190 in the S1 pocket. By crystallography and molecular dynamics simulations, we discovered a novel mechanism involving a water exchange channel at the bottom of the S1 pocket, modulated by the variation of residue 190. Additionally, the specificity of 12 towards the S190-presenting S1 pocket is dependent on this water channel. This study not only introduces novel P1 residues to engineer inhibitory potency and specificity of peptide inhibitors targeting serine proteases, but also unveils a water-mediated molecular mechanism of targeting serine proteases.

Synthesis, biological evaluation, and molecular modelling of substituted thiazolyl thiourea derivatives: A new class of prolyl oligopeptidase inhibitors.

Prolyl oligopeptidase (POP) is a compelling therapeutic target associated with aging and neurodegenerative disorders due to its pivotal role in neuropeptide processing. Despite initial promise demonstrated by early-stage POP inhibitors, their progress in clinical trials has been halted at Phase I or II. This impediment has prompted the pursuit of novel inhibitors. The current study seeks to contribute to the identification of efficacious POP inhibitors through the design, synthesis, and comprehensive evaluation (both in vitro and in silico) of thiazolyl thiourea derivatives (5a-r). In vitro experimentation exhibited that the compounds displayed significant higher potency as POP inhibitors. Compound 5e demonstrated an IC50 value of 16.47 ± 0.54 µM, representing a remarkable potency. A meticulous examination of the structure-activity relationship indicated that halogen and methoxy substituents were the most efficacious. In silico investigations delved into induced fit docking, pharmacokinetics, and molecular dynamics simulations to elucidate the intricate interactions, orientation, and conformational changes of these compounds within the active site of the enzyme. Moreover, our pharmacokinetic assessments confirmed that the majority of the synthesized compounds possess attributes conducive to potential drug development.

Identification of highly potent and selective HTRA1 inhibitors.

High temperature requirement A serine peptidase 1 (HTRA1) is a serine protease involved in an array of signaling pathways. It is also responsible for the regulation of protein aggregates via refolding, translocation, and degradation. It has subsequently been found that runaway proteolytic HTRA1 activity plays a role in a variety of diseases, including Age-Related Macular Degeneration (AMD), osteoarthritis, and Rheumatoid Arthritis. Selective inhibition of serine protease HTRA1 therefore offers a promising new strategy for the treatment of these diseases. Herein we disclose structure-activity-relationship (SAR) studies which identify key interactions responsible for binding affinity of small molecule inhibitors to HTRA1. The study results in highly potent molecules with IC50's less than 15 nM and excellent selectivity following a screen of 35 proteases.

Identification and characterization of aptameric inhibitors of human neutrophil elastase.

Human neutrophil elastase (HNE) plays a pivotal role in innate immunity, inflammation, and tissue remodeling. Aberrant proteolytic activity of HNE contributes to organ destruction in various chronic inflammatory diseases including emphysema, asthma, and cystic fibrosis. Therefore, elastase inhibitors could alleviate the progression of these disorders. Here, we used the systematic evolution of ligands by exponential enrichment to develop ssDNA aptamers that specifically target HNE. We determined the specificity of the designed inhibitors and their inhibitory efficacy against HNE using biochemical and in vitro methods, including an assay of neutrophil activity. Our aptamers inhibit the elastinolytic activity of HNE with nanomolar potency and are highly specific for HNE and do not target other tested human proteases. As such, this study provides lead compounds suitable for the evaluation of their tissue-protective potential in animal models.

[Boronic Acid as a Promising Class of Chemical Entity for Development of Clinical Medicine for Targeted Therapy of Cancer].

The first medicine containing the boron element, bortezomib, was approved for clinical use just 18 years ago. The boronic acid substructure in bortezomib serves as an electrophilic functionality with high affinity for hydroxy groups, which are frequently found in catalytic sites of proteolytic enzymes, to create reversible covalent bonds with a slow dissociation rate. Today, boronic acid is considered an important molecule in the medicinal chemistry toolbox, which was promoted by the success of bortezomib and pioneering approaches to use boronic acid in the molecular design of serine protease inhibitors in the 1980s. In this review article, we first provide an overview of the development of bortezomib, and then summarize our achievements to construct boronic acid analogs of tyropeptin A, a naturally occurring proteasome inhibitor, with potent in vivo efficacy. Representative stereoselective synthetic methods of α-aminoboronic acid are also showcased.

Elastase Inhibitor Cyclotheonellazole A: Total Synthesis and In Vivo Biological Evaluation for Acute Lung Injury.

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is one of the most common complications in COVID-19. Elastase has been recognized as an important target to prevent ALI/ARDS in the patient of COVID-19. Cyclotheonellazole A (CTL-A) is a natural macrocyclic peptide reported to be a potent elastase inhibitor. Herein, we completed the first total synthesis of CTL-A in 24 linear steps. The key reactions include three-component MAC reactions and two late-stage oxidations. We also provided seven CTL-A analogues and elucidated preliminary structure-activity relationships. The in vivo ALI mouse model further suggested that CTL-A alleviated acute lung injury with reductions in lung edema and pathological deterioration, which is better than sivelestat, one approved elastase inhibitor. The activity of CTL-A against elastase, along with its cellular safety and well-established synthetic route, warrants further investigation of CTL-A as a candidate against COVID-19 pathogeneses.

Site-Specific Conjugation of a Selenopolypeptide to Alpha-1-antitrypsin Enhances Oxidation Resistance and Pharmacological Properties.

Human alpha-1-antitrypsin (A1AT), a native serine-protease inhibitor that protects tissue damage from excessive protease activities, is used as an augmentation therapy to treat A1AT-deficienct patients. However, A1AT is sensitive to oxidation-mediated deactivation and has a short circulating half-life. Currently, there is no method that can effectively protect therapeutic proteins from oxidative damage in vivo. Here we developed a novel biocompatible selenopolypeptide and site-specifically conjugated it with A1AT. The conjugated A1AT fully retained its inhibitory activity on neutrophil elastase, enhanced oxidation resistance, extended the serum half-life, and afforded long-lasting protective efficacy in a mouse model of acute lung injury. These results demonstrated that conjugating A1AT with the designed selenopolymer is a viable strategy to improve its pharmacological properties, which could potentially further be applied to a variety of oxidation sensitive biotherapeutics.

1,5,6,7-Tetrahydro-4H-indazol-4-ones as human neutrophil elastase (HNE) inhibitors.

Human neutrophil elastase (HNE) is a serine protease that is expressed in polymorphonuclear neutrophils. It has been recognized as an important therapeutic target for treating inflammatory diseases, especially related to the respiratory system, but also for various types of cancer. Thus, compounds able to inhibit HNE are of great interest in medicinal chemistry. In the present paper, we report the synthesis and biological evaluation of a new series of HNE inhibitors with an innovative 1,5,6,7-tetrahydro-4H-indazol-4-one core that was developed as a molecular modification of our previously reported indazole-based HNE inhibitors. Since the 1,5,6,7-tetrahydro-4H-indazol-4-one scaffold can occur in two possible tautomeric forms, the acylation/alkylation reactions resulted in a mixture of the two isomers, often widely unbalanced in favor of one form. Using analytical techniques and NMR spectroscopy, we characterized and separated the isomer pairs and confirmed the compounds used in biological testing. Analysis of the compounds for HNE inhibitory activity showed that they were potent inhibitors, with Ki values in the low nanomolar range (6-35 nM). They also had reasonable stability in aqueous buffer, with half-lives over 1 h. Overall, our results indicate that the 1,5,6,7-tetrahydro-4H-indazol-4-one core is suitable for the synthesis of potent HNE inhibitors that could be useful in the development of new therapeutics for treating diseases involving excessive HNE activity.

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.

Optimization of peptide-based inhibitors targeting the HtrA serine protease in Chlamydia: Design, synthesis and biological evaluation of pyridone-based and N-Capping group-modified analogues.

The obligate intracellular bacterium Chlamydia trachomatis (C. trachomatis) is responsible for the most common bacterial sexually transmitted infection and is the leading cause of preventable blindness, representing a major global health burden. While C. trachomatis infection is currently treatable with broad-spectrum antibiotics, there would be many benefits of a chlamydia-specific therapy. Previously, we have identified a small-molecule lead compound JO146 [Boc-Val-Pro-ValP(OPh)2] targeting the bacterial serine protease HtrA, which is essential in bacterial replication, virulence and survival, particularly under stress conditions. JO146 is highly efficacious in attenuating infectivity of both human (C. trachomatis) as well as koala (C. pecorum) species in vitro and in vivo, without host cell toxicity. Herein, we present our continuing efforts on optimizing JO146 by modifying the N-capping group as well as replacing the parent peptide structure with the 2-pyridone scaffold at P3/P2. The drug optimization process was guided by molecular modelling, enzyme and cell-based assays. Compound 18b from the pyridone series showed improved inhibitory activity against CtHtrA by 5-fold and selectivity over human neutrophil elastase (HNE) by 109-fold compared to JO146, indicating that 2-pyridone is a suitable bioisostere of the P3/P2 amide/proline for developing CtHtrA inhibitors. Most pyridone-based inhibitors showed superior anti-chlamydial potency to JO146 especially at lower doses (25 and 50 µM) in C. trachomatis and C. pecorum cell culture assays. Modifications of the N-capping group of the peptidyl inhibitors did not have much influence on the anti-chlamydial activities, providing opportunities for more versatile alterations and future optimization. In summary, we present 2-pyridone based analogues as a new generation of non-peptidic CtHtrA inhibitors, which hold better promise as anti-chlamydial drug candidates.

Potent Lys Patch-Containing Stapled Peptides Targeting PCSK9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), identified as a regulator of low-density lipoprotein receptor (LDLR), plays a major role in cardiovascular diseases (CVD). Recently, Pep2-8, a small peptide with discrete three-dimensional structure, was found to inhibit the PCSK9/LDLR interaction. In this paper, we describe the modification of this peptide using stapled peptide and SIP technologies. Their combination yielded potent compounds such as 18 that potently inhibited the binding of PCSK9 to LDLR (KD = 6 ± 1 nM) and restored in vitro LDL uptake by HepG2 cells in the presence of PCSK9 (EC50 = 175 ± 40 nM). The three-dimensional structures of key peptides were extensively studied by circular dichroism and nuclear magnetic resonance, and molecular dynamics simulations allowed us to compare their binding mode to tentatively rationalize structure-activity relationships (SAR).

Synthesis, Structure-Activity Relationships, and Antiviral Activity of Allosteric Inhibitors of Flavivirus NS2B-NS3 Protease.

Flaviviruses, including Zika, dengue, and West Nile viruses, are important human pathogens. The highly conserved NS2B-NS3 protease of Flavivirus is essential for viral replication and therefore a promising drug target. Through compound screening, followed by medicinal chemistry studies, a novel series of 2,5,6-trisubstituted pyrazine compounds are found to be potent, allosteric inhibitors of Zika virus protease (ZVpro) with IC50 values as low as 130 nM. Their structure-activity relationships are discussed. The ZVpro inhibitors also inhibit homologous proteases of dengue and West Nile viruses, and their inhibitory activities are correlated. The most potent compounds 47 and 103 potently inhibited Zika virus replication in cells with EC68 values of 300-600 nM and in a mouse model of Zika infection. These compounds represent novel pharmacological leads for drug development against Flavivirus infections.

Exploration of nitrogen heterocycle scaffolds for the development of potent human neutrophil elastase inhibitors.

Human neutrophil elastase (HNE) is a potent protease that plays an important physiological role in many processes but is also involved in a variety of pathologies that affect the pulmonary system. Thus, compounds able to inhibit HNE proteolytic activity could represent effective therapeutics. We present here a new series of pyrazolopyridine and pyrrolopyridine derivatives as HNE inhibitors designed as modifications of our previously synthesized indazoles and indoles in order to evaluate effects of the change in position of the nitrogen and/or the insertion of an additional nitrogen in the scaffolds on biological activity and chemical stability. We obtained potent HNE inhibitors with IC50 values in the low nanomolar range (10-50 nM), and some compounds exhibited improved chemical stability in phosphate buffer (t1/2 > 6 h). Molecular modeling studies demonstrated that inhibitory activity was strictly dependent on the formation of a Michaelis complex between the OH group of HNE Ser195 and the carbonyl carbon of the inhibitor. Moreover, in silico ADMET calculations predicted that most of the new compounds would be optimally absorbed, distributed, metabolized, and excreted. Thus, these new and potent HNE inhibitors represent novel leads for future therapeutic development.

Discovery of BMS-986144, a Third-Generation, Pan-Genotype NS3/4A Protease Inhibitor for the Treatment of Hepatitis C Virus Infection.

The discovery of a pan-genotypic hepatitis C virus (HCV) NS3/4A protease inhibitor based on a P1-P3 macrocyclic tripeptide motif is described. The all-carbon tether linking the P1-P3 subsites of 21 is functionalized with alkyl substituents, which are shown to effectively modulate both potency and absorption, distribution, metabolism, and excretion (ADME) properties. The CF3Boc-group that caps the P3 amino moiety was discovered to be an essential contributor to metabolic stability, while positioning a methyl group at the C1 position of the P1' cyclopropyl ring enhanced plasma trough values following oral administration to rats. The C7-fluoro, C6-CD3O substitution pattern of the P2* isoquinoline heterocycle of 21 was essential to securing the targeted potency, pharmacokinetic (PK), and toxicological profiles. The C6-CD3O redirected metabolism away from a problematic pathway, thereby circumventing the time-dependent cytochrome P (CYP) 450 inhibition observed with the C6-CH3O prototype.

Peptidyl Fluoromethyl Ketones and Their Applications in Medicinal Chemistry.

Peptidyl fluoromethyl ketones occupy a pivotal role in the current scenario of synthetic chemistry, thanks to their numerous applications as inhibitors of hydrolytic enzymes. The insertion of one or more fluorine atoms adjacent to a C-terminal ketone moiety greatly modifies the physicochemical properties of the overall substrate, especially by increasing the reactivity of this functionalized carbonyl group toward nucleophiles. The main application of these peptidyl α-fluorinated ketones in medicinal chemistry relies in their ability to strongly and selectively inhibit serine and cysteine proteases. These compounds can be used as probes to study the proteolytic activity of the aforementioned proteases and to elucidate their role in the insurgence and progress on several diseases. Likewise, if the fluorinated methyl ketone moiety is suitably connected to a peptidic backbone, it may confer to the resulting structure an excellent substrate peculiarity and the possibility of being recognized by a specific subclass of human or pathogenic proteases. Therefore, peptidyl fluoromethyl ketones are also currently highly exploited for the target-based design of compounds for the treatment of topical diseases such as various types of cancer and viral infections.

1H-Imidazole-2,5-Dicarboxamides as NS4A Peptidomimetics: Identification of a New Approach to Inhibit HCV-NS3 Protease.

The nonstructural (NS) protein NS3/4A protease is a critical factor for hepatitis C virus (HCV) maturation that requires activation by NS4A. Synthetic peptide mutants of NS4A were found to inhibit NS3 function. The bridging from peptide inhibitors to heterocyclic peptidomimetics of NS4A has not been considered in the literature and, therefore, we decided to explore this strategy for developing a new class of NS3 inhibitors. In this report, a structure-based design approach was used to convert the bound form of NS4A into 1H-imidazole-2,5-dicarboxamide derivatives as first generation peptidomimetics. This scaffold mimics the buried amino acid sequence Ile-25` to Arg-28` at the core of NS4A21`-33` needed to activate the NS3 protease. Some of the synthesized compounds (Coded MOC) were able to compete with and displace NS4A21`-33` for binding to NS3. For instance, N5-(4-guanidinobutyl)-N2-(n-hexyl)-1H-imidazole-2,5-dicarboxamide (MOC-24) inhibited the binding of NS4A21`-33` with a competition half maximal inhibitory concentration (IC50) of 1.9 ± 0.12 µM in a fluorescence anisotropy assay and stabilized the denaturation of NS3 by increasing the aggregation temperature (40% compared to NS4A21`-33`). MOC-24 also inhibited NS3 protease activity in a fluorometric assay. Molecular dynamics simulations were conducted to rationalize the differences in structure-activity relationship (SAR) between the active MOC-24 and the inactive MOC-26. Our data show that MOC compounds are possibly the first examples of NS4A peptidomimetics that have demonstrated promising activities against NS3 proteins.

Targeting Fibroblast Activation Protein: Radiosynthesis and Preclinical Evaluation of an 18F-Labeled FAP Inhibitor.

Fibroblast activation protein (FAP) has emerged as an interesting molecular target used in the imaging and therapy of various types of cancers. 68Ga-labeled chelator-linked FAP inhibitors (FAPIs) have been successfully applied to PET imaging of various tumor types. To broaden the spectrum of applicable PET tracers for extended imaging studies of FAP-dependent diseases, we herein report the radiosynthesis and preclinical evaluation of an 18F-labeled glycosylated FAPI ([18F]FGlc-FAPI). Methods: An alkyne-bearing precursor was synthesized and subjected to click chemistry-based radiosynthesis of [18F]FGlc-FAPI by 2-step 18F-fluoroglycosylation. FAP-expressing HT1080hFAP cells were used to study competitive binding to FAP, cellular uptake, internalization, and efflux of [18F]FGlc-FAPI in vitro. Biodistribution studies and in vivo small-animal PET studies of [18F]FGlc-FAPI compared with [68Ga]Ga-FAPI-04 were conducted in nude mice bearing HT1080hFAP tumors or U87MG xenografts. Results: [18F]FGlc-FAPI was synthesized with a 15% radioactivity yield and a high radiochemical purity of more than 99%. In HT1080hFAP cells, [18F]FGlc-FAPI showed specific uptake, a high internalized fraction, and low cellular efflux. Compared with FAPI-04 (half maximal inhibitory concentration [IC50] = 32 nM), the glycoconjugate, FGlc-FAPI (IC50 = 167 nM), showed slightly lower affinity for FAP in vitro, whereas plasma protein binding was higher for [18F]FGlc-FAPI. Biodistribution studies revealed significant hepatobiliary excretion of [18F]FGlc-FAPI; however, small-animal PET studies in HT1080hFAP xenografts showed higher specific tumor uptake of [18F]FGlc-FAPI (4.5 percentage injected dose per gram of tissue [%ID/g]) than of [68Ga]Ga-FAPI-04 (2 %ID/g). In U87MG tumor-bearing mice, both tracers showed similar tumor uptake, but [18F]FGlc-FAPI showed a higher tumor retention. Interestingly, [18F]FGlc-FAPI demonstrated high specific uptake in bone structures and joints. Conclusion: [18F]FGlc-FAPI is an interesting candidate for translation to the clinic, taking advantage of the longer half-life and physical imaging properties of 18F. The availability of [18F]FGlc-FAPI may allow extended PET studies of FAP-related diseases, such as cancer, but also arthritis, heart diseases, or pulmonary fibrosis.

Detection of Active Granzyme A in NK92 Cells with Fluorescent Activity-Based Probe.

Cytotoxic T-lymphocytes (CTLs) and natural killer cells (NKs) kill compromised cells to defend against tumor and viral infections. Both effector cell types use multiple strategies to induce target cell death including Fas/CD95 activation and the release of perforin and a group of lymphocyte granule serine proteases called granzymes. Granzymes have relatively broad and overlapping substrate specificities and may hydrolyze a wide range of peptidic epitopes; it is therefore challenging to identify their natural and synthetic substrates and to distinguish their localization and functions. Here, we present a specific and potent substrate, an inhibitor, and an activity-based probe of Granzyme A (GrA) that can be used to follow functional GrA in cells.

Orally bioavailable amine-linked macrocyclic inhibitors of factor XIa.

The discovery of orally bioavailable FXIa inhibitors has been a challenge. Herein, we describe our efforts to address this challenge by optimization of our imidazole-based macrocyclic series. Our optimization strategy focused on modifications to the P2 prime, macrocyclic amide linker, and the imidazole scaffold. Replacing the amide of the macrocyclic linker with amide isosteres led to the discovery of substituted amine linkers which not only maintained FXIa binding affinity but also improved oral exposure in rats. Combining the optimized macrocyclic amine linker with a pyridine scaffold afforded compounds 23 and 24 that were orally bioavailable, single-digit nanomolar FXIa inhibitors with excellent selectivity against relevant blood coagulation enzymes.