Search for Non-Protein Protease Inhibitors Constituted with an Indole and Acetylene Core.

A possible inhibitor of proteases, which contains an indole core and an aromatic polar acetylene, was designed and synthesized. This indole derivative has a molecular architecture kindred to biologically relevant species and was obtained through five synthetic steps with an overall yield of 37% from the 2,2'-(phenylazanediyl)di(ethan-1-ol). The indole derivative was evaluated through docking assays using the main protease (SARS-CoV-2-Mpro) as a molecular target, which plays a key role in the replication process of this virus. Additionally, the indole derivative was evaluated as an inhibitor of the enzyme kallikrein 5 (KLK5), which is a serine protease that can be considered as an anticancer drug target.

Targeted delivery of cytotoxic proteins to prostate cancer via conjugation to small molecule urea-based PSMA inhibitors.

Prostate cancer cells are characterized by a remarkably low proliferative rate and the production of high levels of prostate-specific proteases. Protein-based toxins are attractive candidates for prostate cancer therapy because they kill cells via proliferation-independent mechanisms. However, the non-specific cytotoxicity of these potent cytotoxins must be redirected to avoid toxicity to normal tissues. Prostate-Specific Membrane Antigen (PSMA) is membrane-bound carboxypeptidase that is highly expressed by prostate cancer cells. Potent dipeptide PSMA inhibitors have been developed that can selectively deliver and concentrate imaging agents within prostate cancer cells based on continuous PSMA internalization and endosomal cycling. On this basis, we conjugated a PSMA inhibitor to the apoptosis-inducing human protease Granzyme B and the potent Pseudomonas exotoxin protein toxin fragment, PE35. We assessed selective PSMA binding and entrance into tumor cell to induce cell death. We demonstrated these agents selectively bound to PSMA and became internalized. PSMA-targeted PE35 toxin was selectively toxic to PSMA producing cells in vitro. Intratumoral and intravenous administration of this toxin produced marked tumor killing of PSMA-producing xenografts with minimal host toxicity. These studies demonstrate that urea-based PSMA inhibitors represent a simpler, less expensive alternative to antibodies as a means to deliver cytotoxic proteins to prostate cancer cells.

Lutetium-177-PSMA-617 for Metastatic Castration-Resistant Prostate Cancer.

BACKGROUND: Metastatic castration-resistant prostate cancer remains fatal despite recent advances. Prostate-specific membrane antigen (PSMA) is highly expressed in metastatic castration-resistant prostate cancer. Lutetium-177 (177Lu)-PSMA-617 is a radioligand therapy that delivers beta-particle radiation to PSMA-expressing cells and the surrounding microenvironment. METHODS: We conducted an international, open-label, phase 3 trial evaluating 177Lu-PSMA-617 in patients who had metastatic castration-resistant prostate cancer previously treated with at least one androgen-receptor-pathway inhibitor and one or two taxane regimens and who had PSMA-positive gallium-68 (68Ga)-labeled PSMA-11 positron-emission tomographic-computed tomographic scans. Patients were randomly assigned in a 2:1 ratio to receive either 177Lu-PSMA-617 (7.4 GBq every 6 weeks for four to six cycles) plus protocol-permitted standard care or standard care alone. Protocol-permitted standard care excluded chemotherapy, immunotherapy, radium-223 (223Ra), and investigational drugs. The alternate primary end points were imaging-based progression-free survival and overall survival, which were powered for hazard ratios of 0.67 and 0.73, respectively. Key secondary end points were objective response, disease control, and time to symptomatic skeletal events. Adverse events during treatment were those occurring no more than 30 days after the last dose and before subsequent anticancer treatment. RESULTS: From June 2018 to mid-October 2019, a total of 831 of 1179 screened patients underwent randomization. The baseline characteristics of the patients were balanced between the groups. The median follow-up was 20.9 months. 177Lu-PSMA-617 plus standard care significantly prolonged, as compared with standard care, both imaging-based progression-free survival (median, 8.7 vs. 3.4 months; hazard ratio for progression or death, 0.40; 99.2% confidence interval [CI], 0.29 to 0.57; P<0.001) and overall survival (median, 15.3 vs. 11.3 months; hazard ratio for death, 0.62; 95% CI, 0.52 to 0.74; P<0.001). All the key secondary end points significantly favored 177Lu-PSMA-617. The incidence of adverse events of grade 3 or above was higher with 177Lu-PSMA-617 than without (52.7% vs. 38.0%), but quality of life was not adversely affected. CONCLUSIONS: Radioligand therapy with 177Lu-PSMA-617 prolonged imaging-based progression-free survival and overall survival when added to standard care in patients with advanced PSMA-positive metastatic castration-resistant prostate cancer. (Funded by Endocyte, a Novartis company; VISION ClinicalTrials.gov number, NCT03511664.).

A Suite of Activity-Based Probes To Dissect the KLK Activome in Drug-Resistant Prostate Cancer.

Kallikrein-related peptidases (KLKs) are a family of secreted serine proteases, which form a network (the KLK activome) with an important role in proteolysis and signaling. In prostate cancer (PCa), increased KLK activity promotes tumor growth and metastasis through multiple biochemical pathways, and specific quantification and tracking of changes in the KLK activome could contribute to validation of KLKs as potential drug targets. Herein we report a technology platform based on novel activity-based probes (ABPs) and inhibitors enabling simultaneous orthogonal analysis of KLK2, KLK3, and KLK14 activity in hormone-responsive PCa cell lines and tumor homogenates. Importantly, we identifed a significant decoupling of KLK activity and abundance and suggest that KLK proteolysis should be considered as an additional parameter, along with the PSA blood test, for accurate PCa diagnosis and monitoring. Using selective inhibitors and multiplexed fluorescent activity-based protein profiling (ABPP), we dissect the KLK activome in PCa cells and show that increased KLK14 activity leads to a migratory phenotype. Furthermore, using biotinylated ABPs, we show that active KLK molecules are secreted into the bone microenvironment by PCa cells following stimulation by osteoblasts suggesting KLK-mediated signaling mechanisms could contribute to PCa metastasis to bone. Together our findings show that ABPP is a powerful approach to dissect dysregulation of the KLK activome as a promising and previously underappreciated therapeutic target in advanced PCa.

The Role of Extracellular Proteases in Tumor Progression and the Development of Innovative Metal Ion Chelators that Inhibit their Activity.

The crucial role of extracellular proteases in cancer progression is well-known, especially in relation to the promotion of cell invasion through extracellular matrix remodeling. This also occurs by the ability of extracellular proteases to induce the shedding of transmembrane proteins at the plasma membrane surface or within extracellular vesicles. This process results in the regulation of key signaling pathways by the modulation of kinases, e.g., the epidermal growth factor receptor (EGFR). Considering their regulatory roles in cancer, therapeutics targeting various extracellular proteases have been discovered. These include the metal-binding agents di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), which increase c-MET degradation by multiple mechanisms. Both the direct and indirect inhibition of protease expression and activity can be achieved through metal ion depletion. Considering direct mechanisms, chelators can bind zinc(II) that plays a catalytic role in enzyme activity. In terms of indirect mechanisms, Dp44mT and DpC potently suppress the expression of the kallikrein-related peptidase-a prostate-specific antigen-in prostate cancer cells. The mechanism of this activity involves promotion of the degradation of the androgen receptor. Additional suppressive mechanisms of Dp44mT and DpC on matrix metalloproteases (MMPs) relate to their ability to up-regulate the metastasis suppressors N-myc downstream regulated gene-1 (NDRG1) and NDRG2, which down-regulate MMPs that are crucial for cancer cell invasion.

Structural Studies on the Inhibitory Binding Mode of Aromatic Coumarinic Esters to Human Kallikrein-Related Peptidase 7.

The serine protease kallikrein-related peptidase 7 (KLK7) is a member of the human tissue kallikreins. Its dysregulation leads to pathophysiological inflammatory processes in the skin. Furthermore, it plays a role in several types of cancer. For the treatment of KLK7-associated diseases, coumarinic esters have been developed as small-molecule enzyme inhibitors. To characterize the inhibition mode of these inhibitors, we analyzed structures of the inhibited protease by X-ray crystallography. Electron density shows the inhibitors covalently attached to His57 of the catalytic triad. This confirms the irreversible character of the inhibition process. Upon inhibitor binding, His57 undergoes an outward rotation; thus, the catalytic triad of the protease is disrupted. Besides, the halophenyl moiety of the inhibitor was absent in the final enzyme-inhibitor complex due to the hydrolysis of the ester linkage. With these results, we analyze the structural basis of KLK7 inhibition by the covalent attachment of aromatic coumarinic esters.

Loss-of-Function Variants in SERPINA12 Underlie Autosomal Recessive Palmoplantar Keratoderma.

Inherited palmoplantar keratodermas refer to a large and heterogeneous group of conditions resulting from abnormal epidermal differentiation and featuring thickening of the skin of the palms and soles. Here, we aimed at delineating the genetic basis of an autosomal recessive form of palmoplantar keratodermas manifesting with erythematous hyperkeratotic plaques over the palms and soles, extending to non-palmoplantar areas. Whole-exome sequencing in affected individuals revealed homozygous nonsense variants in the SERPINA12 gene. SERPINA12 encodes the visceral adipose tissue-derived serpin A12, a serine protease inhibitor. The pathogenic variants were found to result in reduced visceral adipose tissue-derived serpin A12 expression in patients' skin biopsies in comparison to healthy controls. In addition, SERPINA12 downregulation in three-dimensional skin equivalents was associated with marked epidermal acanthosis and hyperkeratosis, replicating the human phenotype. Moreover, decreased SERPINA12 expression resulted in reduced visceral adipose tissue-derived serpin A12-mediated inhibition of kallikrein 7 activity as well as decreased levels of desmoglein-1 and corneodesmosin, two known kallikrein 7 substrates, which are required for normal epidermal differentiation. The present data, taken collectively, demarcate a unique type of autosomal recessive palmoplantar keratodermas, attribute to visceral adipose tissue-derived serpin A12 a role in skin biology, and emphasize the importance of mechanisms regulating proteolytic activity for normal epidermal differentiation.

[Recent progress of positron radionuclide labeled small molecule inhibitors of prostate specific membrane antigen for PET/CT imaging of prostate cancer].

Prostate cancer is the most common tumor of the urinary system, and its mortality rate is second only to lung cancer. With the specific and high expression on the surface of prostate cancer cells, prostate-specific membrane antigen (PSMA) has been an ideal theranostic target of prostate cancer with great clinical significance and research value. Positron emission tomography/computed tomography (PET/CT), a new modality of molecular imaging combining functional metabolic information and anatomical structure, provides high diagnostic performance for cancer detection. This paper mainly reviewed recent progress of PSMA inhibitors labeled by positron-emitting radionuclides for early diagnosis, preoperative staging, response assessment, restaging and metastasis detection of prostate cancer.

Biological therapy in hereditary angioedema: transformation of a rare disease.

Introduction: Hereditary angioedema, a disabling condition, with high mortality when untreated, is caused by C1 inhibitor deficiency and other regulatory disorders of bradykinin production or metabolism. This review covers the remarkable progress made in biological therapies for this rare disorder.Areas covered: Over the past 10 years, several evidence-based parenteral treatments have been licensed, including two plasma-derived C1 inhibitor replacement therapies and one recombinant C1 inhibitor replacement for acute treatment of angioedema attacks and synthetic peptides for inhibition of kallikrein or bradykinin B2 receptors, with oral small molecule treatments currently in clinical trial. Moreover, recent advances in prophylaxis by subcutaneous C1 inhibitor to restore near-normal plasma function or by humanized antibody inhibition of kallikrein have resulted in freedom from symptoms for a high proportion of those treated.Expert opinion: This plethora of treatment possibilities has come about as a result of recent scientific advances. Collaboration between patient groups, basic and clinical scientists, physicians, nurses, and the pharmaceutical industry has underpinned this translation of basic science into treatments and protocols. These in their turn have brought huge improvements in prognosis, quality of life and economic productivity to patients, their families, and the societies in which they live.

Toward the Discovery and Development of PSMA Targeted Inhibitors for Nuclear Medicine Applications.

BACKGROUND: The rising incidence rate of prostate cancer (PCa) has promoted the development of new diagnostic and therapeutic radiopharmaceuticals during the last decades. Promising improvements have been achieved in clinical practice using prostate specific membrane antigen (PSMA) labeled agents, including specific antibodies and small molecular weight inhibitors. Focusing on molecular docking studies, this review aims to highlight the progress in the design of PSMA targeted agents for a potential use in nuclear medicine. RESULTS: Although the first development of radiopharmaceuticals able to specifically recognize PSMA was exclusively oriented to macromolecule protein structure such as radiolabeled monoclonal antibodies and derivatives, the isolation of the crystal structure of PSMA served as the trigger for the synthesis and the further evaluation of a variety of low molecular weight inhibitors. Among the nuclear imaging probes and radiotherapeutics that have been developed and tested till today, labeled Glutamate-ureido inhibitors are the most prevalent PSMA-targeting agents for nuclear medicine applications. CONCLUSION: PSMA represents for researchers the most attractive target for the detection and treatment of patients affected by PCa using nuclear medicine modalities. [99mTc]MIP-1404 is considered the tracer of choice for SPECT imaging and [68Ga]PSMA-11 is the leading diagnostic for PET imaging by general consensus. [18F]DCFPyL and [18F]PSMA-1007 are clearly the emerging PET PSMA candidates for their great potential for a widespread commercial distribution. After paving the way with new imaging tools, academic and industrial R&Ds are now focusing on the development of PSMA inhibitors labeled with alpha or beta minus emitters for a theragnostic application.

Synthesis and Structure-Activity Relationships of N-(4-Benzamidino)-Oxazolidinones: Potent and Selective Inhibitors of Kallikrein-Related Peptidase 6.

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease that belongs to the family of tissue kallikreins. Aberrant expression of KLK6 has been found in different cancers and neurodegenerative diseases, and KLK6 is currently studied as a potential target in these pathologies. We report a novel series of KLK6 inhibitors discovered in a high-throughput screen within the European Lead Factory program. Structure-guided design based on docking studies enabled rapid progression of a hit cluster to inhibitors with improved potency, selectivity and pharmacokinetic properties. In particular, inhibitors 32 ((5R)-3-(4-carbamimidoylphenyl)-N-((S)-1-(naphthalen-1-yl)propyl)-2-oxooxazolidine-5-carboxamide) and 34 ((5R)-3-(6-carbamimidoylpyridin-3-yl)-N-((1S)-1-(naphthalen-1-yl)propyl)-2-oxooxazolidine-5-carboxamide) have single-digit nanomolar potency against KLK6, with over 25-fold and 100-fold selectivities against the closely related enzyme trypsin, respectively. The most potent compound, 32, effectively reduces KLK6-dependent invasion of HCT116 cells. The high potency in combination with good solubility and low clearance of 32 make it a good chemical probe for KLK6 target validation in vitro and potentially in vivo.

Characterization of a KLK2-FGFR2 fusion gene in two cases of metastatic prostate cancer.

BACKGROUND: The fibroblast growth factor receptor (FGFR) signaling pathway is activated in multiple tumor types through gene amplifications, single base substitutions, or gene fusions. Multiple small molecule kinase inhibitors targeting FGFR are currently being evaluated in clinical trials for patients with FGFR chromosomal translocations. Patients with novel gene fusions involving FGFR may represent candidates for kinase inhibitors. METHODS: A targeted RNA-sequencing assay identified a KLK2-FGFR2 fusion gene in two patients with metastatic prostate cancer. NIH3T3 cells were transduced to express the KLK2-FGFR2 fusion. Migration assays, Western blots, and drug sensitivity assays were performed to functionally characterize the fusion. RESULTS: Expression of the KLK2-FGFR2 fusion protein in NIH3T3 cells induced a profound morphological change promoting enhanced migration and activation of downstream proteins in FGFR signaling pathways. The KLK2-FGFR2 fusion protein was determined to be highly sensitive to the selective FGFR inhibitors AZD-4547, BGJ398, JNJ-42756943, the irreversible inhibitor TAS-120, and the non-selective inhibitor Ponatinib. The KLK2-FGFR2 fusion did not exhibit sensitivity to the non-selective inhibitor Dovitinib. CONCLUSIONS: Importantly, the KLK2-FGFR2 fusion represents a novel target for precision therapies and should be screened for in men with prostate cancer.

Visualizing protein-ligand binding with chemical energy-wise decomposition (CHEWD): application to ligand binding in the kallikrein-8 S1 Site.

Kallikrein-8, a serine protease, is a target for structure-based drug design due to its therapeutic potential in treating Alzheimer's disease and is also useful as a biomarker in ovarian cancer. We present a binding assessment of ligands to kallikrein-8 using a residue-wise decomposition of the binding energy. Binding of four putative inhibitors of kallikrein-8 is investigated through molecular dynamics simulation and ligand binding energy evaluation with two methods (MM/PBSA and WaterSwap). For visualization of the residue-wise decomposition of binding energies, chemical energy-wise decomposition or CHEWD is introduced as a plugin to UCSF Chimera and Pymol. CHEWD allows easy comparison between ligands using individual residue contributions to the binding energy. Molecular dynamics simulations indicate one ligand binds stably to the kallikrein-8 S1 binding site. Comparison with other members of the kallikrein family shows that residues responsible for binding are specific to kallikrein-8. Thus, ZINC02927490 is a promising lead for development of novel kallikrein-8 inhibitors.

Effects of two protease inhibitors from Bauhinia bauhinoides with different specificity towards gut enzymes of Nasutitermes corniger and its survival.

Two recombinant protease inhibitors from Bauhinia bauhinioides, rBbKI (kallikrein inhibitor) and rBbCI (cruzipain inhibitor) were evaluated for insecticidal activity against workers and soldiers of Nasutitermes corniger (order: Isoptera; family: Termitidae) through the inhibitors' effect on the insect's gut enzymes. The inhibitor rBbKI was more effective than rBbCI in inhibiting the termite's gut enzymes. The kallikrein inhibitor showed termiticidal activity in workers with an LC50 of 0.9 mg mL-1 after 4 days. Conversely, rBbKI did not affect the survival of soldiers and rBbCI did not show termiticidal activity against N. corniger. The two inhibitors showed different specificity towards the termite's gut enzymes, representing interesting tools to characterize N. corniger enzymes. The different effects of rBbKI and rBbCI on the termite's enzymes and survival may be linked to slight structural differences between these inhibitors.

Depsipeptides Featuring a Neutral P1 Are Potent Inhibitors of Kallikrein-Related Peptidase 6 with On-Target Cellular Activity.

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease that belongs to the family of tissue kallikreins (KLKs). Many KLKs are investigated as potential biomarkers for cancer as well as therapeutic drug targets for a number of pathologies. KLK6, in particular, has been implicated in neurodegenerative diseases and cancer, but target validation has been hampered by a lack of selective inhibitors. This work introduces a class of depsipeptidic KLK6 inhibitors, discovered via high-throughput screening, which were found to function as substrate mimics that transiently acylate the catalytic serine of KLK6. Detailed structure-activity relationship studies, aided by in silico modeling, uncovered strict structural requirements for potency, stability, and acyl-enzyme complex half-life. An optimized scaffold, DKFZ-251, demonstrated good selectivity for KLK6 compared to other KLKs, and on-target activity in a cellular assay. Moreover, DKFZ-633, an inhibitor-derived activity-based probe, could be used to pull down active endogenous KLK6.

Design and synthesis of tailored human caseinolytic protease P inhibitors.

Human caseinolytic protease P (hClpP) is important for degradation of misfolded proteins in the mitochondrial unfolded protein response. We here introduce tailored hClpP inhibitors that utilize a steric discrimination in their core naphthofuran scaffold to selectively address the human enzyme. This novel inhibitor generation exhibited superior activity compared to previously introduced beta-lactones, optimized for bacterial ClpP. Further insights into the bioactivity and binding to cellular targets were obtained via chemical proteomics as well as proliferation- and migration studies in cancer cells.

Current and emerging therapies to prevent hereditary angioedema attacks.

Hereditary angioedema (HAE) is a rare genetic disease defined by recurrent attacks of edema, causing a substantial burden for patients, with morbidity, mortality, and reduced quality of life. This burden is increased by delayed diagnosis, inappropriate treatment, and suboptimal follow-up and patient education. Several novel therapeutics have recently been approved or are currently under evaluation for prevention of HAE attacks.

Discovery, Synthesis, Pharmacological Profiling, and Biological Characterization of Brintonamides A-E, Novel Dual Protease and GPCR Modulators from a Marine Cyanobacterium.

Five novel modified linear peptides named brintonamides A-E (1-5) were discovered from a marine cyanobacterial sample collected from Brinton Channel, Florida Keys. The total synthesis of 1-5 in addition to two other structurally related analogues (6 and 7) was achieved, which provided more material to allow rigorous biological evaluation and SAR studies. Compounds were subjected to cancer-focused phenotypic cell viability and migration assays and orthogonal target-based pharmacological screening platforms to identify their protease and GPCR modulatory activity profiles. The cancer related serine protease kallikrein 7 (KLK7) was inhibited to similar extents with an IC50 near 20 µM by both representative members 1 and 4, which differed in the presence or lack of the N-terminal unit. In contrast to the biochemical protease profiling study, clear SAR was observed in the functional GPCR screens, where five GPCRs in antagonist mode (CCR10, OXTR, SSTR3, TACR2) and agonist mode (CXCR7) were modulated by compounds 1-7 to varying extents. Chemokine receptor type 10 (CCR10) was potently modulated by brintonamide D (4) with an IC50 of 0.44 µM. We performed in silico modeling to understand the structural basis underlying the differences in the antagonistic activity among brintonamides toward CCR10. Because of the significance of KLK7 and CCR10 in cancer progression and metastasis, we demonstrated the ability of brintonamide D (4) at 10 µM to significantly target downstream cellular substrates of KLK7 (Dsg-2 and E-cad) in vitro and to inhibit CCL27-induced CCR10-mediated proliferation and the migration of highly invasive breast cancer cells.

A potent, proteolysis-resistant inhibitor of kallikrein-related peptidase 6 (KLK6) for cancer therapy, developed by combinatorial engineering.

Human tissue kallikrein (KLK) proteases are hormone-like signaling molecules with important functions in cancer pathophysiology. KLK-related peptidase 6 (KLK6), specifically, is highly up-regulated in several types of cancer, where its increased activity promotes cancer invasion and metastasis. This characteristic suggests KLK6 as an attractive target for therapeutic interventions. However, inhibitors that specifically target KLK6 have not yet been reported, possibly because KLK6 shares a high sequence homology and structural similarity with other serine proteases and resists inhibition by many polypeptide inhibitors. Here, we present an innovative combinatorial approach to engineering KLK6 inhibitors via flow cytometry-based screening of a yeast-displayed mutant library of the human amyloid precursor protein Kunitz protease inhibitor domain (APPI), an inhibitor of other serine proteases, such as anionic and cationic trypsins. On the basis of this screening, we generated APPIM17L,I18F,S19F,F34V (APPI-4M), an APPI variant with a KLK6 inhibition constant (Ki ) of 160 pm and a turnover time of 10 days. To the best of our knowledge, APPI-4M is the most potent KLK6 inhibitor reported to date, displaying 146-fold improved affinity and 13-fold improved proteolytic stability compared with WT APPI (APPIWT). We further demonstrate that APPI-4M acts as a functional inhibitor in a cell-based model of KLK6-dependent breast cancer invasion. Finally, the crystal structures of the APPIWT/KLK6 and APPI-4M/KLK6 complexes revealed the structural and mechanistic bases for the improved KLK6 binding and proteolytic resistance of APPI-4M. We anticipate that APPI-4M will have substantial translational potential as both imaging agent and therapeutic.