Identification of Dual Inhibitors Targeting Main Protease (Mpro) and Cathepsin L as Potential Anti-SARS-CoV-2 Agents.

In this structure-activity relationship (SAR) study, we report the development of dual inhibitors with antiviral properties targeting the SARS-CoV-2 main protease (Mpro) and human cathepsin L (hCatL). The novel molecules differ in the aliphatic amino acids at the P2 site and the fluorine position on the phenyl ring at the P3 site. The identified dual inhibitors showed Ki values within 1.61 and 10.72 µM against SARS-CoV-2 Mpro; meanwhile, Ki values ranging from 0.004 to 0.701 µM toward hCatL were observed. A great interdependency between the nature of the side chain at the P2 site and the position of the fluorine atom was found. Three dual-targeting inhibitors exhibited antiviral activity in the low micromolar range with CC50 values >100 µM. Docking simulations were executed to gain a deeper understanding of the SAR profile. The findings herein collected should be taken into consideration for the future development of dual SARS-CoV-2 Mpro/hCatL inhibitors.

Development of Novel Peptidyl Nitriles Targeting Rhodesain and Falcipain-2 for the Treatment of Sleeping Sickness and Malaria.

In recent decades, neglected tropical diseases and poverty-related diseases have become a serious health problem worldwide. Among these pathologies, human African trypanosomiasis, and malaria present therapeutic problems due to the onset of resistance, toxicity problems and the limited spectrum of action. In this drug discovery process, rhodesain and falcipain-2, of Trypanosoma brucei rhodesiense and Plasmodium falciparum, are currently considered the most promising targets for the development of novel antitrypanosomal and antiplasmodial agents, respectively. Therefore, in our study we identified a novel lead-like compound, i.e., inhibitor 2b, which we proved to be active against both targets, with a Ki = 5.06 µM towards rhodesain and an IC50 = 40.43 µM against falcipain-2.

The Inhibition of NS2B/NS3 Protease: A New Therapeutic Opportunity to Treat Dengue and Zika Virus Infection.

In the global pandemic scenario, dengue and zika viruses (DENV and ZIKV, respectively), both mosquito-borne members of the flaviviridae family, represent a serious health problem, and considering the absence of specific antiviral drugs and available vaccines, there is a dire need to identify new targets to treat these types of viral infections. Within this drug discovery process, the protease NS2B/NS3 is considered the primary target for the development of novel anti-flavivirus drugs. The NS2B/NS3 is a serine protease that has a dual function both in the viral replication process and in the elusion of the innate immunity. To date, two main classes of NS2B/NS3 of DENV and ZIKV protease inhibitors have been discovered: those that bind to the orthosteric site and those that act at the allosteric site. Therefore, this perspective article aims to discuss the main features of the use of the most potent NS2B/NS3 inhibitors and their impact at the social level.

Theranostic Applications of 2D Graphene-Based Materials for Solid Tumors Treatment.

Solid tumors are a leading cause of cancer-related deaths globally, being characterized by rapid tumor growth and local and distant metastases. The failures encountered in cancer treatment are mainly related to the complicated biology of the tumor microenvironment. Nanoparticles-based (NPs) approaches have shown the potential to overcome the limitations caused by the pathophysiological features of solid cancers, enabling the development of multifunctional systems for cancer diagnosis and therapy and allowing effective inhibition of tumor growth. Among the different classes of NPs, 2D graphene-based nanomaterials (GBNs), due to their outstanding chemical and physical properties, easy surface multi-functionalization, near-infrared (NIR) light absorption and tunable biocompatibility, represent ideal nanoplatforms for the development of theranostic tools for the treatment of solid tumors. Here, we reviewed the most recent advances related to the synthesis of nano-systems based on graphene, graphene oxide (GO), reduced graphene oxide (rGO), and graphene quantum dots (GQDs), for the development of theranostic NPs to be used for photoacoustic imaging-guided photothermal-chemotherapy, photothermal (PTT) and photodynamic therapy (PDT), applied to solid tumors destruction. The advantages in using these nano-systems are here discussed for each class of GBNs, taking into consideration the different chemical properties and possibility of multi-functionalization, as well as biodistribution and toxicity aspects that represent a key challenge for their translation into clinical use.

Virtual Screening Strategy and In Vitro Tests to Identify New Inhibitors of the Immunoproteasome.

Immunoproteasome inhibition is a promising strategy for the treatment of hematological malignancies, autoimmune diseases, and inflammatory diseases. The design of non-covalent inhibitors of the immunoproteasome ß1i/ß5i catalytic subunits could be a novel approach to avoid the drawbacks of the known covalent inhibitors, such as toxicity due to off-target binding. In this work, we report the biological evaluation of thirty-four compounds selected from a commercially available collection. These hit compounds are the outcomes of a virtual screening strategy including a dynamic pharmacophore modeling approach onto the ß1i subunit and a pharmacophore/docking approach onto the ß5i subunit. The computational studies were first followed by in vitro enzymatic assays at 100 µM. Only compounds capable of inhibiting the enzymatic activity by more than 50% were characterized in detail using Tian continuous assays, determining the dissociation constant (Ki) of the non-covalent complex where Ki is also the measure of the binding affinity. Seven out of thirty-four hits showed to inhibit ß1i and/or ß5i subunit. Compound 3 is the most active on the ß1i subunit with Ki = 11.84 ± 1.63 µM, and compound 17 showed Ki = 12.50 ± 0.77 µM on the ß5i subunit. Compound 2 showed inhibitory activity on both subunits (Ki = 12.53 ± 0.18 and Ki = 31.95 ± 0.81 on the ß1i subunit and ß5i subunit, respectively). The induced fit docking analysis revealed interactions with Thr1 and Phe31 of ß1i subunit and that represent new key residues as reported in our previous work. Onto ß5i subunit, it interacts with the key residues Thr1, Thr21, and Tyr169. This last hit compound identified represents an interesting starting point for further optimization of ß1i/ß5i dual inhibitors of the immunoproteasome.

Blunting Neuroinflammation by Targeting the Immunoproteasome with Novel Amide Derivatives.

Neuroinflammation is an inflammatory response of the nervous tissue mediated by the production of cytokines, chemokines, and reactive oxygen species. Recent studies have shown that an upregulation of immunoproteasome is highly associated with various diseases and its inhibition attenuates neuroinflammation. In this context, the development of non-covalent immunoproteasome-selective inhibitors could represent a promising strategy for treating inflammatory diseases. Novel amide derivatives, KJ3 and KJ9, inhibit the ß5 subunit of immunoproteasome and were used to evaluate their possible anti-inflammatory effects in an in vitro model of TNF-α induced neuroinflammation. Differentiated SH-SY5Y and microglial cells were challenged with 10 ng/mL TNF-α for 24 h and treated with KJ3 (1 µM) and KJ9 (1 µM) for 24 h. The amide derivatives showed a significant reduction of oxidative stress and the inflammatory cascade triggered by TNF-α reducing p-ERK expression in treated cells. Moreover, the key action of these compounds on the immunoproteasome was further confirmed by halting the IkB-α phosphorylation and the consequent inhibition of NF-kB. As downstream targets, IL-1ß and IL-6 expression resulted also blunted by either KJ3 and KJ9. These preliminary results suggest that the effects of these two compounds during neuroinflammatory response relies on the reduced expression of pro-inflammatory targets.

Cysteine Proteases as Validated Targets for the Treatment of Neglected and Poverty-Related Parasitic Diseases.

Neglected tropical diseases (NTDs) include 20 diverse infections mainly prevalent in tropical areas that mostly affect disadvantaged communities and women and children [...].

Influence of amino acid size at the P3 position of N-Cbz-tripeptide Michael acceptors targeting falcipain-2 and rhodesain for the treatment of malaria and human african trypanosomiasis.

In recent decades, several structure-activity relationship (SAR) studies provided potent inhibitors of the cysteine proteases falcipain-2 (FP-2) and rhodesain (RD) from Plasmodium falciparum and Trypanosoma brucei rhodesiense, respectively. Whilst the roles of the warhead and residues targeting the P1 and P2 pockets of the proteases were extensively investigated, the roles of the amino acids occupying the S3 pocket were not widely assessed. Herein we report the synthesis and biological evaluation of a set of novel Michael acceptors bearing amino acids of increasing size at the P3 site (1a-g/2a-g, SPR20-SPR33) against FP-2, RD, P. falciparum, and T. brucei. Overall, the Michael acceptors bearing small amino acids at the P3 site exhibited the most potent inhibitory properties towards FP-2. In contrast, analogues with bulky residues at the P3 position were very potent rhodesain inhibitors. In cell based assays, single-digit micromolar EC50 values against the two protozoa were observed. These findings can be a starting point for the development of peptide-based FP-2 and RD inhibitors.

Dipeptide Nitrile CD34 with Curcumin: A New Improved Combination Strategy to Synergistically Inhibit Rhodesain of Trypanosoma brucei rhodesiense.

Rhodesain is the main cysteine protease of Trypanosoma brucei rhodesiense, the parasite causing the acute lethal form of Human African Trypanosomiasis. Starting from the dipeptide nitrile CD24, the further introduction of a fluorine atom in the meta position of the phenyl ring spanning in the P3 site and the switch of the P2 leucine with a phenylalanine led to CD34, a synthetic inhibitor that shows a nanomolar binding affinity towards rhodesain (Ki = 27 nM) and an improved target selectivity with respect to the parent dipeptide nitrile CD24. In the present work, following the Chou and Talalay method, we carried out a combination study of CD34 with curcumin, a nutraceutical obtained from Curcuma longa L. Starting from an affected fraction (fa) of rhodesain inhibition of 0.5 (i.e., the IC50), we observed an initial moderate synergistic action, which became a synergism for fa values ranging from 0.6 to 0.7 (i.e., 60-70% inhibition of the trypanosomal protease). Interestingly, at 80-90% inhibition of rhodesain proteolytic activity, we observed a strong synergism, resulting in 100% enzyme inhibition. Overall, in addition to the improved target selectivity of CD34 with respect to CD24, the combination of CD34 + curcumin resulted in an increased synergistic action with respect to CD24 + curcumin, thus suggesting that it is desirable to use CD34 and curcumin in combination.

Dimeric polyphenols to pave the way for new antimalarial drugs.

Because of the threat of resistant Plasmodium sp., new orally active antimalarials are urgently needed. Inspired by the structure of ellagic acid, exhibiting potent in vivo and in vitro antiplasmodial effects, polyphenolic structures possessing a similar activity-safety profile were synthesized. Indeed, most exhibited a marked in vitro effect (IC50 < 4 µM) on resistant P. falciparum, without any detrimental effects reported during the toxicity assays (hemolysis, cytotoxicity, in vivo). In addition, they possessed a greater hydrosolubility (from 7 µM to 2.7 mM) compared to ellagic acid. Among them, 30 is the most promising for antimalarial purposes since it displayed a significant parasitaemia reduction after oral administration in mice (50 mg kg-1) compared to the orally ineffective ellagic acid. In conclusion, our investigations led to the identification of a promising scaffold, which could bring new insights for malaria treatment.

Monoclonal Antibodies: The Greatest Resource to Treat Multiple Myeloma.

Multiple myeloma (MM) is a currently incurable hematologic cancer. This disease is characterized by immunological alterations of myeloid cells and lymphocytes. The first-line therapy involves the use of classic chemotherapy; however, many patients have a relapsed form that could evolve into a refractory MM. The new therapeutic frontiers involve the use of new monoclonal antibodies (Mab) such as daratumumab, isatuximab, and elotuzumab. In addition to monoclonal antibodies, new immunotherapies based on modern bispecific antibodies and chimeric antigen receptor (CAR) T cell therapy have been investigated. For this reason, immunotherapy represents the greatest hope for the treatment of MM. This review intends to focus the attention on the new approved antibody targets. The most important are: CD38 (daratumumab and isatuximab), SLAM7 (elotuzumab), and BCMA (belantamab mafodotin) for the treatment of MM currently used in clinical practice. Although the disease is still incurable, the future perspective is to find the best therapeutic combination among all available drugs.

Alarmins and MicroRNAs, a New Axis in the Genesis of Respiratory Diseases: Possible Therapeutic Implications.

It is well ascertained that airway inflammation has a key role in the genesis of numerous respiratory pathologies, including asthma, chronic obstructive pulmonary disease, and acute respiratory distress syndrome. Pulmonary tissue inflammation and anti-inflammatory responses implicate an intricate relationship between local and infiltrating immune cells and structural pulmonary cells. Alarmins are endogenic proteins discharged after cell injury in the extracellular microenvironment. The purpose of our review is to highlight the alterations in respiratory diseases involving some alarmins, such as high mobility group box 1 (HMGB1) and interleukin (IL)-33, and their inter-relationships and relationships with genetic non-coding material, such as microRNAs. The role played by these alarmins in some pathophysiological processes confirms the existence of an axis composed of HMGB1 and IL-33. These alarmins have been implicated in ferroptosis, the onset of type 2 inflammation and airway alterations. Moreover, both factors can act on non-coding genetic material capable of modifying respiratory function. Finally, we present an outline of alarmins and RNA-based therapeutics that have been proposed to treat respiratory pathologies.

Structure-based lead optimization of peptide-based vinyl methyl ketones as SARS-CoV-2 main protease inhibitors.

Despite several major achievements in the development of vaccines and antivirals, the fight against SARS-CoV-2 and the health problems accompanying COVID-19 are still ongoing. SARS-CoV-2 main protease (Mpro), an essential viral cysteine protease, is a crucial target for the development of antiviral agents. A virtual screening analysis of in-house cysteine protease inhibitors against SARS-CoV-2 Mpro allowed us to identify two hits (i.e., 1 and 2) bearing a methyl vinyl ketone warhead. Starting from these compounds, we herein report the development of Michael acceptors targeting SARS-CoV-2 Mpro, which differ from each other for the warhead and for the amino acids at the P2 site. The most promising vinyl methyl ketone-containing analogs showed sub-micromolar activity against the viral protease. SPR38, SPR39, and SPR41 were fully characterized, and additional inhibitory properties towards hCatL, which plays a key role in the virus entry into host cells, were observed. SPR39 and SPR41 exhibited single-digit micromolar EC50 values in a SARS-CoV-2 infection model in cell culture.

The Impact of Curcumin on Immune Response: An Immunomodulatory Strategy to Treat Sepsis.

Primary and secondary immunodeficiencies cause an alteration in the immune response which can increase the rate of infectious diseases and worsened prognoses. They can also alter the immune response, thus, making the infection even worse. Curcumin is the most biologically active component of the turmeric root and appears to be an antimicrobial agent. Curcumin cooperates with various cells such as macrophages, dendritic cells, B, T, and natural killer cells to modify the body's defence capacity. Curcumin also inhibits inflammatory responses by suppressing different metabolic pathways, reduces the production of inflammatory cytokines, and increases the expression of anti-inflammatory cytokines. Curcumin may also affect oxidative stress and the non-coding genetic material. This review analyses the relationships between immunodeficiency and the onset of infectious diseases and discusses the effects of curcumin and its derivatives on the immune response. In addition, we analyse some of the preclinical and clinical studies that support its possible use in prophylaxis or in the treatment of infectious diseases. Lastly, we examine how nanotechnologies can enhance the clinical use of curcumin.

Drug Combination Studies of the Dipeptide Nitrile CD24 with Curcumin: A New Strategy to Synergistically Inhibit Rhodesain of Trypanosoma brucei rhodesiense.

Rhodesain is a cysteine protease that is crucial for the life cycle of Trypanosoma brucei rhodesiense, a parasite causing the lethal form of Human African Trypanosomiasis. CD24 is a recently developed synthetic inhibitor of rhodesain, characterized by a nanomolar affinity towards the trypanosomal protease (Ki = 16 nM), and acting as a competitive inhibitor. In the present work, we carried out a combination study of CD24 with curcumin, the multitarget nutraceutical obtained from Curcuma longa L., which we demonstrated to inhibit rhodesain in a non-competitive manner. By applying the Chou and Talalay method, we obtained an initial additive effect at IC50 (fa = 0.5, Combination Index = 1), while for the most relevant fa values, ranging from 0.6 to 1, i.e., from 60% to 100% of rhodesain inhibition, we obtained a combination index < 1, thus suggesting that an increasingly synergistic action occurred for the combination of the synthetic inhibitor CD24 and curcumin. Furthermore, the combination of the two inhibitors showed an antitrypanosomal activity better than that of CD24 alone (EC50 = 4.85 µM and 10.1 µM for the combination and CD24, respectively), thus suggesting the use of the two inhibitors in combination is desirable.

Corrigendum: Exploiting curcumin synergy with natural products using quantitative analysis of dose-effect relationships in an experimental in vitro model of osteoarthritis.

[This corrects the article DOI: 10.3389/fphar.2019.01347.].

A Proteomic Platform Unveils the Brain Glycogen Phosphorylase as a Potential Therapeutic Target for Glioblastoma Multiforme.

In the last few years, several efforts have been made to identify original strategies against glioblastoma multiforme (GBM): this requires a more detailed investigation of the molecular mechanism of GBM so that novel targets can be identified for new possible therapeutic agents. Here, using a combined biochemical and proteomic approach, we evaluated the ability of a blood-brain barrier-permeable 2,3-benzodiazepin-4-one, called 1g, to interfere with the activity and the expression of brain glycogen phosphorylase (PYGB) on U87MG cell line in parallel with the capability of this compound to inhibit the cell growth and cycle. Thus, our results highlighted PYGB as a potential therapeutic target in GBM prompting 1g as a capable anticancer drug thanks to its ability to negatively modulate the uptake and metabolism of glucose, the so-called "Warburg effect", whose increase is considered a common feature of cancer cells in respect of their normal counterparts.

Development of Urea-Bond-Containing Michael Acceptors as Antitrypanosomal Agents Targeting Rhodesain.

Human African Trypanosomiasis (HAT) is a neglected tropical disease widespread in sub-Saharan Africa. Rhodesain, a cysteine protease of Trypanosoma brucei rhodesiense, has been identified as a valid target for the development of anti-HAT agents. Herein, we report a series of urea-bond-containing Michael acceptors, which were demonstrated to be potent rhodesain inhibitors with K i values ranging from 0.15 to 2.51 nM, and five of them showed comparable k 2nd values to that of K11777, a potent antitrypanosomal agent. Moreover, most of the urea derivatives exhibited single-digit micromolar activity against the protozoa, and the presence of substituents at the P3 position appears to be essential for the antitrypanosomal effect. Replacement of Phe with Leu at the P2 site kept unchanged the inhibitory properties. Compound 7 (SPR7) showed the best compromise in terms of rhodesain inhibition, selectivity, and antiparasitic activity, thus representing a new lead compound for future SAR studies.

Development of Reduced Peptide Bond Pseudopeptide Michael Acceptors for the Treatment of Human African Trypanosomiasis.

Human African Trypanosomiasis (HAT) is an endemic protozoan disease widespread in the sub-Saharan region that is caused by T. b. gambiense and T. b. rhodesiense. The development of molecules targeting rhodesain, the main cysteine protease of T. b. rhodesiense, has led to a panel of inhibitors endowed with micro/sub-micromolar activity towards the protozoa. However, whilst impressive binding affinity against rhodesain has been observed, the limited selectivity towards the target still remains a hard challenge for the development of antitrypanosomal agents. In this paper, we report the synthesis, biological evaluation, as well as docking studies of a series of reduced peptide bond pseudopeptide Michael acceptors (SPR10-SPR19) as potential anti-HAT agents. The new molecules show Ki values in the low-micro/sub-micromolar range against rhodesain, coupled with k2nd values between 1314 and 6950 M-1 min-1. With a few exceptions, an appreciable selectivity over human cathepsin L was observed. In in vitro assays against T. b. brucei cultures, SPR16 and SPR18 exhibited single-digit micromolar activity against the protozoa, comparable to those reported for very potent rhodesain inhibitors, while no significant cytotoxicity up to 70 µM towards mammalian cells was observed. The discrepancy between rhodesain inhibition and the antitrypanosomal effect could suggest additional mechanisms of action. The biological characterization of peptide inhibitor SPR34 highlights the essential role played by the reduced bond for the antitrypanosomal effect. Overall, this series of molecules could represent the starting point for further investigations of reduced peptide bond-containing analogs as potential anti-HAT agents.

New Insights into Cold Shock Proteins Effects in Human Cancer: Correlation with Susceptibility, Prognosis and Therapeutical Perspectives.

The microenvironment of the tumor cells is central to its phenotypic modification. One of the essential elements of this milieu is thermal regulation. An augment in local temperature has been reported to augment the tumor cell's responsiveness to chemoand radiation treatment. Cold shock proteins are RNA/DNA binding proteins identified by the existence of one or more cold shock domains. In humans, the best studied components of this group of proteins are called Y-box binding proteins, such as Y-box binding protein-1 (YB-1), but several other proteins have been recognized. Biological functions of these proteins extend from the control of transcription, translation and splicing to the regulation of exosomal RNA content. Several findings correlate an altered cold shock protein expression profile with tumor diseases. In this review we summarize the data for a causative participation of cold shock proteins in cancer onset and diffusion. Furthermore, the possible use of cold shock proteins for diagnostics, prognosis, and as targets for cancer treatment is exposed.