Optimization of α-amido boronic acids via cryo-electron microscopy analysis: Discovery of a novel highly selective immunoproteasome subunit LMP7 (ß5i)/LMP2 (ß1i) dual inhibitor.

The immunoproteasome subunit LMP7 (ß5i)/LMP2 (ß1i) dual blockade has been reported to suppress B cell differentiation and activation, suggesting that the dual inhibition of LMP7/LMP2 is a promising approach for treating autoimmune diseases. In contrast, the inhibition of the constitutive proteasome subunit ß5c correlates with cytotoxicity against non-immune cells. Therefore, LMP7/LMP2 dual inhibitors with high selectivity over ß5c may be desirable for treating autoimmune diseases. In this study, we present the optimization and discovery of α-amido boronic acids using cryo-electron microscopy (cryo-EM). The exploitation of structural differences between the proteasome subunits led to the identification of a highly selective LMP7/LMP2 dual inhibitor 19. Molecular dynamics simulation based on cryo-EM structures of the proteasome subunits complexed with 19 explained the inhibitory activity profile. In mice immunized with 4-hydroxy-3-nitrophenylacetyl conjugated to ovalbumin, results indicate that 19 is orally bioavailable and shows promise as potential treatment for autoimmune diseases.

Development of small molecule-drug conjugates based on derivatives of natural proteasome inhibitors that exhibit selectivity for PSMA-expressing cancer cells.

In this study, we have developedsmall molecule drug conjugates (SMDCs)consisting ofa prostate specific membrane antigen (PSMA) ligandand syringolin derivatives, which are potent proteasome inhibitors, to selectively deliver syringolin derivatives to prostate cancer cells. Two parent compounds were used for syringolin derivatives with different linkage sites. These SMDCs exhibited PSMA-expressing cell-selective cytotoxicity and they could potentially be used for safer treatment of cancer.

Modulation of proteasome subunit selectivity of syringolins.

Development of selective or dual proteasome subunit inhibitors based on syringolin B as a scaffold is described. We focused our efforts on a structure-activity relationship study of inhibitors with various substituents at the 3-position of the macrolactam moiety of syringolin B analogue to evaluate whether this would be sufficient to confer subunit selectivity by using sets of analogues with hydrophobic, basic and acidic substituents, which were designed to target Met45, Glu53 and Arg45 embedded in the S1 subsite, respectively. The structure-activity relationship study using systematic analogues provided insight into the origin of the subunit-selective inhibitory activity. This strategy would be sufficient to confer subunit selectivity regarding ß5 and ß2 subunits.

Bortezomib Inhibits Open Configurations of the 20S Proteasome.

Bortezomib, a small dipeptide-like molecule, is a proteasome inhibitor used widely in the treatment of myeloma and lymphoma. This molecule reacts with threonine side chains near the center of the 20S proteasome and disrupts proteostasis by blocking enzymatic sites that are responsible for protein degradation. In this work, we use novel mass-spectrometry-based techniques to examine the influence of bortezomib on the structures and stabilities of the 20S core particle. These studies indicate that bortezomib binding dramatically favors compact 20S structures (in which the axial gate is closed) over larger structures (in which the axial gate is open)─suppressing gate opening by factors of at least ∼400 to 1300 over the temperature range that is studied. Thus, bortezomib may also restrict degradation in the 20S proteasome by preventing substrates from entering the catalytic pore. That bortezomib influences structures at the entrance region of the pore at such a long distance (∼65 to 75 Å) from its binding sites raises a number of interesting biophysical issues.

Research strategies of small molecules as chemotherapeutics to overcome multiple myeloma resistance.

Multiple myeloma (MM), a cancer of plasma cells, is the second most common hematological malignancy which is characterized by aberrant plasma cells infiltration in the bone marrow and complex heterogeneous cytogenetic abnormalities. Over the past two decades, novel treatment strategies such as proteasome inhibitors, immunomodulators, and monoclonal antibodies have significantly improved the relative survival rate of MM patients. However, the development of drug resistance results in the majority of MM patients suffering from relapse, limited treatment options and uncontrolled disease progression after relapse. There are urgent needs to develop and explore novel MM treatment strategies to overcome drug resistance and improve efficacy. Here, we review the recent small molecule therapeutic strategies for MM, and introduce potential new targets and corresponding modulators in detail. In addition, this paper also summarizes the progress of multi-target inhibitor therapy and protein degradation technology in the treatment of MM.

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.

Stability and potential degradation of the α',ß'-epoxyketone pharmacophore on ZnO nanocarriers: insights from reactive molecular dynamics and density functional theory calculations.

We investigate the structure and dynamics of a zinc oxide nanocarrier loaded with Carfilzomib, an epoxyketone proteasome inhibitor developed for treating multiple myeloma. We demonstrate that, even though both bare and functionalized zinc oxide supports have been used for drug delivery, their interactions with the reactive functional groups of the ligands could be detrimental. This is because pharmacophores like α',ß'-epoxyketones should preserve the groups required for the drug activity and be capable of leaving the vehicle at the target site. Earlier studies showed that even when ZnO is functionalized with oleic acid surfactants, the drug could reach parts of the surface and remain stably adsorbed. Herein, we have used reactive molecular dynamics simulations and quantum chemistry calculations to explore the potential interactions of the Carfilzomib functional groups with the typical surfaces of ZnO supports. We have found that Carfilzomib can adsorb on the (0001)Zn-terminated polar surface through the carbonyl oxygens and the epoxyketone moiety. These strong connections could prevent the drug release and induce the epoxy ring opening with its consequential inactivation. Therefore, regulating the dosage to maintain the desired level of drug bioavailability is paramount. These findings emphasize the need for appropriate carrier functionalizations to efficiently entrap, transport, and release the cargo at the target sites and the crucial role played by predictive/descriptive computational techniques to complement and drive experiments to the most appropriate selections of the materials to optimize drug delivery.

Exploration of novel four-membered-heterocycle constructed peptidyl proteasome inhibitors with improved metabolic stability for cancer treatment.

Peptides have limitations as active pharmaceutical agents due to rapid hydrolysis by proteases and poor cell permeability. To overcome these limitations, a series of peptidyl proteasome inhibitors embedded with four-membered heterocycles were designed to enhance their metabolic stabilities. All synthesized compounds were screened for their inhibitory activities against human 20S proteasome, and 12 target compounds displayed potent efficacy with IC50 values lower than 20 nM. Additionally, these compounds exhibited strong anti-proliferative activities against multiple myeloma (MM) cell lines (MM1S: 72, IC50 = 4.86 ± 1.34 nM; RPMI-8226: 67, IC50 = 12.32 ± 1.44). Metabolic stability assessments of SGF, SIF, plasma and blood were conducted, and the representative compound 73 revealed long half-lives (Plasma: T1/2 = 533 min; Blood: T1/2 > 1000 min) and good proteasome inhibitory activity in vivo. These results suggest that compound 73 serve as a lead compound for the development of more novel proteasome inhibitors.

Design and discovery of novel dipeptide boronic acid ester proteasome inhibitors, an oral slowly-released prodrug for the treatment of multiple myeloma.

Multiple myeloma (MM), the second most common hematological malignancy, is a disease characterized by a clonal expansion of malignant plasma cells that accumulate in the bone marrow. Ixazomib citrate was the first commercially available oral proteasome inhibitor for the treatment of MM. However, it immediately hydrolyzed into the active form on exposure to aqueous solution and so it was a pseudo prodrug. Herein, a series of dipeptide boronic acid esters as novel oral proteasome inhibitors were designed, synthesized and biologically investigated for the inhibition of the ß5 subunit of 20S proteasome. Based on the enzymatic results, structure-activity relationships (SAR) were discussed in detail. Some potent compounds were further evaluated to inhibit the proliferation of MM cell line RPMI-8226. The results showed that some compounds were active against RPMI-8226 with IC50 values of less than 10 nM. The solution stability showed that ixazomib citrate was completely hydrolyzed to its active form ixazomib within 2 min in the simulated gastric juice. However, among the screened compounds, prodrug 18u was stable enough in simulated gastric juice and simulated intestinal juice, and its hydrolysis rate was 59.7% and 3.6% after 2 h, respectively. In addition, 18u exhibited good microsome stabilities and pharmacokinetic properties and displayed strong antiproliferative activity against the RPMI-8226 cell line (5.6 nM). Furthermore, compound 18u exhibited strong in vivo anticancer efficacy in human MM (RPMI-8226) xenograft mouse model.

Design, synthesis, and biological evaluation of some novel naphthoquinone-glycine/ß-alanine anilide derivatives as noncovalent proteasome inhibitors.

A series of novel noncovalent glycine/ß-alanine anilide derivatives possessing 2-chloronaphthoquinone structure as a pharmacophoric unit were designed, synthesized, and evaluated for their antiproliferative and antiproteasomal activities against MCF-7 cell line, in vitro. According to biological activity results, all the target compounds showed antiproliferative activity in the range of IC50  = 7.10 ± 0.10-41.08 ± 0.14 µM and most of them exhibited inhibitory efficacy with varying ratios against the three catalytic subunits (ß1, ß2, and ß5) presenting caspase-like (C-L), trypsin-like (T-L) and chymotrypsin-like (ChT-L) activities of proteasome. The antiproteasomal activity evaluations revealed that compounds preferentially inhibited the ß5 subunit compared with ß1 and ß2 subunits of the proteasome. Among the compounds, compounds 7 and 9 showed the highest antiproliferative activity with an IC50 value of 7.10 ± 0.10 and 7.43 ± 0.25 µM, respectively. Additionally, compound 7 displayed comparable potency to PI-083 lead compound in terms of ß5 antiproteasomal activity with an inhibition percentage of 34.67 at 10 µM. This compound showed an IC50 value of 32.30 ± 0.45 µM against ß5 subunit. Furthermore, molecular modeling studies of the most active compound 7 revealed key interactions with ß5 subunit. The results suggest that this class of compounds may be beneficial for the development of new potent proteasome inhibitors.

Discovery of Highly Selective Inhibitors of the Human Constitutive Proteasome ß5c Chymotryptic Subunit.

We describe our discovery and development of potent and highly selective inhibitors of human constitutive proteasome chymotryptic activity (ß5c). Structure-activity relationship studies of the novel class of inhibitors focused on optimization of N-cap, C-cap, and side chain of the chemophore asparagine. Compound 32 is the most potent and selective ß5c inhibitor in this study. A docking study provides a structure rationale for potency and selectivity. Kinetic studies show a reversible and noncompetitive inhibition mechanism. It enters the cells to engage the proteasome target, potently and selectively kills multiple myeloma cells, and does so by synergizing with a ß5i-selective inhibitor.

From Nature to Synthetic Compounds: Novel 1(N),2,3 Trisubstituted-5-oxopyrrolidines Targeting Multiple Myeloma Cells.

The insurgence of drug resistance in treating Multiple Myeloma (MM) still represents a major hamper in finding effective treatments, although over the past decades new classes of drugs, such as proteasome inhibitors and immunomodulatory drugs, have been discovered. Recently, our research team, within a Nature-Aided Drug Discovery project, isolated from Hibiscus Sabdariffa L. calyces the secondary metabolite called Hib-ester which possesses antiproliferative properties against human multiple myeloma RPMI 8226 cells, reduces migration and cell invasion and inhibits proteasome without neurotoxic effects. In the present study, we explored the chemical spaces of the hit compound Hib-ester. We explored the structure-activity relationships (SAR), and we optimized the scaffold through sequentially modifying Hib-ester subunits. Compound screening was performed based on cytotoxicity against the RPMI 8226 cells to assess the potential efficacy toward human MM. The ability of the most effective molecules to inhibit the proteasome was evaluated and the binding mode of the most promising compounds in the proteasome chymotrypsin binding pocket was deciphered through molecular modeling simulations. Compounds 13 and 14 are more potent than Hib-ester, demonstrating that our strategy was suitable for the identification of a novel chemotype for developing possible drug candidates and hopefully widening the drug armamentarium against MM.

Susceptibilities of Ugandan Plasmodium falciparum Isolates to Proteasome Inhibitors.

The proteasome is a promising target for antimalarial chemotherapy. We assessed ex vivo susceptibilities of fresh Plasmodium falciparum isolates from eastern Uganda to seven proteasome inhibitors: two asparagine ethylenediamines, two macrocyclic peptides, and three peptide boronates; five had median IC50 values <100 nM. TDI8304, a macrocylic peptide lead compound with drug-like properties, had a median IC50 of 16 nM. Sequencing genes encoding the ß2 and ß5 catalytic proteasome subunits, the predicted targets of the inhibitors, and five additional proteasome subunits, identified two mutations in ß2 (I204T, S214F), three mutations in ß5 (V2I, A142S, D150E), and three mutations in other subunits. The ß2 S214F mutation was associated with decreased susceptibility to two peptide boronates, with IC50s of 181 nM and 2635 nM against mutant versus 62 nM and 477 nM against wild type parasites for MMV1579506 and MMV1794229, respectively, although significance could not be formally assessed due to the small number of mutant parasites with available data. The other ß2 and ß5 mutations and mutations in other subunits were not associated with susceptibility to tested compounds. Against culture-adapted Ugandan isolates, two asparagine ethylenediamines and the peptide proteasome inhibitors WLW-vinyl sulfone and WLL-vinyl sulfone (which were not studied ex vivo) demonstrated low nM activity, without decreased activity against ß2 S214F mutant parasites. Overall, proteasome inhibitors had potent activity against P. falciparum isolates circulating in Uganda, and genetic variation in proteasome targets was uncommon.

Discovery and Mechanistic Study of Mycobacterium tuberculosis PafA Inhibitors.

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis (Mtb) and is ranked as the second killer infectious disease after COVID-19. Proteasome accessory factor A (PafA) is considered an attractive target because of its low sequence conservation in humans and its role in virulence. In this study, we designed a mutant of Mtb PafA that enabled large-scale purification of active PafA. Using a devised high-throughput screening assay, two PafA inhibitors were discovered. ST1926 inhibited Mtb PafA by binding in the Pup binding groove, but it was less active against Corynebacterium glutamicum PafA because the ST1926-binding residues are not conserved. Bithionol bound to the conserved ATP-binding pocket, thereby, inhibits PafA in an ATP-competitive manner. Both ST1926 and bithionol inhibited the growth of an attenuated Mtb strain (H37Ra) at micromolar concentrations. Our work thus provides new tools for tuberculosis research and a foundation for future PafA-targeted drug development for treating tuberculosis.

Discovery of Novel Epoxyketone Peptides as Lipase Inhibitors.

Obesity is the most common nutritional disorder in the developed world and is associated with important comorbidities. Pancreatic lipase (PL) inhibitors play a key role in the metabolism of human fat. A series of novel epoxyketones peptide derivatives were investigated for their pancreatic lipase inhibitory activity. The epoxyketone moiety is a well-known reactive electrophile group that has been used as part of proteasome inhibitors in cancer therapy, and it is widely believed that these are very selective for targeting the proteasome active site. Here we investigated various peptide derivatives with an epoxide warhead for their anti-lipase activity. The assessment of these novel epoxyketones was performed by an in-house method that we developed for rapid screening and identification of lipase inhibitors using GC-FID. Herein, we present a novel anti-lipase pharmacophore based on epoxyketone peptide derivatives that showed potent anti-lipase activity. Many of these derivatives had comparable or more potent activity than the clinically used lipase inhibitors such as orlistat. In addition, the lipase appears to be inhibited by a wide range of epoxyketone analogues regardless of the configuration of the epoxide in the epoxyketone moiety. The presented data in this study shows the first example of the use of epoxyketone peptides as novel lipase inhibitors.

Identification of N, C-capped di- and tripeptides as selective immunoproteasome inhibitors.

A series of N, C-capped di- and tripeptides were designed as selective immunoproteasome inhibitors based on the known inhibitor 4-CA. Forty-eight new compounds were synthesized and evaluated, and the structure-activity relationship (SAR) of this compound class as ß5i selective inhibitors were explored. Most of these compounds showed significant inhibition against the ß5i subunit of the immunoproteasome and the most potent ß5i inhibitor (15) showed an IC50 of 0.94 nM. A selective ß5i inhibitor (54) with over 500-fold ß5i/ß5c selectivity was identified. Three of the inhibitors were found to selectively inhibit ß5i and ß5c, and showed no noticeable inhibition against the other four subunits. Six inhibitors with significant inhibitory activity against the HCT-116 cells were recognized, and the most active inhibitors, 14 and 50, showed IC50 values of 0.46 µM and 0.16 µM, respectively. Some selective ß5i inhibitors exhibited significant inhibitory effects on the release of the cytokines TNF-α and IL-6. The results not only afford effective chemical tools to elucidate the relationships between subunit selectivity and pharmacological profiles, but also offer useful clues for further optimization and development of selective immunoproteasome inhibitors.

Cell-based screening of extracts of natural sources to search for inhibitors of the ubiquitin-proteasome system and identification of proteasome inhibitors from the fungus Remotididymella sp.

The ubiquitin-proteasome system (UPS) regulates selective protein degradation to maintain protein homeostasis. Small molecules that inhibit the UPS-dependent protein degradation are promising anti-tumor agents. We report a cell-based luminescent assay using HeLa cells expressing luciferase-fused oxygen-dependent destruction domain (ODD) of hypoxia-inducible factor 1 α (HIF-1 α). ODD is degraded by the UPS and this assay system can aid in the identification of natural products that inhibit either process of the UPS, including ubiquitination/deubiquitination and proteasomal degradation. This reporter assay can exclude the influences of coloring or fluorescent compounds in extracts, thereby leading to effective high-throughput processing. The screening of 15,025 extracts of natural sources identified the culture extract of the fungus Remotididymella sp. (18F02908). Bioassay-guided isolation yielded two new polyketides, mellains A (1) and B (2), together with leptosphaerodione (3) and its acetone adduct 4. Compound 1 was revealed to have an unprecedented benzo[g]isoquinoline-8,10-dione skeleton. Evaluation of the biological activities demonstrated that these polyketides inhibit the proteasomal proteolysis. This is the first report of the identification of proteasome inhibitors from natural sources using a cell-based reporter assay targeting UPS inhibitors.

A review on the treatment of multiple myeloma with small molecular agents in the past five years.

Multiple myeloma is currently incurable, and the incidence rate is increasing year by year worldwide. Although in recent years the combined treatment plan based on proteasome inhibitors and immunomodulatory drugs has greatly improved the treatment effect of multiple myeloma, most patients still relapse and become resistant to current treatments. To solve this problem, scientists are committed to developing drugs with higher specificity, such as iberdomide, which is highly specific to ikaros and aiolos. This review aims to focus on the small molecular agents that are being researched/clinically used for the treatment of multiple myeloma, including the target mechanism, structure-activity relationship and application prospects of small molecular agents.

Resensitising proteasome inhibitor-resistant myeloma with sphingosine kinase 2 inhibition.

The introduction of the proteasome inhibitor bortezomib into treatment regimens for myeloma has led to substantial improvement in patient survival. However, whilst bortezomib elicits initial responses in many myeloma patients, this haematological malignancy remains incurable due to the development of acquired bortezomib resistance. With other patients presenting with disease that is intrinsically bortezomib resistant, it is clear that new therapeutic approaches are desperately required to target bortezomib-resistant myeloma. We have previously shown that targeting sphingolipid metabolism with the sphingosine kinase 2 (SK2) inhibitor K145 in combination with bortezomib induces synergistic death of bortezomib-naïve myeloma. In the current study, we have demonstrated that targeting sphingolipid metabolism with K145 synergises with bortezomib and effectively resensitises bortezomib-resistant myeloma to this proteasome inhibitor. Notably, these effects were dependent on enhanced activation of the unfolded protein response, and were observed in numerous separate myeloma models that appear to have different mechanisms of bortezomib resistance, including a new bortezomib-resistant myeloma model we describe which possesses a clinically relevant proteasome mutation. Furthermore, K145 also displayed synergy with the next-generation proteasome inhibitor carfilzomib in bortezomib-resistant and carfilzomib-resistant myeloma cells. Together, these findings indicate that targeting sphingolipid metabolism via SK2 inhibition may be effective in combination with a broad spectrum of proteasome inhibitors in the proteasome inhibitor resistant setting, and is an approach worth clinical exploration.

Development of isoquinolinone derivatives as immunoproteasome inhibitors.

The inhibition of immunoproteasome is considered nowadays a promising strategy for the treatment of hematologic malignancies. In this paper we report the design, synthesis, and biological evaluation as immunoproteasome inhibitors of a new series of isoquinolinone derivatives characterized by a (E)-prop-1-ene fragment that connects the heterocycle to a distal amide functionality. Among all the synthesized compounds, we identified an inhibitor with Ki values in the low micromolar or submicromolar range towards the chymotrypsin-like activities of both proteasome and immunoproteasome (ß5c, ß5i and ß1i subunits). Molecular modeling studies suggest that the most potent compound of the series may act a single-site binder. In particular, through its isopentyl group, it might dock into P1 site in the case of the ß1i catalytic subunit, while in the case of ß5c and ß5i subunits, the P3 site might be the preferred binding site.