RESUMO
The proteasome is a proteolytic enzyme complex essential for protein homeostasis in mammalian cells and protozoan parasites like Trichomonas vaginalis (Tv), the cause of the most common, non-viral sexually transmitted disease. Tv and other protozoan 20S proteasomes have been validated as druggable targets for antimicrobials. However, low yields and purity of the native proteasome have hindered studies of the Tv 20S proteasome (Tv20S). We address this challenge by creating a recombinant protozoan proteasome by expressing all seven α and seven ß subunits of Tv20S alongside the Ump-1 chaperone in insect cells. The recombinant Tv20S displays biochemical equivalence to its native counterpart, confirmed by various assays. Notably, the marizomib (MZB) inhibits all catalytic subunits of Tv20S, while the peptide inhibitor carmaphycin-17 (CP-17) specifically targets ß2 and ß5. Cryo-electron microscopy (cryo-EM) unveils the structures of Tv20S bound to MZB and CP-17 at 2.8 Å. These findings explain MZB's low specificity for Tv20S compared to the human proteasome and demonstrate CP-17's higher specificity. Overall, these data provide a structure-based strategy for the development of specific Tv20S inhibitors to treat trichomoniasis.
Assuntos
Microscopia Crioeletrônica , Complexo de Endopeptidases do Proteassoma , Inibidores de Proteassoma , Proteínas Recombinantes , Trichomonas vaginalis , Trichomonas vaginalis/efeitos dos fármacos , Trichomonas vaginalis/genética , Trichomonas vaginalis/enzimologia , Trichomonas vaginalis/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Humanos , Animais , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas de Protozoários/química , Modelos MolecularesRESUMO
Computational pharmacogenomics can potentially identify new indications for already approved drugs and pinpoint compounds with similar mechanism-of-action. Here, we used an integrated drug repositioning approach based on transcriptomics data and structure-based virtual screening to identify compounds with gene signatures similar to three known proteasome inhibitors (PIs; bortezomib, MG-132, and MLN-2238). In vitro validation of candidate compounds was then performed to assess proteasomal proteolytic activity, accumulation of ubiquitinated proteins, cell viability, and drug-induced expression in A375 melanoma and MCF7 breast cancer cells. Using this approach, we identified six compounds with PI properties ((-)-kinetin-riboside, manumycin-A, puromycin dihydrochloride, resistomycin, tegaserod maleate, and thapsigargin). Although the docking scores pinpointed their ability to bind to the ß5 subunit, our in vitro study revealed that these compounds inhibited the ß1, ß2, and ß5 catalytic sites to some extent. As shown with bortezomib, only manumycin-A, puromycin dihydrochloride, and tegaserod maleate resulted in excessive accumulation of ubiquitinated proteins and elevated HMOX1 expression. Taken together, our integrated drug repositioning approach and subsequent in vitro validation studies identified six compounds demonstrating properties similar to proteasome inhibitors.
Assuntos
Bortezomib , Reposicionamento de Medicamentos , Inibidores de Proteassoma , Humanos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Reposicionamento de Medicamentos/métodos , Bortezomib/farmacologia , Transcriptoma , Complexo de Endopeptidases do Proteassoma/metabolismo , Linhagem Celular Tumoral , Células MCF-7 , Simulação de Acoplamento Molecular , Antineoplásicos/farmacologia , Antineoplásicos/química , Puromicina/farmacologia , Perfilação da Expressão Gênica , Sobrevivência Celular/efeitos dos fármacosRESUMO
Protein homeostasis is a tightly conserved process that is regulated through the ubiquitin proteasome system (UPS) in a ubiquitin-independent or ubiquitin-dependent manner. Over the past two decades, the proteasome has become an excellent therapeutic target through inhibition of the catalytic core particle, inhibition of subunits responsible for recognizing and binding ubiquitinated proteins, and more recently, through targeted protein degradation using proteolysis targeting chimeras (PROTACs). The majority of the developed inhibitors of the proteasome's core particle rely on gaining selectivity through binding interactions within the unprimed substrate channel. Although this has allowed for selective inhibitors and chemical probes to be generated for the different proteasome isoforms, much remains unknown about the interactions that could be harnessed within the primed substrate channel to increase potency or selectivity. Herein, we discuss small molecules that interact with the primed substrate pocket and how their differences may give rise to altered activity. Taking advantage of additional interactions with the primed substrate pocket of the proteasome could allow for the generation of improved chemical tools for perturbing or monitoring proteasome activity.
Assuntos
Complexo de Endopeptidases do Proteassoma , Complexo de Endopeptidases do Proteassoma/metabolismo , Humanos , Especificidade por Substrato , Ligação Proteica , Proteólise , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Ubiquitina/metabolismo , AnimaisRESUMO
Malaria remains a global health concern as drug resistance threatens treatment programs. We identified a piperidine carboxamide (SW042) with anti-malarial activity by phenotypic screening. Selection of SW042-resistant Plasmodium falciparum (Pf) parasites revealed point mutations in the Pf_proteasome ß5 active-site (Pfß5). A potent analog (SW584) showed efficacy in a mouse model of human malaria after oral dosing. SW584 had a low propensity to generate resistance (minimum inoculum for resistance [MIR] >109) and was synergistic with dihydroartemisinin. Pf_proteasome purification was facilitated by His8-tag introduction onto ß7. Inhibition of Pfß5 correlated with parasite killing, without inhibiting human proteasome isoforms or showing cytotoxicity. The Pf_proteasome_SW584 cryoelectron microscopy (cryo-EM) structure showed that SW584 bound non-covalently distal from the catalytic threonine, in an unexplored pocket at the ß5/ß6/ß3 subunit interface that has species differences between Pf and human proteasomes. Identification of a reversible, species selective, orally active series with low resistance propensity provides a path for drugging this essential target.
Assuntos
Antimaláricos , Piperidinas , Plasmodium falciparum , Inibidores de Proteassoma , Piperidinas/química , Piperidinas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Animais , Antimaláricos/farmacologia , Antimaláricos/química , Humanos , Camundongos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Inibidores de Proteassoma/síntese química , Administração Oral , Complexo de Endopeptidases do Proteassoma/metabolismo , Malária/tratamento farmacológico , Malária/parasitologia , Amidas/química , Amidas/farmacologia , Amidas/síntese química , Malária Falciparum/tratamento farmacológico , Feminino , Estrutura MolecularRESUMO
There is an urgent need to develop new, safer, and more effective drugs against Chagas disease (CD) as well as related kinetoplastid diseases. Targeting and inhibiting the Trypanosoma cruzi proteasome has emerged as a promising therapeutic approach in this context. To expand the chemical space for this class of inhibitors, we performed virtual screening campaigns with emphasis on shape-based similarity and ADMET prioritization. We describe the ideation and application of robustly validated shape queries for these campaigns, which furnished 44 compounds for biological evaluation. Five hit compounds demonstrated in vitro antitrypanosomal activity by potential inhibition of T. cruzi proteasome and notable chemical diversities, particularly, LCQFTC11. Structural insights were achieved by homology modeling, sequence/structure alignment, proteasome-species comparison, docking, molecular dynamics, and MMGBSA binding affinity estimations. These methods confirmed key interactions as well as the stability of LCQFTC11 at the ß4/ß5 subunits' binding site of the T. cruzi proteasome, consistent with known inhibitors. Our results warrant future assay confirmation of our hit as a T. cruzi proteasome inhibitor. Importantly, we also shed light into dynamic details for a proteasome inhibition mechanism that shall be further investigated. We expect to contribute to the development of viable CD drug candidates through such a relevant approach.
Assuntos
Simulação de Acoplamento Molecular , Complexo de Endopeptidases do Proteassoma , Inibidores de Proteassoma , Trypanosoma cruzi , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Simulação de Dinâmica Molecular , Tripanossomicidas/farmacologia , Tripanossomicidas/química , Sítios de Ligação , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade , Ligação ProteicaRESUMO
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.
Assuntos
Antígenos de Superfície , Antineoplásicos , Glutamato Carboxipeptidase II , Inibidores de Proteassoma , Humanos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Inibidores de Proteassoma/síntese química , Glutamato Carboxipeptidase II/antagonistas & inibidores , Glutamato Carboxipeptidase II/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Antígenos de Superfície/metabolismo , Relação Estrutura-Atividade , Estrutura Molecular , Ensaios de Seleção de Medicamentos Antitumorais , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
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.
Assuntos
Ácidos Borônicos , Microscopia Crioeletrônica , Complexo de Endopeptidases do Proteassoma , Inibidores de Proteassoma , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Animais , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Inibidores de Proteassoma/síntese química , Camundongos , Ácidos Borônicos/química , Ácidos Borônicos/farmacologia , Ácidos Borônicos/síntese química , Humanos , Relação Estrutura-Atividade , Cisteína Endopeptidases/metabolismo , Estrutura Molecular , Simulação de Dinâmica Molecular , Descoberta de DrogasRESUMO
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)âsuppressing gate opening by factors of at least â¼400 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 (â¼65 to 75 Å) from its binding sites raises a number of interesting biophysical issues.
Assuntos
Bortezomib , Complexo de Endopeptidases do Proteassoma , Inibidores de Proteassoma , Bortezomib/farmacologia , Bortezomib/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , HumanosRESUMO
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.
Assuntos
Complexo de Endopeptidases do Proteassoma , Inibidores de Proteassoma , Relação Estrutura-Atividade , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Inibidores de Proteassoma/síntese química , Humanos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Subunidades Proteicas/química , Estrutura MolecularRESUMO
Previously, we demonstrated that linear peptide epoxyketones targeting the immunoproteasome (iP) could ameliorate cognitive deficits in mouse models of Alzheimer's disease (AD) independently of amyloid deposition. We also reported the first iP-targeting macrocyclic peptide epoxyketones, which exhibit improved metabolic stability compared with their linear counterparts. Here, we prepared additional macrocyclic peptide epoxyketones and compared them with existing macrocyclic iP inhibitors by assessing Caco2 cell-based permeability and microsomal stability, providing the four best macrocyclic iP inhibitors. We then evaluated the four compounds using the Ames test and the potency assays in BV2 cells, selecting compound 5 as our AD drug lead. When 5 was administered intravenously (40 mg/kg) or orally (150 mg/kg) into healthy BALB/c mice, we observed considerable iP inhibition in the mouse brain, indicating good blood-brain barrier permeability and target engagement. Combined results suggest that 5 is a promising AD drug lead that may need further investigation.
Assuntos
Doença de Alzheimer , Barreira Hematoencefálica , Encéfalo , Camundongos Endogâmicos BALB C , Animais , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Humanos , Barreira Hematoencefálica/metabolismo , Camundongos , Células CACO-2 , Encéfalo/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Permeabilidade , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/farmacocinética , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Compostos Macrocíclicos/farmacocinética , Cetonas/química , Cetonas/farmacologia , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Antineoplásicos , Resistencia a Medicamentos Antineoplásicos , Mieloma Múltiplo , Bibliotecas de Moléculas Pequenas , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Humanos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Inibidores de Proteassoma/uso terapêutico , Estrutura MolecularRESUMO
The proteasome of the malaria parasite Plasmodium falciparum (Pf20S) is an advantageous drug target because its inhibition kills P. falciparum in multiple stages of its life cycle and synergizes with artemisinins. We recently developed a macrocyclic peptide, TDI-8304, that is highly selective for Pf20S over human proteasomes and is potent in vitro and in vivo against P. falciparum. A mutation in the Pf20S ß6 subunit, A117D, confers resistance to TDI-8304, yet enhances both enzyme inhibition and anti-parasite activity of a tripeptide vinyl sulfone ß2 inhibitor, WLW-vs. Here we present the high-resolution cryo-EM structures of Pf20S with TDI-8304, of human constitutive proteasome with TDI-8304, and of Pf20Sß6A117D with WLW-vs that give insights into the species selectivity of TDI-8304, resistance to it, and the collateral sensitivity associated with resistance, including that TDI-8304 binds ß2 and ß5 in wild type Pf20S as well as WLW-vs binds ß2 and ß5 in Pf20Sß6A117D. We further show that TDI-8304 kills P. falciparum as quickly as chloroquine and artemisinin and is active against P. cynomolgi at the liver stage. This increases interest in using these structures to facilitate the development of Pf20S inhibitors that target multiple proteasome subunits and limit the emergence of resistance.
Assuntos
Antimaláricos , Malária Falciparum , Humanos , Plasmodium falciparum/genética , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Sensibilidade Colateral a Medicamentos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Antimaláricos/farmacologia , Antimaláricos/química , Resistência a Medicamentos/genética , Proteínas de Protozoários/genéticaRESUMO
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.
Assuntos
Doenças Autoimunes , Inibidores de Proteassoma , Humanos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Domínio Catalítico , Fagocitose , Técnicas In Vitro , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
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.
Assuntos
Simulação de Dinâmica Molecular , Óxido de Zinco , Farmacóforo , Teoria da Densidade Funcional , Inibidores de Proteassoma/químicaRESUMO
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.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Relação Estrutura-Atividade , Desenho de Fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proliferação de Células , Linhagem Celular TumoralRESUMO
In this issue of Cell Chemical Biology, Zhan et al. report dual-pharmacophore molecules ("artezomibs"), combining an artemisinin and proteasome inhibitor that exhibit potent activity against both wild-type and drug-resistant malarial parasites.1 This study indicates that artezomibs offer a promising approach to combat drug resistance encountered by current antimalarial therapies.
Assuntos
Antimaláricos , Antimaláricos/química , Complexo de Endopeptidases do Proteassoma , Resistência a Medicamentos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/químicaRESUMO
The Plasmodium falciparum proteasome constitutes a promising antimalarial target, with multiple chemotypes potently and selectively inhibiting parasite proliferation and synergizing with the first-line artemisinin drugs, including against artemisinin-resistant parasites. We compared resistance profiles of vinyl sulfone, epoxyketone, macrocyclic peptide, and asparagine ethylenediamine inhibitors and report that the vinyl sulfones were potent even against mutant parasites resistant to other proteasome inhibitors and did not readily select for resistance, particularly WLL that displays covalent and irreversible binding to the catalytic ß2 and ß5 proteasome subunits. We also observed instances of collateral hypersensitivity, whereby resistance to one inhibitor could sensitize parasites to distinct chemotypes. Proteasome selectivity was confirmed using CRISPR/Cas9-edited mutant and conditional knockdown parasites. Molecular modeling of proteasome mutations suggested spatial contraction of the ß5 P1 binding pocket, compromising compound binding. Dual targeting of P. falciparum proteasome subunits using covalent inhibitors provides a potential strategy for restoring artemisinin activity and combating the spread of drug-resistant malaria.
Assuntos
Antimaláricos , Artemisininas , Malária Falciparum , Plasmodium , Humanos , Antimaláricos/farmacologia , Antimaláricos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Plasmodium/metabolismo , Artemisininas/química , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/químicaRESUMO
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.
Assuntos
Antineoplásicos , Naftoquinonas , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Complexo de Endopeptidases do Proteassoma , Glicina/farmacologia , Naftoquinonas/farmacologia , Naftoquinonas/química , beta-Alanina/farmacologia , Anilidas/farmacologia , Relação Estrutura-Atividade , Estrutura Molecular , Proliferação de Células , Antineoplásicos/farmacologiaRESUMO
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.
Assuntos
Antineoplásicos , Mieloma Múltiplo , Pró-Fármacos , Humanos , Camundongos , Animais , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Ácidos Borônicos/química , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Dipeptídeos/farmacologia , Dipeptídeos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Citratos/uso terapêutico , Antineoplásicos/química , Linhagem Celular TumoralRESUMO
Advances in DNA sequencing technology and bioinformatics have revealed the enormous potential of microbes to produce structurally complex specialized metabolites with diverse uses in medicine and agriculture. However, these molecules typically require structural modification to optimize them for application, which can be difficult using synthetic chemistry. Bioengineering offers a complementary approach to structural modification but is often hampered by genetic intractability and requires a thorough understanding of biosynthetic gene function. Expression of specialized metabolite biosynthetic gene clusters (BGCs) in heterologous hosts can surmount these problems. However, current approaches to BGC cloning and manipulation are inefficient, lack fidelity, and can be prohibitively expensive. Here, we report a yeast-based platform that exploits transformation-associated recombination (TAR) for high efficiency capture and parallelized manipulation of BGCs. As a proof of concept, we clone, heterologously express and genetically analyze BGCs for the structurally related nonribosomal peptides eponemycin and TMC-86A, clarifying remaining ambiguities in the biosynthesis of these important proteasome inhibitors. Our results show that the eponemycin BGC also directs the production of TMC-86A and reveal contrasting mechanisms for initiating the assembly of these two metabolites. Moreover, our data shed light on the mechanisms for biosynthesis and incorporation of 4,5-dehydro-l-leucine (dhL), an unusual nonproteinogenic amino acid incorporated into both TMC-86A and eponemycin.