scholarly journals Deciphering the Molecular Mechanism of HCV Protease Inhibitor Fluorination as a General Approach to Avoid Drug Resistance

2021 ◽  
Author(s):  
Jacqueto Zephyr ◽  
Desaboini Nageswara Rao ◽  
Sang V Vo ◽  
Mina Henes ◽  
Klajdi Kosovrasti ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Crystal Structures ◽  
Structural Changes ◽  
Molecular Mechanisms ◽  
Specific Inhibitor ◽  
Matched Pairs ◽  
Resistant Variant ◽  
Resistance Profile ◽  
Design And Synthesis ◽  
Hcv Protease

Third generation Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs), glecaprevir and voxilaprevir, are highly effective across genotypes and against many resistant variants. Unlike earlier PIs, these compounds have fluorine substitutions on the P2-P4 macrocycle and P1 moieties. Fluorination has long been used in medicinal chemistry as a strategy to improve physicochemical properties and potency. However, the molecular basis by which fluorination improves potency and resistance profile of HCV NS3/4A PIs is not well understood. To systematically analyze the contribution of fluorine substitutions to inhibitor potency and resistance profile, we used a multi-disciplinary approach involving inhibitor design and synthesis, enzyme inhibition assays, co-crystallography, and structural analysis. A panel of inhibitors in matched pairs were designed with and without P4 cap fluorination, tested against WT protease and the D168A resistant variant, and a total of 22 high-resolution co-crystal structures were determined. While fluorination did not significantly improve potency against the WT protease, PIs with fluorinated P4 caps retained much better potency against the D168A protease variant. Detailed analysis of the co-crystal structures revealed that PIs with fluorinated P4 caps can sample alternate binding conformations that enable adapting to structural changes induced by the D168A substitution. Our results elucidate molecular mechanisms of fluorine-specific inhibitor interactions that can be leveraged in avoiding drug resistance.

scholarly journals Transcriptomic Profiling Reveals AKR1C1 and AKR1C3 Mediate Cisplatin Resistance in Signet Ring Cell Gastric Carcinoma via Autophagic Cell Death

2021 ◽  
Vol 22 (22) ◽  
pp. 12512
Author(s):  
Nang Lae Lae Phoo ◽  
Pornngarm Dejkriengkraikul ◽  
Patompong Khaw-On ◽  
Supachai Yodkeeree
Keyword(s):  
Drug Resistance ◽  
Cell Death ◽  
Gastric Carcinoma ◽  
Combination Treatment ◽  
Molecular Mechanisms ◽  
Cisplatin Resistance ◽  
Specific Inhibitor ◽  
Signet Ring Cell ◽  
Signet Ring ◽  
Ring Cell

Signet ring cell gastric carcinoma (SRCGC) is a lethal malignancy that has developed drug resistance to cisplatin therapies. The aim of this study was to characterize the acquisition of the cisplatin-resistance SRCGC cell line (KATO/DDP cells) and to understand the molecular mechanisms underlying cisplatin resistance. Transcriptomic and bioinformatic analyses were used to identify the candidate gene. This was confirmed by qPCR and Western blot. Aldoketoreductase1C1 and 1C3 (AKR1C1 and AKR1C3) were the most promising molecules in KATO/DDP cells. A specific inhibitor of AKR1C1 (5PBSA) and AKR1C3 (ASP9521) was used to enhance cisplatin-induced KATO/DPP cell death. Although cisplatin alone induced KATO/DDP apoptosis, a combination treatment of cisplatin and the AKR1C inhibitors had no influence on percent cell apoptosis. In conjunction with the autophagy inhibitor, 3MA, attenuated the effects of 5PBSA or ASP9521 to enhance cisplatin-induced cell death. These results indicated that AKR1C1 and 1C3 regulated cisplatin-induced KATO/DDP cell death via autophagy. Moreover, cisplatin in combination with AKR1C inhibitors and N-acetyl cysteine increased KATO/DDP cells’ viability when compared with a combination treatment of cisplatin and the inhibitors. Taken together, our results suggested that AKR1C1 and 1C3 play a crucial role in cisplatin resistance of SRCGC by regulating redox-dependent autophagy.

scholarly journals Roles of the hydroxy group of tyrosine in crystal structures of Sulfurisphaera tokodaii O 6-methylguanine-DNA methyltransferase

2021 ◽  
Vol 77 (12) ◽  
Author(s):  
Makiko Kikuchi ◽  
Takahiro Yamauchi ◽  
Yasuhito Iizuka ◽  
Masaru Tsunoda
Keyword(s):  
Crystal Structure ◽  
Crystal Structures ◽  
Hydroxy Group ◽  
Active Site ◽  
Dna Methyltransferase ◽  
Structural Changes ◽  
Molecular Mechanisms ◽  
Alkylating Agents ◽  
Substrate Analogs ◽  
Methylguanine Dna Methyltransferase

O 6-Methylguanine-DNA methyltransferase (MGMT) removes cytotoxic O 6-alkyl adducts on the guanine base and protects the cell from genomic damage induced by alkylating agents. Although there are reports of computational studies on the activity of the enzyme with mutations at tyrosine residues, no studies concerning the crystal structure of its mutants have been found. In this study, the function of Tyr91 was investigated in detail by comparing the crystal structures of mutants and their complexes with substrate analogs. In this study, tyrosine, a conserved amino acid near the active-site loop in the C-terminal domain of Sulfurisphaera tokodaii MGMT (StoMGMT), was mutated to phenylalanine to produce a Y91F mutant, and the cysteine which is responsible for receiving the methyl group in the active site was mutated to a serine to produce a C120S mutant. A Y91F/C120S double-mutant StoMGMT was also created. The function of tyrosine is discussed based on the crystal structure of Y91F mutant StoMGMT. The crystal structures of StoMGMT were determined at resolutions of 1.13–2.60 Å. They showed no structural changes except in the mutated part. No electron density for deoxyguanosine or methyl groups was observed in the structure of Y91F mutant crystals immersed in O 6-methyl-2′-deoxyguanosine, nor was the group oxidized in wild-type StoMGMT. Therefore, the hydroxy group of Tyr91 may prevent the oxidant from entering the active site. This suggests that tyrosine, which is highly conserved at the N-terminus of the helix–turn–helix motif across species, protects the active site of MGMTs, which are deactivated after repairing only one alkyl adduct. Overall, the results may provide a basis for understanding the molecular mechanisms by which high levels of conserved amino acids play a role in ensuring the integrity of suicide enzymes, in addition to promoting their activity.

Structural Studies of Fibrinolysis by Electron and Light Microscopy

1999 ◽  
Vol 82 (08) ◽  
pp. 277-282 ◽  
Author(s):  
Yuri Veklich ◽  
Jean-Philippe Collet ◽  
Charles Francis ◽  
John W. Weisel
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Plasminogen Activator ◽  
Structural Changes ◽  
Molecular Mechanisms ◽  
Degradation Products ◽  
Molecular Model ◽  
Three Dimensional ◽  
Tissue Type ◽  
Type Plasminogen Activator

IntroductionMuch is known about the fibrinolytic system that converts fibrin-bound plasminogen to the active protease, plasmin, using plasminogen activators, such as tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator. Plasmin then cleaves fibrin at specific sites and generates soluble fragments, many of which have been characterized, providing the basis for a molecular model of the polypeptide chain degradation.1-3 Soluble degradation products of fibrin have also been characterized by transmission electron microscopy, yielding a model for their structure.4 Moreover, high resolution, three-dimensional structures of certain fibrinogen fragments has provided a wealth of information that may be useful in understanding how various proteins bind to fibrin and the overall process of fibrinolysis (Doolittle, this volume).5,6 Both the rate of fibrinolysis and the structures of soluble derivatives are determined in part by the fibrin network structure itself. Furthermore, the activation of plasminogen by t-PA is accelerated by the conversion of fibrinogen to fibrin, and this reaction is also affected by the structure of the fibrin. For example, clots made of thin fibers have a decreased rate of conversion of plasminogen to plasmin by t-PA, and they generally are lysed more slowly than clots composed of thick fibers.7-9 Under other conditions, however, clots made of thin fibers may be lysed more rapidly.10 In addition, fibrin clots composed of abnormally thin fibers formed from certain dysfibrinogens display decreased plasminogen binding and a lower rate of fibrinolysis.11-13 Therefore, our increasing knowledge of various dysfibrinogenemias will aid our understanding of mechanisms of fibrinolysis (Matsuda, this volume).14,15 To account for these diverse observations and more fully understand the molecular basis of fibrinolysis, more knowledge of the physical changes in the fibrin matrix that precede solubilization is required. In this report, we summarize recent experiments utilizing transmission and scanning electron microscopy and confocal light microscopy to provide information about the structural changes occurring in polymerized fibrin during fibrinolysis. Many of the results of these experiments were unexpected and suggest some aspects of potential molecular mechanisms of fibrinolysis, which will also be described here.

E6 and E7 oncoproteins: Potential targets of cervical cancer

2020 ◽  
Vol 27 ◽  
Author(s):  
Ramarao Malla ◽  
Mohammad Amjad Kamal
Keyword(s):  
Cervical Cancer ◽  
Drug Resistance ◽  
Host Cell ◽  
Molecular Mechanisms ◽  
Immune Therapy ◽  
Cervical Lesions ◽  
Diagnostic Strategies ◽  
E6 And E7 Oncoproteins ◽  
E6 And E7

: Cervical cancer (CC) is the fourth leading cancer in women in the age group 15-44 globally. Experimental as well as epidemiological studies identified that type16 and 18 HPV cause 70% of precancerous cervical lesions as well as cervical cancer worldwide by bringing about genetic as well as epigenetic changes in the host genome. The insertion of the HPV genome triggers various defense mechanisms including the silencing of tumor suppressor genes as well as activation of oncogenes associated with cancer metastatic pathway. E6 and E7 are small oncoproteins consisting of 150 and 100 amino acids respectively. These oncoproteins affect the regulation of the host cell cycle by interfering with p53 and pRb. Further these oncoproteins adversely affect the normal functions of the host cell by binding to their signaling proteins. Recent studies demonstrated that E6 and E7 oncoproteins are potential targets for CC. Therefore, this review discusses the role of E6 and E7 oncoproteins in metastasis and drug resistance as well as their regulation, early oncogene mediated signaling pathways. This review also uncovers the recent updates on molecular mechanisms of E6 and E7 mediated phytotherapy, gene therapy, immune therapy, and vaccine strategies as well as diagnosis through precision testing. Therefore, understanding the potential role of E6/E7 in metastasis and drug resistance along with targeted treatment, vaccine, and precision diagnostic strategies could be useful for the prevention and treatment of cervical cancer.

Small Molecular Gemcitabine Prodrugs for Cancer Therapy

2020 ◽  
Vol 27 (33) ◽  
pp. 5562-5582 ◽  
Author(s):  
He Miao ◽  
Xuehong Chen ◽  
Yepeng Luan
Keyword(s):  
Drug Resistance ◽  
Side Effects ◽  
Anticancer Drug ◽  
Drug Stability ◽  
Effective Means ◽  
Active Form ◽  
Deoxycytidine Kinase ◽  
High Efficacy ◽  
Pyrimidine Nucleoside ◽  
Design And Synthesis

Gemcitabine as a pyrimidine nucleoside analog anticancer drug has high efficacy for a broad spectrum of solid tumors. Gemcitabine is activated within tumor cells by sequential phosphorylation carried out by deoxycytidine kinase to mono-, di-, and triphosphate nucleotides with the last one as the active form. But the instability, drug resistance and toxicity severely limited its utilization in clinics. In the field of medicinal chemistry, prodrugs have proven to be a very effective means for elevating drug stability and decrease undesirable side effects including the nucleoside anticancer drug such as gemcitabine. Many works have been accomplished in design and synthesis of gemcitabine prodrugs, majority of which were summarized in this review.

scholarly journals Hypoxia, Metabolic Reprogramming, and Drug Resistance in Liver Cancer

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1715
Author(s):  
Macus Hao-Ran Bao ◽  
Carmen Chak-Lui Wong
Keyword(s):  
Hepatocellular Carcinoma ◽  
Drug Resistance ◽  
Liver Cancer ◽  
Effective Treatment ◽  
Molecular Mechanisms ◽  
Hypoxia Inducible Factor ◽  
Metabolic Reprogramming ◽  
Combination Therapies ◽  
Low Oxygen ◽  
Treatment Regimens

Hypoxia, low oxygen (O2) level, is a hallmark of solid cancers, especially hepatocellular carcinoma (HCC), one of the most common and fatal cancers worldwide. Hypoxia contributes to drug resistance in cancer through various molecular mechanisms. In this review, we particularly focus on the roles of hypoxia-inducible factor (HIF)-mediated metabolic reprogramming in drug resistance in HCC. Combination therapies targeting hypoxia-induced metabolic enzymes to overcome drug resistance will also be summarized. Acquisition of drug resistance is the major cause of unsatisfactory clinical outcomes of existing HCC treatments. Extra efforts to identify novel mechanisms to combat refractory hypoxic HCC are warranted for the development of more effective treatment regimens for HCC patients.

scholarly journals Crystal structures of an E1–E2–ubiquitin thioester mimetic reveal molecular mechanisms of transthioesterification

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lingmin Yuan ◽  
Zongyang Lv ◽  
Melanie J. Adams ◽  
Shaun K. Olsen
Keyword(s):  
Complex Formation ◽  
Crystal Structures ◽  
Molecular Mechanisms ◽  
Conformational State ◽  
Biochemical Data ◽  
Conformational States ◽  
Product Release ◽  
Closed Conformation ◽  
Open Conformation ◽  
Conjugating Enzymes

AbstractE1 enzymes function as gatekeepers of ubiquitin (Ub) signaling by catalyzing activation and transfer of Ub to tens of cognate E2 conjugating enzymes in a process called E1–E2 transthioesterification. The molecular mechanisms of transthioesterification and the overall architecture of the E1–E2–Ub complex during catalysis are unknown. Here, we determine the structure of a covalently trapped E1–E2–ubiquitin thioester mimetic. Two distinct architectures of the complex are observed, one in which the Ub thioester (Ub(t)) contacts E1 in an open conformation and another in which Ub(t) instead contacts E2 in a drastically different, closed conformation. Altogether our structural and biochemical data suggest that these two conformational states represent snapshots of the E1–E2–Ub complex pre- and post-thioester transfer, and are consistent with a model in which catalysis is enhanced by a Ub(t)-mediated affinity switch that drives the reaction forward by promoting productive complex formation or product release depending on the conformational state.

scholarly journals Molecular Mechanisms of Drug Resistance in Glioblastoma

2021 ◽  
Vol 22 (12) ◽  
pp. 6385
Author(s):  
Maya A. Dymova ◽  
Elena V. Kuligina ◽  
Vladimir A. Richter
Keyword(s):  
Drug Resistance ◽  
Brain Tumor ◽  
Molecular Mechanisms ◽  
Primary Brain Tumor ◽  
Therapeutic Resistance ◽  
Molecular Characteristics ◽  
Blood Brain ◽  
Conventional Radiation ◽  
The Brain ◽  
Made In

Glioblastoma multiforme (GBM) is the most common and fatal primary brain tumor, is highly resistant to conventional radiation and chemotherapy, and is not amenable to effective surgical resection. The present review summarizes recent advances in our understanding of the molecular mechanisms of therapeutic resistance of GBM to already known drugs, the molecular characteristics of glioblastoma cells, and the barriers in the brain that underlie drug resistance. We also discuss the progress that has been made in the development of new targeted drugs for glioblastoma, as well as advances in drug delivery across the blood–brain barrier (BBB) and blood–brain tumor barrier (BBTB).

scholarly journals Comparative Study of Structural Changes of Polylactide and Poly(ethylene terephthalate) in the Presence of Trichoderma viride

2021 ◽  
Vol 22 (7) ◽  
pp. 3491
Author(s):  
Grażyna B. Dąbrowska ◽  
Zuzanna Garstecka ◽  
Ewa Olewnik-Kruszkowska ◽  
Grażyna Szczepańska ◽  
Maciej Ostrowski ◽  
...  
Keyword(s):  
Structural Changes ◽  
Ethylene Terephthalate ◽  
Molecular Mechanisms ◽  
Blot Hybridization ◽  
Trichoderma Viride ◽  
Cost Effective ◽  
Plastic Pollution ◽  
Scanning Calorimetry ◽  
Poly Ethylene Terephthalate ◽  
Poly Ethylene

Plastic pollution is one of the crucial global challenges nowadays, and biodegradation is a promising approach to manage plastic waste in an environment-friendly and cost-effective way. In this study we identified the strain of fungus Trichoderma viride GZ1, which was characterized by particularly high pectinolytic activity. Using differential scanning calorimetry, Fourier-transform infrared spectroscopy techniques, and viscosity measurements we showed that three-month incubation of polylactide and polyethylene terephthalate in the presence of the fungus lead to significant changes of the surface of polylactide. Further, to gain insight into molecular mechanisms underneath the biodegradation process, western blot hybridization was used to show that in the presence of poly(ethylene terephthalate) (PET) in laboratory conditions the fungus produced hydrophobin proteins. The mycelium adhered to the plastic surface, which was confirmed by scanning electron microscopy, possibly due to the presence of hydrophobins. Further, using atomic force microscopy we demonstrated for the first time the formation of hydrophobin film on the surface of aliphatic polylactide (PLA) and PET by T. viride GZ1. This is the first stage of research that will be continued under environmental conditions, potentially leading to a practical application.

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