Enzyme inhibitor | EPFL Graph Search

An enzyme inhibitor is a molecule that binds to an enzyme and blocks its activity. Enzymes are proteins that speed up chemical reactions necessary for life, in which substrate molecules are converted into products. An enzyme facilitates a specific chemical reaction by binding the substrate to its active site, a specialized area on the enzyme that accelerates the most difficult step of the reaction. An enzyme inhibitor stops ("inhibits") this process, either by binding to the enzyme's active site (thus preventing the substrate itself from binding) or by binding to another site on the enzyme such that the enzyme's catalysis of the reaction is blocked. Enzyme inhibitors may bind reversibly or irreversibly. Irreversible inhibitors form a chemical bond with the enzyme such that the enzyme is inhibited until the chemical bond is broken. By contrast, reversible inhibitors bind non-covalently and may spontaneously leave the enzyme, allowing the enzyme to resume its function. Reversible inhi

Investigation of Carboxylic Acid Isosteres and Prodrugs for Inhibition of the Human SIRT5 Lysine Deacylase Enzyme

Alexander Lund Nielsen

Sirtuin 5 (SIRT5) is a protein lysine deacylase enzyme that regulates diverse biology by hydrolyzing epsilon-N-carboxyacyllysine posttranslational modifications in the cell. Inhibition of SIRT5 has been linked to potential treatment of several cancers but potent compounds with activity in cells have been lacking. Here we developed mechanism-based inhibitors that incorporate isosteres of a carboxylic acid residue that is important for high-affinity binding to the enzyme active site. By masking of the tetrazole moiety of the most potent candidate from our initial SAR study, we achieved potent and cytoselective growth inhibition for the treatment of SIRT5-dependent leukemic cancer cell lines in culture. Thus, we provide an efficient, cellularly active small molecule that targets SIRT5, which can help elucidate its function and potential as a future drug target. This work shows that masked isosteres of carboxylic acids are viable chemical motifs for the development of inhibitors that target mitochondrial enzymes, which may have applications beyond the sirtuin field.

2022

The role of TIF1 gamma during adult murine hematopoiesis

Konstantin Shakhbazov

Transforming Growth Factor beta (TGFβ) signaling plays an important role in a variety of cellular processes during embryonic development, adult tissue homeostasis and cancer. TGFβI ligand is a potent inhibitor of early hematopoietic progenitor [1] cells in vitro, however the exact role and mechanisms of this pathway during adult hematopoietic homeostasis remains poorly understood. The Transcriptional Intermediary Factor 1 gamma (Tif1γ) gene encodes a recently uncovered nuclear protein in the TGFβ transduction pathway. Tif1γ is defective in the zebrafish moonshine mutant, which exhibits primitive and definitive hematopoiesis defects [2]. In addition, Tif1γ has been shown to influence differentiation of human erythroid cells in vitro [3]. Recent work in our laboratory has provided genetic evidence that Tif1γ is required for the generation and survival of hematopoietic stem/progenitor cells during murine development (C.Adolphe, K.Shakhbazov et al., manuscript under submission). Given that Tif1γ function is required for embryonic development and null mutants are embryonic lethal [4] (R.Losson unpublished data), we took advantage of a conditional Tif1γ flox allele (R.Losson unpublished data) and combined it with the inducible MxCre line [5] to generate mice in which Tif1γ function can be specifically ablated during adult hematopoiesis. MxCre:Tif1γKO/flox mutants exhibit a decrease in erythroblasts and dramatic increase in granulocytes within the bone marrow. This phenotype is attributable to a massive increase in granulocyte-monocyte progenitors (GMPs) at the expense of common myeloid progenitors (CMPs) and megakaryocyte- erythrocyte progenitors (MEPs), which are severely decreased in Tif1γ mutant bone marrow. In addition, pre-B cells were dramatically decreased in numbers, attributable to an increase in cell death, indicating a pro-survival role for Tif1γ in B lymphocytes. In addition, we performed deletion of Tif1γ in non-competitive and competitive bone marrow transplantation settings, which indicated that only the CMP/MEP phenotype is a non-cell autonomous effect. Two distinct models have been reported suggesting how Tif1γ functions within the TGFβ pathway: one indicating that Tif1γ binds and acts through Smad2/3 and the other that Tif1γ functions as a Smad4 mono-ubiquitin ligase [3, 6, 7]. To genetically address this apparent discrepancy, we generated double Tif1γ/Smad4 hematopoietic specific mutant mice, whose phenotype was surprisingly similar to that observed in Tif1γ single mutants. These data suggest that Tif1γ function is independent of Smad4 in hematopoietic cells. To address the mechanism by which Tif1γ functions, we performed microarray profiling of normal and mutant progenitors, which revealed prostaglandin D2 synthase as a hub gene linking Tif1γ and TGFβ signaling. Finally, we were able to mimic the hematopoietic-Tif1γ null phenotype by providing 16,16-dimethyl prostaglandin D2 to wild-type hematopoietic cells in vitro. In summary, our data provide genetic evidence for a key role of Tif1γ during early myeloid development as well as pre-B cell survival. In addition, we have identified that the majority of Tif1γ function is Smad4 independent. Finally, we identified a novel link between Tif1γ function and prostaglandin metabolism/signaling as a likely mechanism by which Tif1γ regulates hematopoiesis.

EPFL2009

How adeno-associated virus Rep78 relocalises Cdc25B to arrest the cell cycle

Florence Magnin

Adeno-associated virus (AAV) is a small DNA virus belonging to the family of the parvoviridae. AAV infects the human population, however no disease has been associated with this virus. On the contrary, AAV was reported to have oncosuppressive activities. The AAV genome contains two genes: the Rep gene encoding four non-structural proteins (Rep78, 68, 52 and 40) and the Cap gene encoding the three capsid proteins. Rep78 is implicated in transcription, replication and site-specific integration of the viral DNA. It has been shown to block the cell cycle in G1, G2 and S phase with a complete inhibition of DNA synthesis. To be able to induce such a strong cell cycle arrest, Rep78 affects several proteins implicated in the regulation of the cell cycle, such as the family of the cell division cycle 25 (Cdc25) phosphatases. Rep78 has been shown to inactivate Cdc25A by binding to it and preventing it from accessing its substrates. Cdc25B shares conserved domains with Cdc25A and moreover has an important role in cell cycle regulation, especially in giving the signal for entry into mitosis. Thus, in this work, the interaction between Cdc25B and Rep78 was examined in more detail. Rep78 was found to bind to Cdc25B and to alter its intracellular localisation. Cdc25B is nuclear, but has to migrate into the cytoplasm at the end of the G2 phase to activate proteins needed for the entry into mitosis. The expression of Rep78, which is nuclear, leads to the sequestration of Cdc25B in the nucleus. Moreover, Rep78 by interacting with Cdc25B not only changes its localisation, but also inhibits its phosphatase activity. Our model is that Rep78 interacts with Cdc25B and thus retains it in the nucleus. This sequestration of Cdc25B in the nucleus prevents it from activating the proteins in the cytoplasm needed to give the entry signal into mitosis, thus contributing to the G2/M arrest induced by Rep78. Moreover, the inhibition of Cdc25B phosphatase activity by Rep78 may also contribute to the cell cycle block. Rep78 also interacts with a number of cellular proteins implicated in DNA replication, DNA repair or recombination. Interactions between Rep78 and mini-chromosome maintenance 3 (MCM3), DNA polymerases α and δ, proliferating cell nuclear antigen (PCNA), Rad50 and Rad51 were identified in this work as well as possible interactions between Rep78 and Ataxia telangiectasia mutated (ATM) or Ataxia telangiectasia and Rad3 related (ATR) proteins. These interactions may contribute to AAV genome replication as well as its integration into the host DNA.

EPFL2009

How adeno-associated virus Rep78 relocalises Cdc25B to arrest the cell cycle

Florence Magnin

EPFL2009

The role of TIF1 gamma during adult murine hematopoiesis

Konstantin Shakhbazov

EPFL2009