Insights Into the Binding Mechanism of GC7 to Deoxyhypusine Synthase in Sulfolobus solfataricus: A Thermophilic Model for the Design of New Hypusination Inhibitors

Abstract

Translation factor 5A (eIF5A) is one of the most conserved proteins involved in protein synthesis. It plays a key role during the elongation of polypeptide chains, and its activity is critically dependent on hypusination, a post-translational modification of a specific lysine residue through two consecutive enzymatic steps carried out by deoxyhypusine synthase (DHS), with spermidine as substrate, and deoxyhypusine hydroxylase (DOHH). It is well-established that eIF5A is overexpressed in several cancer types, and it is involved in various diseases such as HIV-1 infection, malaria, and diabetes; therefore, the development of inhibitors targeting both steps of the hypusination process is considered a promising and challenging therapeutic strategy. One of the most efficient inhibitors of the hypusination process is the spermidine analog N1-guanyl-1,7-diaminoheptane (GC7). GC7 interacts in a specific binding pocket of the DHS completely blocking its activity; however, its therapeutic use is limited by poor selectivity and restricted bioavailability. Here we have performed a comparative study between human DHS (hDHS) and archaeal DHS from crenarchaeon Sulfolobus solfataricus (aDHS) to understand the structural and dynamical features of the GC7 inhibition. The advanced metadynamics (MetaD) classical molecular dynamics simulations show that the GC7 interaction is less stable in the thermophilic enzyme compared to hDHS that could underlie a lower inhibitory capacity of the hypusination process in Sulfolobus solfataricus. To confirm this hypothesis, we have tested GC7 activity on S. solfataricus by measuring cellular growth, and results have shown the lack of inhibition of aIF5A hypusination in contrast to the established effect on eukaryotic cellular growth. These results provide, for the first time, detailed molecular insights into the binding mechanism of GC7 to aDHS generating the basis for the design of new and more specific DHS inhibitors.

Polyamines and eIF5A hypusination facilitate SREBP2 translation and cholesterol synthesis to enhance enterovirus attachment and infection

“…One such drug is the spermidine analog N1-guanyl-1,7-diaminoheptane (GC7). GC7 inhibits DHPS by directly binding to the active site and prevents spermidine from being attached to eIF5A 6 . Additionally, deferiprone (DEF) is an iron-chelator which has broad effects on the cell and also inhibits DOHH 7 .…”

PathDetect-SOM: A Neural Network Approach for the Identification of Pathways in Ligand Binding Simulations

“…In a recent work by some of the authors, we investigated the unbinding of GC7 from DHS using an approach inspired to infrequent MetaD. 65 We used the number of contacts between the ligand and the protein binding site atoms as a single CV in 30 replicas of infrequent MetaD that were stopped when the ligand reached an unbound state.…”

“…Compared to the previous case, this graph is more densely connected due to the bidirectionality of the sampling during the MetaD simulation. Most of the simulations (70%) evolve through branch 1 (Pathway A in the original work 65 ) in which the ligand escapes from the side of its guanidine group. The remaining replicas (30%) proceed through an opposite pathway, indicated as branch 2 in the graph, in which the ligand exits from the side of its aminogroup (Pathway B in the original work 65 ).…”

SARS-CoV-2 multi-variant graphene biosensor based on engineered dimeric ACE2 receptor

“…After a 1-hour incubation, beads were washed three times with 500 µl PBST buffer and the bound proteins were eluted using 50 µl of elution buffer (PBS added of 2.5 mM biotin (Sigma)). The samples were then subjected to SDS-PAGE and analysed by western blotting using an anti-Histidine antibody (Thermo Scientific) to detect Spike and ACE2, an anti-rabbit antibody (Santa Cruz Biotechnology) to detect Ab-CR3022 and an anti-ACE2 antibody (EMP Millipore Corp) to detect ACE2-Fc by chemiluminescent revelation 39 . The same protocol, using empty beads (without Spike), was performed as a negative control for each system.…”