Cancer research experiments
Characterization of partially ordered states in the intrinsically disordered N-terminal domain of p53 using millisecond molecular dynamics simulations.
The exploration of intrinsically disordered proteins in isolation is a crucial step to understand their complex dynamical behavior. In particular, the emergence of partially ordered states has not been explored in depth. The experimental characterization of such partially ordered states remains elusive due to their transient nature. Molecular dynamics mitigates this limitation thanks to its capability to explore biologically relevant timescales while retaining atomistic resolution. Here, millisecond unbiased molecular dynamics simulations were performed in the exemplar N-terminal region of p53. In combination with state-of-the-art Markov state models, simulations revealed the existence of several partially ordered states accounting for [Formula: see text] 40% of the equilibrium population. Some of the most relevant states feature helical conformations similar to the bound structure of p53 to Mdm2, as well as novel [Formula: see text]-sheet elements. This highlights the potential complexity underlying the energy surface of intrinsically disordered proteins.
- Herrera-Nieto P, Pérez A, De Fabritiis G, Characterization of partially ordered states in the intrinsically disordered N-terminal domain of p53 using millisecond molecular dynamics simulations. Scientific reports 2020. doi:10.1038/s41598-020-69322-2
|1||GPUGRID Role account||109,002,400.00|
|5||Rick A. Sponholz||48,480,200.00|
Computational and experimental characterization of NF023, a candidate anticancer compound inhibiting cIAP2/TRAF2 assembly.
Protein-protein interactions are the basis of many important physiological processes, and are currently promising, yet difficult, targets for drug discovery. In this context, inhibitors of apoptosis (IAPs)-mediated interactions are pivotal for cancer cell survival; the interaction of the BIR1 domain of cIAP2 with TRAF2 was shown to lead the recruitment of cIAPs to the TNF-receptor, promoting the activation of NF-κB survival pathway. In this work, using a combined in silico-in vitro approach we identified a drug-like molecule, NF023, able to disrupt cIAP2 interaction with TRAF2. We demonstrated in vitro its ability to interfere with the assembly of the cIAP2-BIR1/TRAF2 complex and performed a thorough characterization of the compound's mode of action through 248 parallel unbiased molecular dynamics simulations of 300 ns (totaling almost 75 µs of all-atom sampling), which identified multiple binding modes to the BIR1 domain of cIAP2 via clustering and ensemble docking. NF023 is, thus, a promising protein-protein interaction disruptor, representing a starting point to develop modulators of NF-κB-mediated cell survival in cancer. This study represents a model-procedure that shows the use of large-scale molecular dynamics methods to typify promiscuous interactors.
- Cossu F, Sorrentino L, Fagnani E, Zaffaroni M, Milani M, Giorgino T, Mastrangelo E, Computational and experimental characterization of NF023, a candidate anticancer compound inhibiting cIAP2/TRAF2 assembly. Journal of chemical information and modeling 2020. doi:10.1021/acs.jcim.0c00518
|8||Thomas M. Taylor||206,250.00|
Dopamine D3 receptor antagonist reveals a cryptic pocket in aminergic GPCRs.
The recent increase in the number of X-ray crystal structures of G-protein coupled receptors (GPCRs) has been enabling for structure-based drug design (SBDD) efforts. These structures have revealed that GPCRs are highly dynamic macromolecules whose function is dependent on their intrinsic flexibility. Unfortunately, the use of static structures to understand ligand binding can potentially be misleading, especially in systems with an inherently high degree of conformational flexibility. Here, we show that docking a set of dopamine D3 receptor compounds into the existing eticlopride-bound dopamine D3 receptor (D3R) X-ray crystal structure resulted in poses that were not consistent with results obtained from site-directed mutagenesis experiments. We overcame the limitations of static docking by using large-scale high-throughput molecular dynamics (MD) simulations and Markov state models (MSMs) to determine an alternative pose consistent with the mutation data. The new pose maintains critical interactions observed in the D3R/eticlopride X-ray crystal structure and suggests that a cryptic pocket forms due to the shift of a highly conserved residue, F
- Ferruz N, Doerr S, Vanase-Frawley MA, Zou Y, Chen X, Marr ES, Nelson RT, Kormos BL, Wager TT, Hou X, Villalobos A, Sciabola S, De Fabritiis G, Dopamine D3 receptor antagonist reveals a cryptic pocket in aminergic GPCRs. Scientific reports 2018. doi:10.1038/s41598-018-19345-7
The pathway of ligand entry from the membrane bilayer to a lipid G protein-coupled receptor.
The binding process through the membrane bilayer of lipid-like ligands to a protein target is an important but poorly explored recognition process at the atomic level. In this work we succeeded in resolving the binding of the lipid inhibitor ML056 to the sphingosine-1-phosphate receptor 1 (S1P1R) using unbiased molecular dynamics simulations with an aggregate sampling of over 800 μs. The binding pathway is a multi-stage process consisting of the ligand diffusing in the bilayer leaflet to contact a "membrane vestibule" at the top of TM 7, subsequently moving from this lipid-facing vestibule to the orthosteric binding cavity through a channel formed by TMs 1 and 7 and the N-terminal of the receptor. Unfolding of the N-terminal alpha-helix increases the volume of the channel upon ligand entry, helping to reach the crystallographic pose that also corresponds to the predicted favorable pose. The relaxation timescales of the binding process show that the binding of the ligand to the "membrane vestibule" is the rate-limiting step in the multi microseconds timescale. We comment on the significance and parallels of the binding process in the context of other binding studies.
- Stanley N, Pardo L, Fabritiis GD, The pathway of ligand entry from the membrane bilayer to a lipid G protein-coupled receptor. Scientific reports 2016. doi:10.1038/srep22639
|6||Grzegorz Roman Granowski||170,047,850.00|
Kinetic modulation of a disordered protein domain by phosphorylation
WU tags: KIDc22
Not all proteins are naturally structured, and those that aren't are called intrinsically disordered proteins (IDPs). While they don't have structure, they do have many important roles in cell biology that are still being fully understood. They are often found mutated in many cancers at places that control the expression of genes and intra-cellular communication. In this work we show that a common chemical modification to IDPs known as phosphorylation can cause one IDP know as KID to change it's behavior. We further show that this could have important consequences for protein interactions, which affects all kinds of things like what genes are expressed and how signals are passed inside the cell.
- N. Stanley. et al. Kinetic modulation of a disordered protein domain by phosphorylation. Nat. Commun. 5:5272 (2014)
|7||Rick A. Sponholz||109,582,625.00|
|8||Grzegorz Roman Granowski||103,799,025.00|
Visualizing the Induced Binding of SH2-Phosphopeptide.
Approximately 100 proteins in the human genome contain an SH2 domain recognizing small flexible phosphopeptides. It is therefore important to understand in atomistic detail the way these peptides bind and the conformational changes that take place upon binding. Here, we obtained several spontaneous binding events between the p56 lck SH2 domain and the pYEEI peptide within 2 Å RMSD from the crystal structure and with kinetic rates compatible with experiments using high-throughput molecular dynamics simulations. Binding is achieved in two phases, fast contacts of the charged phospho-tyrosine and then rearrangement of the ligand involving the stabilization of two important loops in the SH2 domain. These observations provide insights into the binding pathways and induced conformations of the SH2-phosphopeptide complex which, due to the characteristics of SH2 domains, should be relevant for other SH2 recognition peptides.
- Giorgino T, Buch I, De Fabritiis G, Visualizing the Induced Binding of SH2-Phosphopeptide. Journal of chemical theory and computation 2012. doi:10.1021/ct300003f
Computational modeling of an EGFR single-mutation resistance to cetuximab in colorectal cancer treatment
WU tags: EGFR
Extracellular S468R mutation of the epidermal growth factor receptor (EGFR) was recently identified as the cause of resistance to cetuximab, a widely used drug in colorectal cancer treatment. Here, we have determined the binding free energies of cetuximab's Fab V(H)-V(L) domains and endogenous EGF ligand to wild type and S468R EGFR by high-throughput molecular dynamics. This work provides a possible mechanism of resistance in terms of increased competition, an hypothesis that can be further validated experimentally.
- I. Buch, N. Ferruz and G. De Fabritiis, Computational modeling of an EGFR single-mutation resistance to cetuximab in colorectal cancer treatment, J. Chem. Inf. Model., 2013, 53 (12), pp 3123–3126
Molecular simulations of the SH2 and ligand peptide binding affinity
WU tags: pYEEI, SH2
The SH2 is a protein domain involved in protein-protein interactions. This particular domain plays a major role in cell communication on the sigalling processes for cell growth and development. However, the end goal for running such simulations is not to expand the knowldege on this particular system, but to use it as a model for developing methods to calculate protein-protein binding affinities.
Such methods will be very useful, for example, in the study of why certain wrong forms of proteins stop interacting with other partner proteins, as a way to give explanation to diseases in which these sort of mechanisms occur.
- I. Buch, S. K. Sadiq and G. De Fabritiis, Optimized potential of mean force calculations of standard binding free energy, J. Chem. Theory Comput., 7, 1765–1772 (2011)
- I. Buch, M. J. Harvey, T. Giorgino, D. P. Anderson and G. De Fabritiis, High-throughput all-atom molecular dynamics simulations using distributed computing, J. Chem. Inf. and Mod. 50, 397 (2010)