Design of Allosteric Stimulators of the Hsp90 ATPase as New Anticancer Leads

Allosteric compounds that stimulate Hsp90 adenosine triphosphatase (ATPase) activity were rationally designed, showing anticancer potencies in the low micromolar to nanomolar range. In parallel, the mode of action of these compounds was clarified and a quantitative model that links the dynamic ligand-protein cross-talk to observed cellular and in vitro activities was developed. The results support the potential of using dynamics-based approaches to develop original mechanism-based cancer therapeutics

Simulations of DNA topoisomerase 1B bound to supercoiled DNA reveal changes in the flexibility pattern of the enzyme and a secondary protein-DNA binding site

Human topoisomerase 1B has been simulated covalently bound to a negatively supercoiled DNA minicircle, and its behavior compared to the enzyme bound to a simple linear DNA duplex. The presence of the more realistic supercoiled substrate facilitates the formation of larger number of protein-DNA interactions when compared to a simple linear duplex fragment. The number of protein-DNA hydrogen bonds doubles in proximity to the active site, affecting all of the residues in the catalytic pentad. The clamp over the DNA, characterized by the salt bridge between Lys369 and Glu497, undergoes reduced fluctuations when bound to the supercoiled minicircle. The linker domain of the enzyme, which is implicated in the controlled relaxation of superhelical stress, also displays an increased number of contacts with the minicircle compared to linear DNA. Finally, the more complex topology of the supercoiled DNA minicircle gives rise to a secondary DNA binding site involving four residues located on subdomain III. The simulation trajectories reveal significant changes in the interactions between the enzyme and the DNA for the more complex DNA topology, which are consistent with the experimental observation that the protein has a preference for binding to supercoiled DNA

Prikazi: Propovijedi sv. Antuna; Lendićevi "Božji kotači" u Zagrebu

Long-duration comparative molecular dynamics simulations of the DNA-topoisomerase binary and DNA-topoisomerase-indenoisoquinoline ternary complexes have been carried out. The analyses demonstrated the role of the drug in conformationally stabilizing the protein-DNA interaction. In detail, the protein lips, clamping the DNA substrate, interact more tightly in the ternary complex than in the binary one. The drug also reduces the conformational space sampled by the protein linker domain through an increased interaction with the helix bundle proximal to the active site. A similar alteration of linker domain dynamics has been observed in a precedent work for topotecan but the molecular mechanisms were different if compared to those described in this work. Finally, the indenoisoquinoline keeps Lys532 far from the DNA, making it unable to participate in the religation reaction, indicating that both short- and long-range interactions contribute to the drug poisoning effect

Visualizing the Dynamics of a Protein Folding Machinery: The Mechanism of Asymmetric ATP Processing in Hsp90 and its Implications for Client Remodelling

The Hsp90 chaperone system interacts with a wide spectrum of client proteins, forming variable and dynamic multiprotein complexes that involve the intervention of cochaperone partners. Recent results suggest that the role of Hsp90 complexes is to establish interactions that suppress unwanted client activities, allow clients to be protected from degradation and respond to biochemical signals. Cryo-electron microscopy (cryoEM) provided the first key molecular picture of Hsp90 in complex with a kinase, Cdk4, and a cochaperone, Cdc37. Here, we use a combination of molecular dynamics (MD) simulations and advanced comparative analysis methods to elucidate key aspects of the functional dynamics of the complex, with different nucleotides bound at the N-terminal Domain of Hsp90. The results reveal that nucleotide-dependent structural modulations reverberate in a striking asymmetry of the dynamics of Hsp90 and identify specific patterns of long-range coordination between the nucleotide binding site, the client binding pocket, the cochaperone and the client. Our model establishes a direct atomic-resolution cross-talk between the ATP-binding site, the client region that is to be remodeled and the surfaces of the Cdc37-cochaperone

Structural-dynamical properties of the Deinococcus radiodurans topoisomerase IB in absence of DNA: correlation with the human enzyme

A molecular dynamics simulation of the Deinococcus radiodurans topoisomerase IB crystallized in absence of DNA has been carried out. The protein stably maintains its initial conformation with the N- and C-terminal domains slightly oscillating around their initial positions, being their relative orientations stabilized by two inter-domain interactions, Arg4:Glu228 and Glu63:Arg91. A charge perturbation has then been introduced to destabilize the protein from its local minimum. The simulation carried out after the destabilization permits the two domains to change their relative orientation and to move one from the other, acting as independent units that fully maintain their intrinsic secondary and tertiary structure. The orientational rearrangement occurs because of the presence of a hinge, Gln93, located in a segment connecting the N- and C-terminal domains. Despite the large amplitude of motion the active site remains preformed with an orientation similar to that found in the crystal structure. Comparison with a similar study previously carried out on the human enzyme reveals the presence of structural common features between the two enzymes

Topoisomerase 1B as a target against Leishmaniasis

Leishmaniasis affects more than 12 million people in 98 countries, the infection being caused by more than 20 species of protozoan parasites belonging to the genus Leishmania and spread by sandflies bite. Poor sanitary conditions, malnutrition, deforestation and urbanization increase the risk for leishmaniasis. Leishmaniasis is the only tropical disease treated with non-anti-leishmanial drugs, among which liposomal amphotericin B, a combination of pentavalent antimonials and paromomycin and miltefosine, that are highly toxic, represent the most used ones. Drug resistance is now widespread and the search for new molecular targets is open. Topoisomerase 1B, that controls the topological state of DNA and is essential for the parasites viability, has been detected as a promising target for antileishmaniasis therapy. The enzyme presents structural/functional differences with the human counterpart, making it unique among Eukarya. Here we review the structural features of this enzyme and the drugs that can be developed and used for this specific targeting

Structural-dynamical properties of the Deinococcus radiodurans topoisomerase IB in absence of DNA: correlation with the human enzyme

A molecular dynamics simulation of the Deinococcus radiodurans topoisomerase IB crystallized in absence of DNA has been carried out. The protein stably maintains its initial conformation with the N- and C-terminal domains slightly oscillating around their initial positions, being their relative orientations stabilized by two inter-domain interactions, Arg4:Glu228 and Glu63:Arg91. A charge perturbation has then been introduced to destabilize the protein from its local minimum. The simulation carried out after the destabilization permits the two domains to change their relative orientation and to move one from the other, acting as independent units that fully maintain their intrinsic secondary and tertiary structure. The orientational rearrangement occurs because of the presence of a hinge, Gln93, located in a segment connecting the N- and C-terminal domains. Despite the large amplitude of motion the active site remains preformed with an orientation similar to that found in the crystal structure. Comparison with a similar study previously carried out on the human enzyme reveals the presence of structural common features between the two enzymes

Geometrical constraints limiting the poly(ADP-ribose) conformation investigated by molecular dynamics simulation.

Poly(ADP-ribosylation) is a post-transductional modification that regulates protein's function. Most of the proteins subjected to this control mechanism belong to machineries involved in DNA damage repair, or DNA interacting proteins. Poly(ADP-ribose) polymers are long chains of even 100 monomer length that can be branched at several positions but, not withstanding its importance, nothing is known concerning its structure. To understand, which are the geometrical parameters that confer to the polymer the structural constraints that determine its interaction with the target proteins, we have performed molecular dynamics of three chains of different length, made by 5, 25, and 30 units, the last one being branched. Analysis of the simulations allowed us to identify the main intra- and inter-monomer dihedral angles that govern the structure of the polymer that however, does not reach a unique definite conformatio

A unique binding mode of the eukaryotic translation initiation factor 4E for guiding the design of novel peptide inhibitors

The interaction between the eukaryotic translation initiation factor 4E (eIF4E) and eIF4E binding proteins (4E-BP) is a promising template for the inhibition of eIF4E and the treatment of diseases such as cancer and a spectrum of autism disorders, including the Fragile X syndrome (FXS). Here, we report an atomically detailed model of the complex between eIF4E and a peptide fragment of a 4E-BP, the cytoplasmic Fragile X interacting protein (CYFIP1). This model was generated using computer simulations with enhanced sampling from an alchemical replica exchange approach and validated using long molecular dynamics simulations. 4E-BP proteins act as post-transcriptional regulators by binding to eIF4E and preventing mRNA translation. Dysregulation of eIF4E activity has been linked to cancer, FXS, and autism spectrum disorders. Therefore, the study of the mechanism of inhibition of eIF4E by 4E-BPs is key to the development of drug therapies targeting this regulatory pathways. The results obtained in this work indicate that CYFIP1 interacts with eIF4E by an unique mode not shared by other 4E-BP proteins and elucidate the mechanism by which CYFIP1 interacts with eIF4E despite having a sequence binding motif significantly different from most 4E-BPs. Our study suggests an alternative strategy for the design of eIF4E inhibitor peptides with superior potency and specificity than currently available

Ligand Binding, Unbinding, and Allosteric Effects: Deciphering Small-Molecule Modulation of HSP90

The molecular chaperone HSP90 oversees the functional activation of a large number of client proteins. Because of its role in multiple pathways linked to cancer and neurodegeneration, drug discovery targeting HSP90 has been actively pursued. Yet, a number of inhibitors failed to meet expectations due to induced toxicity problems. In this context, allosteric perturbation has emerged as an alternative strategy for the pharmacological modulation of HSP90 functions. Specifically, novel allosteric stimulators showed the interesting capability of accelerating HSP90 closure dynamics and ATPase activities while inducing tumor cell death. Here, we gain atomistic insight into the mechanisms of allosteric ligand recognition and their consequences on the functional dynamics of HSP90, starting from the fully unbound state. We integrate advanced computational sampling methods based on FunnelMetadynamics, with the analysis of internal dynamics of the structural ensembles visited during the simulations. We observe several binding/unbinding events, and from these, we derive an accurate estimation of the absolute binding free energy. Importantly, we show that different binding poses induce different dynamics states. Our work for the first time explicitly correlates HSP90 responses to binding/unbinding of an allosteric ligand to the modulation of functionally oriented protein motions