Pharmacological inactivation of the prion protein by targeting a folding intermediate.

Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. These techniques allowed us to design a drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein levels by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP and identified four different small molecule ligands for this conformer, all capable of selectively lowering the load of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.

New Insights on KCa3.1 Channel Modulation.

The human intermediate conductance calcium-activated potassium channel, KCa3.1, is involved in several pathophysiological conditions playing a critical role in cell secretory machinery and calcium signalling. The recent cryo-EM analysis provides new insights for understanding the modulation by both endogenous and pharmacological agents. A typical feature of this channel is the low open probability in saturating calcium concentrations and its modulation by potassium channel openers (KCOs), such as benzo imidazolone 1-EBIO, without changing calcium-dependent activation. In this paper, we proposed a model of KCOs action in the modulation of channel activity. The KCa3.1 channel has a very rich pharmacological profile with several classes of molecules that selectively interact with different binding sites of the channel. Among them, benzo imidazolones can be openers (positive modulators such as 1-EBIO, DC-EBIO) or blockers (negative modulators such as NS1619). Through computation modelling techniques, we identified the 1,4-benzothiazin-3-one as a promising scaffold to develop new KCa3.1 channel modulators. Further studies are needed to explore the potential use of 1-4 benzothiazine- 3-one in KCa3.1 modulation and its pharmacological application.

Pharmacophore-Based Repositioning of Approved Drugs as Novel Staphylococcus aureus NorA Efflux Pump Inhibitors.

An intriguing opportunity to address antimicrobial resistance is represented by the inhibition of efflux pumps. Focusing on NorA, the most important efflux pump of Staphylococcus aureus, an efflux pump inhibitors (EPIs) library was used for ligand-based pharmacophore modeling studies. By exploitation of the obtained models, an in silico drug repositioning approach allowed for the identification of novel and potent NorA EPIs.

Response of feline immunodeficiency virus (FIV) to tipranavir may provide new clues for development of broad-based inhibitors of retroviral proteases acting on drug-resistant HIV-1.

The feline AIDS model for HIV-1 treatment failed in the 1990s, due to structural features resembling protease inhibitor (PI) resistant HIV-1 variants. Widespread drug-resistance to PIs now invokes the possibility of rescuing feline immunodeficiency virus (FIV) as a model for PI treatment. We here analyzed susceptibility of FIV to second generation PIs, lopinavir, atazanavir, and the structurally unrelated non-peptidic PI tipranavir. We found that FIV protease resembles HIV-1 protease drug resistance mutations limiting binding of lopinavir and atazanavir but not tipranavir. All three PIs were found to inhibit FIV replication in a concentration-dependent manner, but only tipranavir inhibited FIV similarly to HIV-1. This drug inhibited FIV synergistically with ritonavir. Inhibition of protease activity was confirmed by Western blot analysis. In molecular docking simulations, tipranavir displayed energetically favorable interactions with the catalytic cavity of the mature dimeric FIV protease. The calculated hydrogen bond network was similar to that found in HIV-1 protease/tipranavir complexes and involved atoms in the protein backbone. We also modeled the interaction of tipranavir with an immature protease monomer, suggesting that inhibition of protease dimerization may be a secondary modality for FIV inhibition by tipranavir. In conclusion, tipranavir is the first FDA-approved non-reverse transcriptase inhibitor of HIV-1 to show anti-FIV properties. The tipranavir response by FIV may 1) support the idea of using FIV as a small animal model for PI-resistant HIV-1, thus expanding access to animal AIDS models; and 2) pave the way for development of novel broad-based inhibitors for treatment of drug resistant HIV-1.

Molecular dynamics studies of the wild-type and double mutant HIV-1 integrase complexed with the 5CITEP inhibitor: mechanism for inhibition and drug resistance.

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an essential enzyme in the life cycle of the virus and is an attractive target for the development of new drugs useful in acquired immunodeficiency syndrome multidrug therapy. Starting from the crystal structure of the 5CITEP inhibitor bound to the active site in the catalytic domain of the HIV-1 IN, two different molecular dynamics simulations in water have been carried out. In the first simulation the wild-type IN was used, whereas in the second one the double mutation T66I/M154I, described to lead to drug resistance, was introduced in the protein. Compelling differences have been observed in these two structures during analyses of the molecular dynamics trajectories, particularly in the inhibitor binding modes and in the conformational flexibility of the loop (residues 138-149) located near the three catalytic residues in the active site (Asp(64), Asp(116), Glu(152)). Because the conformational flexibility of this region is important for efficient biological activity and its behavior is quite different in the two models, we suggest a hypothetical mechanism for the inhibition and drug resistance of HIV-1 IN. These results can be useful for the rational design of more potent and selective integrase inhibitors and may allow for the design of inhibitors that will be more robust against known resistance mutations.

Design, synthesis, structure-activity relationships, and molecular modeling studies of 2,3-diaryl-1,3-thiazolidin-4-ones as potent anti-HIV agents.

Starting from 1H,3H-thiazolo[3,4-a]benzimidazoles (TBZs), we performed the design, synthesis, and the structure-activity relationship studies of a series of 2,3-diaryl-1,3-thiazolidin-4-ones. Some derivatives proved to be highly effective in inhibiting HIV-1 replication at nanomolar concentrations with minimal cytotoxicity, thereby acting as nonnucleoside HIV-1 RT inhibitors (NNRTIs). Computational studies were used to delineate the ligand-RT interactions and to probe the binding of the ligands to HIV-1 RT.