Andrea Cavalli

Molecular basis of cyclooxygenase enzymes (COXs) selective inhibition

The widely used nonsteroidal anti-inflammatory drugs block the cyclooxygenase enzymes (COXs) and are clinically used for the treatment of inflammation, pain, and cancers. A selective inhibition of the different isoforms, particularly COX-2, is desirable, and consequently a deeper understanding of the molecular basis of selective inhibition is of great demand. Using an advanced computational technique we have simulated the full dissociation process of a highly potent and selective inhibitor, SC-558, in both COX-1 and COX-2. We have found a previously unreported alternative binding mode in COX-2 explaining the time-dependent inhibition exhibited by this class of inhibitors and consequently their long residence time inside this isoform. Our metadynamics-based approach allows us to illuminate the highly dynamical character of the ligand/protein recognition process, thus explaining a wealth of experimental data and paving the way to an innovative strategy for designing new COX inhibitors with tuned selectivity.

Systematic Exploitation of Multiple Receptor Conformations for Virtual Ligand Screening

The role of virtual ligand screening in modern drug discovery is to mine large chemical collections and to prioritize for experimental testing a comparatively small and diverse set of compounds with expected activity against a target. Several studies have pointed out that the performance of virtual ligand screening can be improved by taking into account receptor flexibility. Here, we systematically assess how multiple crystallographic receptor conformations, a powerful way of discretely representing protein plasticity, can be exploited in screening protocols to separate binders from non-binders. Our analyses encompass 36 targets of pharmaceutical relevance and are based on actual molecules with reported activity against those targets. The results suggest that an ensemble receptor-based protocol displays a stronger discriminating power between active and inactive molecules as compared to its standard single rigid receptor counterpart. Moreover, such a protocol can be engineered not only to enrich a higher number of active compounds, but also to enhance their chemical diversity. Finally, some clear indications can be gathered on how to select a subset of receptor conformations that is most likely to provide the best performance in a real life scenario.

New Insight into the Mechanism of Action of the Temporin Antimicrobial Peptides

Temporins constitute a family of amphipathic alpha-helical antimicrobial peptides (AMPs) and contain some of the shortest cytotoxic peptides, comprised of only 10-14 residues. We have recently investigated two members of this family, temporin A (TA) and temporin L (TL), because of their different spectra of antimicrobial activity and toxicity. Consequently, we developed new analogues with promising biological activities named Pro(3)-TL and Gln(3)-TA. In this work, we performed a detailed NMR analysis of the new analogues in SDS and DPC micelles, which mimic bacterial and mammalian membranes, respectively. NMR studies reveal that strongly hemolytic Gln(3)-TA was in a stable helical conformation along the entire sequence, while weakly hemolytic but antimicrobial Pro(3)-TL showed conformational averaging at the N-terminus. Furthermore, molecular dynamics (MD) simulations on TL and Pro(3)-TL were performed in explicit water and DPC micelles. Simulations indicated that both peptides prefer a location at the micelle-water interface; however, Phe(1) of strongly hemolytic TL was embedded more in depth into DPC, and only TL caused a significant distortion of the micelle shape. By combining NMR and computational analyses, we obtained a molecular-level resolution of the interactions between TL and its analogues with membrane mimicking micelles.

Methyl Phosphate Dianion Hydrolysis in Solution Characterized by Path Collective Variables Coupled with DFT-Based Enhanced Sampling Simulations.

Herein, we propose a conceptually innovative approach to investigating reaction mechanisms. This study demonstrates the importance of considering explicitly the effects of large amplitude motions, aside from the intrinsic reaction coordinate, when tuning the free energy landscape of reaction pathways. We couple the path collective variables method with DFT-based enhanced sampling simulations to characterize the associative mechanism of the hydrolysis of the methyl phosphate dianion in solution. Importantly, energetics and mechanistic differences are observed when passing from the potential to the free energy surface.

Synthesis of monomeric derivatives to probe memoquin's bivalent interactions.

Eight monomeric congeners, related to the multitarget lead candidate memoquin, were prepared and evaluated at multiple targets to determine their profile against Alzheimers disease. 2-4 bind to AChE with similar low nanomolar affinities and function as effective inhibitors of amyloid aggregation. The most potent monovalent ligand 2 also inhibits BACE-1 in vitro and APP metabolism in primary chicken telencephalic neurons.

Complementary medicinal chemistry-driven strategies toward new antitrypanosomal and antileishmanial lead drug candidates.

Trypanosomiases and Leishmaniases are neglected tropical diseases that affect the less developed countries. For this reason, they did not and still do not have high visibility in Western societies. The name neglected diseases also refers to the fact that they often received little interest at the level of public investment, research and development. The drug discovery scenario, however, is changing dramatically. After a period in which different socioeconomic factors have prevented massive research efforts in this field, such efforts have increased considerably in the very recent years, with significant scientific advancements. In this context, we have embarked on a new drug discovery project devoted to identification of new small molecules for the treatment of trypanosomal and leishmanial diseases. Two complementary approaches have been pursued and are reported here. The first deals with a structure-based drug design, and a privileged structure-guided synthesis of quinazoline compounds able to modulate trypanothione reductase activity was accomplished. In the second, a combinatorial library, built on a natural product-based strategy, was synthesized. Using whole parasite assays, different quinones have been identified as promising lead compounds. A combination of both approaches to hopefully overcome some of the challenges of anti-trypanosomatid drug discovery has eventually been proposed.

Insights into Ligand–Protein Binding from Local Mechanical Response

Computational studies of ligand–protein binding are crucial for properly designing novel compounds of potential pharmacological interest. In this respect, researchers are increasingly interested in steered molecular dynamics for ligand–protein binding and unbinding studies. In particular, it has been suggested that analyzing the work profiles along the ligand–protein undocking paths could be fruitful. Here, we propose that small portions of work profiles, termed “local mechanical responses” of the system to a steering force, could serve as a universal measure for capturing relevant information about the system under investigation. Specifically, we first collected a high number of steering trajectories using two biological systems of increasing complexity (i.e., alanine dipeptide and (R)-roscovitine/CDK5 complex). Then, we devised a novel postprocessing tool to be applied to the local mechanical responses, to extract structural information related to the biological processes under investigation. Despite the out-of-equilibrium character of the trajectories, the analysis carried out on the work profiles provided pivotal information about the investigated biological processes. This could eventually be applied to drug design.

Cyclin-dependent kinases: bridging their structure and function through computations

Cyclin-dependent kinases (CDKs) are one of the most promising target families for drug discovery for several diseases, such as cancer and neurodegenerative disorders. Over the years, structural insights on CDKs have demonstrated high protein plasticity, with several cases where two or more structures of the same protein adopt different conformations. This has generated a great deal of interest in understanding the relationship between CDK structure and function. Here, we highlight how computer simulations have recently contributed in characterizing some key rare and transient events in CDKs, such as the reaction transition state and activation loop movement. Although not yet fully defined, we can now portray the enzymatic mechanism and plasticity of CDKs at high spatial and temporal resolution. These theoretical studies bridge with experiments and highlight structural determinants that could help in designing specific CDK inhibitors.

SERAPhiC: a benchmark for in silico fragment-based drug design.

Our main objective was to compile a data set of high-quality protein-fragment complexes and make it publicly available. Once assembled, the data set was challenged using docking procedures to address the following questions: (i) Can molecular docking correctly reproduce the experimentally solved structures? (ii) How thorough must the sampling be to replicate the experimental data? (iii) Can commonly used scoring functions discriminate between the native pose and other energy minima? The data set, named SERAPhiC (Selected Fragment Protein Complexes), is publicly available in a ready-to-dock format ( http://www.iit.it/en/drug-discovery-and-development/seraphic.html ). It offers computational medicinal chemists a reliable test set for both in silico protocol assessment and software development.

Sequential Virtual Screening Approach to the Identification of Small Organic Molecules as Potential BACE-1 Inhibitors

In this letter, we report on the sequential application of two different in silico screening approaches combined with bioassays aimed at the identification of small organic molecules as potential BACE‐1 inhibitors. Two hits endowed of micromolar inhibitory potency were selected, and the binding mode of the most potent compound was further characterized through docking simulations.