[Show abstract][Hide abstract]ABSTRACT: Protein kinases are key regulatory nodes in cellular networks and their function has been shown to be intimately coupled with their structural flexibility. However, understanding the key structural mechanisms of large conformational transitions remains a difficult task. CDK2 is a crucial regulator of cell cycle. Its activity is finely tuned by Cyclin E/A and the catalytic segment phosphorylation, whereas its deregulation occurs in many types of cancer. ATP competitive inhibitors have failed to be approved for clinical use due to toxicity issues raised by a lack of selectivity. However, in the last few years type III allosteric inhibitors have emerged as an alternative strategy to selectively modulate CDK2 activity. In this study we have investigated the conformational variability of CDK2. A low dimensional conformational landscape of CDK2 was modeled using classical multidimensional scaling on a set of 255 crystal structures. Microsecond-scale plain and accelerated MD simulations were used to populate this landscape by using an out-of-sample extension of multidimensional scaling. CDK2 was simulated in the apo-form and in complex with the allosteric inhibitor 8-anilino-1-napthalenesulfonic acid (ANS). The apo-CDK2 landscape analysis showed a conformational equilibrium between an Src-like inactive conformation and an active-like form. These two states are separated by different metastable states that share hybrid structural features with both forms of the kinase. In contrast, the CDK2/ANS complex landscape is compatible with a conformational selection picture where the binding of ANS in proximity of the αC helix causes a population shift toward the inactive conformation. Interestingly, the new metastable states could enlarge the pool of candidate structures for the development of selective allosteric CDK2 inhibitors. The method here presented should not be limited to the CDK2 case but could be used to systematically unmask similar mechanisms throughout the human kinome.
[Show abstract][Hide abstract]ABSTRACT: A new somatic mutation in the coding region of KRAS gene (G48A) has been identified in a NSCLC patient. No other mutations were found by screening several genes known to be mutated in NSCLC. The patient responded to first-line therapy and is still under maintenance treatment 18 months from diagnosis. Normal and cancer cells were engineered to express the KRAS(G48A) mutation. KRAS(G48A) overexpression did not change the growth or the response to treatment compared to KRAS(wt) expressing cells. Analysis of the KRAS(G48A) mutant structure predicted altered interactions with other proteins. Analysis of KRAS binding to BRAF showed that the KRAS(G48A) mutant behaves more like a wild-type than a classical KRAS(G12) mutant. In conclusion, this new mutation in the coding region of KRAS, found in NSCLC, does not induce phenotypic changes similar to G12 mutants but presumably affects KRAS binding to proteins other than BRAF.
No preview · Article · Apr 2016 · Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer
[Show abstract][Hide abstract]ABSTRACT: We have recently reported drug-releasing, degradable Tetra-PEG hydrogels as a new drug delivery system. The gels contain two self-cleaving β-eliminative linkers: one that covalently tethers the drug to the gel and releases it at a predictable rate, and another with slower cleavage that is installed in each cross-link of the polymer to control gel degradation. By balancing the two cleavage rates, the system can be designed to discharge most or all of the drug before the gel undergoes significant degradation. If polymer degradation is too rapid, undesirable gel-fragments covalently bound to the drug are released; if too slow, the gel remains in the body as an inert substance for prolonged periods. Here, we describe an analytical theory as well as a Monte Carlo simulation of concurrent drug release from and degradation of Tetra-PEG polymers. Considerations are made for an ideal network as well as networks containing missing bonds and double link defects. The analytical and simulation approaches are in perfect agreement with each other and with experimental data in the regime of interest. Using these models, we are able to (a) compute the time courses of drug release and gel degradation as well as the amount of fragment–drug conjugate present at any time and (b) estimate the rate constants of drug release and gel degradation necessary to control each of the above. We can also account for the size-dependent elimination of gel fragments from a localized semipermeable compartment and hence estimate fragment mass vs time curves in such in vivo compartments. The models described allow design of an optimal Tetra-PEG drug delivery vehicle for a particular use.
[Show abstract][Hide abstract]ABSTRACT: This study describe the discovery of novel dengue virus inhibitors targeting both a crucial viral protein-protein interaction and an essential host cell factor as a strategy to reduce the emergence of drug-resistance. Starting from known c-Src inhibitors, a virtual screening was performed to identify molecules able to interact with a recently discovered allosteric pocket on the dengue virus NS5 polymerase. The selection of cheap-to-produce scaffolds and the exploration of the biologically relevant chemical space around them suggested promising candidates for chemical synthesis. A series of purines emerged as the most interesting candidates able to inhibit virus replication at low micromolar concentrations with no significant toxicity to the host cell. Among the identified antivirals, compound 16i proved to be ten times more potent than ribavirin, showed a better selectivity index and represents the first-in-class DENV-NS5 allosteric inhibitor able to target both the virus NS5-NS3 interaction and the host kinases c-Src/Fyn.
No preview · Article · Jun 2015 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract]ABSTRACT: The design of a single drug molecule able to simultaneously and specifically interact with multiple biological targets is gaining major consideration in drug discovery. However, the rational design of drugs with a desired polypharmacology profile is still a challenging task, especially when these targets are distantly related or unrelated. In this work, we present a computational approach aiming at the identification of suitable target combinations for multitarget drug design within an ensemble of biologically relevant proteins. The target selection relies on the analysis of activity annotations present in molecular databases and on ligand-based virtual screening. A few target combinations were also inspected with structure-based methods to demonstrate that the identified dual activity compounds are able to bind target combinations characterized by remote binding site similarities. Our approach was applied to the heat shock protein 90 (Hsp90) interactome, which contains several targets of key importance in cancer. Promising target combinations were identified, providing a basis for the computational design of compounds with dual activity. The approach may be used on any ensemble of proteins of interest for which known inhibitors are available.
No preview · Article · Feb 2015 · Journal of Chemical Information and Modeling
[Show abstract][Hide abstract]ABSTRACT: This study aimed to explore the capability of potentially probiotic bifidobacteria to hydrolyze chlorogenic acid into caffeic acid (CA), and to recognize the enzymes involved in this reaction. Bifidobacterium strains belonging to eight species occurring in the human gut were screened. The hydrolysis seemed peculiar of Bifidobacterium animalis, whereas the other species failed to release CA. Intracellular feruloyl esterase activity capable of hydrolyzing chlorogenic acid was detected only in B. animalis. In silico research among bifidobacteria esterases identified Balat_0669 as the cytosolic enzyme likely responsible of CA release in B. animalis. Comparative modeling of Balat_0669 and molecular docking studies support its role in chlorogenic acid hydrolysis. Expression, purification, and functional characterization of Balat_0669 in Escherichia coli were obtained as further validation. A possible role of B. animalis in the activation of hydroxycinnamic acids was demonstrated and new perspectives were opened in the development of new probiotics, specifically selected for the enhanced bioconversion of phytochemicals into bioactive compounds.
[Show abstract][Hide abstract]ABSTRACT: At present, the legendary magic bullet, i.e. a drug with high potency and selectivity towards a specific biological target, shares the spotlight with an emerging and alternative polypharmacology approach. Polypharmacology suggests that more effective drugs can be developed by specifically modulating multiple targets. It is generally thought that complex diseases such as cancer and central nervous system diseases may require complex therapeutic approaches. In this respect, a drug that "hits" multiple sensitive nodes belonging to a network of interacting targets offers the potential for higher efficacy, and may limit drawbacks generally arising from the use of a single-target drug or a combination of multiple drugs. In this article, we will compare advantages and disadvantages of multi-target versus combination therapies, discuss potential drug promiscuity arising from off-target effects, comment on drug repurposing, and introduce approaches to the computational design of multi-target drugs.
No preview · Article · Jun 2014 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract]ABSTRACT: Allosteric targeting of protein kinases via displacement of the structural αC helix with type III allosteric inhibitors is currently gaining a foothold in drug discovery. Recently, the first crystal structure of CDK2 with an open allosteric pocket adjacent to the αC helix has been described, prospecting new opportunities to design more selective inhibitors, but the structure has not yet been exploited for the structure-based design of type III allosteric inhibitors. In this work we report the results of a virtual screening campaign that resulted in the discovery of the first-in-class type III allosteric ligands of CDK2. Using a combination of docking and post-docking analyses made with our tool BEAR, 7 allosteric ligands (hit rate of 20%) with micromolar affinity for CDK2 were identified, some of them inhibiting the growth of breast cancer cell lines in the micromolar range. Competition experiments performed in the presence of the ATP-competitive inhibitor staurosporine confirmed that the 7 ligands are truly allosteric, in agreement with their design. Of these, compound 2 bound CDK2 with an EC 50 value of 3 μM and inhibited the proliferation of MDA-MB231 and ZR-75-1 breast cancer cells with IC 50 values of approximately 20 μM, while compound 4 had an EC 50 value of 71 μM and IC 50 values around 4 μM. Remarkably, the most potent compound 4 was able to selectively inhibit CDK2-mediated Retinoblastoma phosphorylation, confirming that its mechanism of action is fully compatible with a selective inhibition of CDK2 phosphorylation in cells. Finally, hit expansion through analog search of the most potent inhibitor 4 revealed an additional ligand 4g with similar in vitro potency on breast cancer cells.
[Show abstract][Hide abstract]ABSTRACT: Dimerization is an essential step of the Hsp90 cycle. This work describes the results of molecular dynamics and dimerization free energy analyses performed on the structure of the human Hsp90 closed dimer. Free energy decomposition on a domain- and residue-basis highlighted different dimerization hot spots within the dimer interface that could provide binding sites for the design of allosteric inhibitors.
No preview · Article · Jun 2014 · Medicinal Chemistry Communication
[Show abstract][Hide abstract]ABSTRACT: G-protein coupled receptors (GPCRs) are highly relevant drug targets. Four GPCRs with known crystal structure were analyzed with docking (AutoDock4) and post-docking (MM-PBSA) in order to evaluate the ability to recognize known antagonists from a larger database of molecular decoys and to predict correct binding modes. Moreover, implications on multitarget drug screening are put forward. The results suggest that these methods may be of interest to the growing field of GPCR structure based virtual screening.
No preview · Article · Mar 2013 · Journal of Chemical Information and Modeling
[Show abstract][Hide abstract]ABSTRACT: Molecular dynamics simulations and the generation of ad hoc chemical libraries are playing an increasingly important and recognized role in structure-based virtual screening. These approaches are important for treating target flexibility and improving the drug discovery pipeline. In this article I will comment on these two topics and put them into perspective.
No preview · Article · Mar 2013 · Pharmaceutical Research
[Show abstract][Hide abstract]ABSTRACT: Displacement of the αC helix in kinases by allosteric modulators is becoming a prominent approach in drug discovery, owing to its potential ability to provide inhibitor selectivity. According to recent evidence, this approach appears to be more generally applicable to a broader number of kinases of the human kinome than was previously expected. Owing to their crucial role in the modulation of cell pathways, protein kinases are important targets for a number of human diseases, including but not limited to cancer. The classic approach of targeting the ATP active site has recently come up against selectivity issues, which can be considerably reduced by following an allosteric modulation approach. Being closely related to protein kinase inactivation, allosteric targeting via displacement of the conserved structural αC helix enables a direct and specific modulation mechanism. A structure-based survey of the allosteric regulation of αC helix conformation in various kinase families is provided, highlighting key allosteric pockets and modulation mechanisms that appear to be more broadly conserved than was previously thought.
No preview · Article · Nov 2012 · Drug discovery today
[Show abstract][Hide abstract]ABSTRACT: In the last decades, molecular docking has emerged as an increasingly useful tool in the modern drug discovery process, but it still needs to overcome many hurdles and limitations such as how to account for protein flexibility and poor scoring function performance. For this reason, it has been recognized that in many cases docking results need to be post-processed to achieve a significant agreement with experimental activities. In this study, we have evaluated the performance of MM-PBSA and MM-GBSA scoring functions, implemented in our post-docking procedure BEAR, in rescoring docking solutions. For the first time, the performance of this post-docking procedure has been evaluated on six different biological targets (namely estrogen receptor, thymidine kinase, factor Xa, adenosine deaminase, aldose reductase, and enoyl ACP reductase) by using i) both a single and a multiple protein conformation approach, and ii) two different software, namely AutoDock and LibDock. The assessment has been based on two of the most important criteria for the evaluation of docking methods, i.e., the ability of known ligands to enrich the top positions of a ranked database with respect to molecular decoys, and the consistency of the docking poses with crystallographic binding modes. We found that, in many cases, MM-PBSA and MM-GBSA are able to yield higher enrichment factors compared to those obtained with the docking scoring functions alone. However, for only a minority of the cases, the enrichment factors obtained by using multiple protein conformations were higher than those obtained by using only one protein conformation.
Full-text · Article · Oct 2012 · European Journal of Medicinal Chemistry
[Show abstract][Hide abstract]ABSTRACT: Nowadays, the improvement of R&D productivity is the primary commitment in pharmaceutical research, both in big pharma and smaller biotech companies. To reduce costs, to speed up the discovery process and to increase the chance of success, advanced methods of rational drug design are very helpful, as demonstrated by several successful applications. Among these, computational methods able to predict the binding affinity of small molecules to specific biological targets are of special interest because they can accelerate the discovery of new hit compounds. Here we provide an overview of the most widely used methods in the field of binding affinity prediction, as well as of our own work in developing BEAR, an innovative methodology specifically devised to overtake some limitations in existing approaches. The BEAR method was successfully validated against different biological targets, and proved its efficacy in retrieving active compounds from virtual screening campaigns. The results obtained so far indicate that BEAR may become a leading tool in the drug discovery pipeline. We primarily discuss advantages and drawbacks of each technique and show relevant examples and applications in drug discovery.
No preview · Article · Aug 2011 · Biotechnology advances