Gabriela Mustata

Discovery of novel Myc-Max heterodimer disruptors with a three-dimensional pharmacophore model.

A three-dimensional pharmacophore model was generated utilizing a set of known inhibitors of c-Myc-Max heterodimer formation. The model successfully identified a set of structurally diverse compounds with potential inhibitory activity against c-Myc. Nine compounds were tested in vitro, and four displayed affinities in the micromolar range and growth inhibitory activity against c-Myc-overexpressing cells. These studies demonstrate the applicability of pharmacophore modeling to the identification of novel and potentially more puissant inhibitors of the c-Myc oncoprotein.

Identification of potent chemotypes targeting Leishmania major using a high-throughput, low-stringency, computationally enhanced, small molecule screen.

Patients with clinical manifestations of leishmaniasis, including cutaneous leishmaniasis, have limited treatment options, and existing therapies frequently have significant untoward liabilities. Rapid expansion in the diversity of available cutaneous leishmanicidal chemotypes is the initial step in finding alternative efficacious treatments. To this end, we combined a low-stringency Leishmania major promastigote growth inhibition assay with a structural computational filtering algorithm. After a rigorous assay validation process, we interrogated ∼200,000 unique compounds for L. major promastigote growth inhibition. Using iterative computational filtering of the compounds exhibiting >50% inhibition, we identified 553 structural clusters and 640 compound singletons. Secondary confirmation assays yielded 93 compounds with EC50s ≤ 1 µM, with none of the identified chemotypes being structurally similar to known leishmanicidals and most having favorable in silico predicted bioavailability characteristics. The leishmanicidal activity of a representative subset of 15 chemotypes was confirmed in two independent assay formats, and L. major parasite specificity was demonstrated by assaying against a panel of human cell lines. Thirteen chemotypes inhibited the growth of a L. major axenic amastigote-like population. Murine in vivo efficacy studies using one of the new chemotypes document inhibition of footpad lesion development. These results authenticate that low stringency, large-scale compound screening combined with computational structure filtering can rapidly expand the chemotypes targeting in vitro and in vivo Leishmania growth and viability.

Development of Small-Molecule PUMA Inhibitors for Mitigating Radiation-Induced Cell Death

PUMA (p53 upregulated modulator of apoptosis) is a Bcl-2 homology 3 (BH3)-only Bcl-2 family member and a key mediator of apoptosis induced by a wide variety of stimuli. PUMA is particularly important in initiating radiation-induced apoptosis and damage in the gastrointestinal and hematopoietic systems. Unlike most BH3-only proteins, PUMA neutralizes all five known antiapoptotic Bcl-2 members though high affinity interactions with its BH3 domain to initiate mitochondria-dependent cell death. Using structural data on the conserved interactions of PUMA with Bcl-2-like proteins, we developed a pharmacophore model that mimics these interactions. In silico screening of the ZINC 8.0 database with this pharmacophore model yielded 142 compounds that could potentially disrupt these interactions. Thirteen structurally diverse compounds with favorable in silico ADME/Toxicity profiles have been retrieved from this set. Extensive testing of these compounds using cell-based and cell-free systems identified lead compounds that confer considerable protection against PUMA-dependent and radiation-induced apoptosis, and inhibit the interaction between PUMA and Bcl-xL.

Comparative molecular dynamics simulations of HIV-1 integrase and the T66I/M154I mutant: Binding modes and drug resistance to a diketo acid inhibitor

HIV‐1 IN is an essential enzyme for viral replication and an interesting target for the design of new pharmaceuticals for use in multidrug therapy of AIDS. L‐731,988 is one of the most active molecules of the class of β‐diketo acids. Individual and combined mutations of HIV‐1 IN at residues T66, S153, and M154 confer important degrees of resistance to one or more inhibitors belonging to this class. In an effort to understand the molecular mechanism of the resistance of T66I/M154I IN to the inhibitor L‐731,988 and its specific binding modes, we have carried out docking studies, explicit solvent MD simulations, and binding free energy calculations. The inhibitor was docked against different protein conformations chosen from prior MD trajectories, resulting in 2 major orientations within the active site. MD simulations have been carried out for the T66I/M154I DM IN, DM IN in complex with L‐731,988 in 2 different orientations, and 1QS4 IN in complex with L‐731,988. The results of these simulations show a similar dynamical behavior between T66I/M154I IN alone and in complex with L‐731,988, while significant differences are observed in the mobility of the IN catalytic loop (residues 138–149). Water molecules bridging the inhibitor to residues from the active site have been identified, and residue Gln62 has been found to play an important role in the interactions between the inhibitor and the protein. This work provides information about the binding modes of L‐731,988, as well as insight into the mechanism of inhibitor–resistance in HIV‐1 integrase. Proteins 2005.

A target-based high throughput screen yields Trypanosoma brucei hexokinase small molecule inhibitors with antiparasitic activity

Background The parasitic protozoan Trypanosoma brucei utilizes glycolysis exclusively for ATP production during infection of the mammalian host. The first step in this metabolic pathway is mediated by hexokinase (TbHK), an enzyme essential to the parasite that transfers the γ-phospho of ATP to a hexose. Here we describe the identification and confirmation of novel small molecule inhibitors of bacterially expressed TbHK1, one of two TbHKs expressed by T. brucei, using a high throughput screening assay. Methodology/Principal Findings Exploiting optimized high throughput screening assay procedures, we interrogated 220,233 unique compounds and identified 239 active compounds from which ten small molecules were further characterized. Computation chemical cluster analyses indicated that six compounds were structurally related while the remaining four compounds were classified as unrelated or singletons. All ten compounds were ∼20-17,000-fold more potent than lonidamine, a previously identified TbHK1 inhibitor. Seven compounds inhibited T. brucei blood stage form parasite growth (0.03≤EC50<3 µM) with parasite specificity of the compounds being demonstrated using insect stage T. brucei parasites, Leishmania promastigotes, and mammalian cell lines. Analysis of two structurally related compounds, ebselen and SID 17387000, revealed that both were mixed inhibitors of TbHK1 with respect to ATP. Additionally, both compounds inhibited parasite lysate-derived HK activity. None of the compounds displayed structural similarity to known hexokinase inhibitors or human African trypanosomiasis therapeutics. Conclusions/Significance The novel chemotypes identified here could represent leads for future therapeutic development against the African trypanosome.

The crystal structure of Trichomonas vaginalis ferredoxin provides insight into metronidazole activation.

Crystallographic studies revealing the three-dimensional structure of the oxidized form of the [2Fe-2S] ferredoxin from Trichomonas vaginalis (TvFd) are presented. TvFd, a member of the hydrogenosomal class of ferredoxins, possesses a unique combination of redox and spectroscopic properties, and is believed to be the biological molecule that activates the drug metronidazole reductively in the treatment of trichomoniasis. It is the first hydrogenosomal ferredoxin to have its structure determined. The structure of TvFd reveals a monomeric, 93 residue protein with a fold similar to that of other known [2Fe-2S] ferredoxins. It contains nine hydrogen bonds to the sulfur atoms of the cluster, which is more than the number predicted on the basis of the spectroscopic data. The TvFd structure contains a large dipole moment like adrenodoxin, and appears to have a similar interaction domain. Our analysis demonstrates that TvFd has a unique cavity near the iron-sulfur cluster that exposes one of the inorganic sulfur atoms of the cluster to solvent. This cavity is not seen in any other [2Fe-2S] ferredoxin with known structure, and is hypothesized to be responsible for the high rate of metronidazole reduction by TvFd.

Drug delivery global summit – evaluating emerging technologies

Two day-long sessions at the Drug delivery global summit, organised by SMi Group Ltd, were devoted to discussion on critical aspects of drug delivery, including advances in drug delivery systems and their applications to new products, with a primary focus on oral systems, but also highlighting recent progress in inhalation, parenteral and transdermal delivery. The event included case studies from big pharma, biotech and drug delivery companies to illustrate emerging delivery technologies and how they can be applied to develop innovative products. The conference created a platform for discussion on a range of topics from scientific issues and challenges to ways of establishing mutually beneficial relationships between technology and pharma companies.

Aromatase inhibitors and antiepileptic drugs: a computational systems biology analysis

BackgroundThe present study compares antiepileptic drugs and aromatase (CYP19) inhibitors for chemical and structural similarity. Human aromatase is well known as an important pharmacological target in anti-breast cancer therapy, but recent research demonstrates its role in epileptic seizures, as well. The current antiepileptic treatment methods cause severe side effects that endanger patient health and often preclude continued use. As a result, less toxic and more tolerable antiepileptic drugs (AEDs) are needed, especially since every individual responds differently to given treatment options.MethodsThrough a pharmacophore search, this study shows that a model previously designed to search for new classes of aromatase inhibitors is able to identify antiepileptic drugs from the set of drugs approved by the Food and Drug Administration. Chemical and structural similarity analyses were performed using five potent AIs, and these studies returned a set of AEDs that the model identifies as hits.ResultsThe pharmacophore model returned 73% (19 out of 26) of the drugs used specifically to treat epilepsy and approximately 82% (51 out of 62) of the compounds with anticonvulsant properties. Therefore, this study supports the possibility of identifying AEDs with a pharmacophore model that had originally been designed to identify new classes of aromatase inhibitors. Potential candidates for anticonvulsant therapy identified in this manner are also reported. Additionally, the chemical and structural similarity between antiepileptic compounds and aromatase inhibitors is proved using similarity analyses.ConclusionsThis study demonstrates that a pharmacophore search using a model based on aromatase inhibition and the enzymes structural features can be used to screen for new candidates for antiepileptic therapy. In fact, potent aromatase inhibitors and current antiepileptic compounds display significant - over 70% - chemical and structural similarity, and the similarity analyses performed propose a number of antiepileptic compounds with high potential for aromatase inhibition.