Noncanonical-NF-κB activation and DDX3 inhibition reduces the HIV-1 reservoir by elimination of latently infected cells ex-vivo.

IMPORTANCE: HIV-1 continues to be a major global health challenge. Current HIV-1 treatments are effective but need lifelong adherence. An HIV-1 cure should eliminate the latent viral reservoir that persists in people living with HIV-1. Different methods have been investigated that focus on reactivation and subsequent elimination of the HIV-1 reservoir, and it is becoming clear that a combination of compounds with different mechanisms of actions might be more effective. Here, we target two host factors, inhibitor of apoptosis proteins that control apoptosis and the DEAD-box helicase DDX3, facilitating HIV mRNA transport/translation. We show that targeting of these host factors with SMAC mimetics and DDX3 inhibitors induce reversal of viral latency and eliminate HIV-1-infected cells in vitro and ex vivo.

The FHP01 DDX3X Helicase Inhibitor Exerts Potent Anti-Tumor Activity In Vivo in Breast Cancer Pre-Clinical Models.

Inhibition of DDX3X expression or activity reduces proliferation in cells from various tumor tissues, in particular in breast cancer, and its expression often correlates to tumor aggressiveness. This makes DDX3X a prominent candidate for the design of drugs for novel personalized therapeutic strategies. Starting from an in silico drug discovery approach, a group of molecules has been selected by molecular docking at the RNA binding site of DDX3X. Here, the most promising among them, FHP01, was evaluated in breast cancer preclinical models. Specifically, FHP01 exhibited very effective antiproliferative and killing activity against different breast cancer cell types, among which those from triple-negative breast cancer (TNBC). Interestingly, FHP01 also inhibited WNT signaling, a key tumorigenic pathway already correlated to DDX3X functions in breast cancer model cell lines. Ultimately, FHP01 also caused a significant reduction, in vivo, in the growth of MDA MB 231-derived TNBC xenograft models. Importantly, FHP01 showed good bioavailability and no toxicity on normal peripheral blood mononuclear cells in vitro and on several mouse tissues in vivo. Overall, our data suggest that the use of FHP01 and its related compounds may represent a novel therapeutic approach with high potential against breast cancer, including the triple-negative subtype usually correlated to the most unfavorable outcomes because of the lack of available targeted therapies.

Optimization of linear and cyclic peptide inhibitors of KEAP1-NRF2 protein-protein interaction.

Inhibition of KEAP1-NRF2 protein-protein interaction is considered a promising strategy to selectively and effectively activate NRF2, a transcription factor which is involved in several pathologies such as Huntington's disease (HD). A library of linear peptides based on the NRF2-binding motifs was generated on the nonapeptide lead Ac-LDEETGEFL-NH2 spanning residues 76-84 of the Neh2 domain of NRF2 with the aim to replace E78, E79 and E82 with non-acidic amino acids. A deeper understanding of the features and accessibility of the T80 subpocket was also targeted by structure-based design. Approaches to improve cell permeability were investigated using both different classes of cyclic peptides and conjugation to cell-penetrating peptides. This insight will guide future design of macrocycles, peptido-mimetics and, most importantly, small neutral brain-penetrating molecules to evaluate whether NRF2 activators have utility in HD.

Combined Peptide and Small-Molecule Approach toward Nonacidic THIQ Inhibitors of the KEAP1/NRF2 Interaction.

The NRF2-ARE pathway is an intrinsic mechanism of defense against oxidative stress. Inhibition of the interaction between NRF2 and its main negative regulator KEAP1 is an attractive strategy toward neuroprotective agents. We report here the identification of nonacidic tetrahydroisoquinolines (THIQs) that inhibit the KEAP1/NRF2 protein-protein interaction. Peptide SAR at one residue is utilized as a tool to probe structural changes within a specific pocket of the KEAP1 binding site. We used structural information from peptide screening at the P2 pocket, noncovalent small-molecules inhibitors, and the outcome from an explorative SAR at position 5 of THIQs to identify a series of neutral THIQ analogs that bind to KEAP1 in the low micromolar range. These analogs establish new H-bond interactions at the P3 and P2 pockets allowing the replacement of the carboxylic acid functionality by a neutral primary carboxamide. X-ray crystallographic studies reveal the novel binding mode of these molecules to KEAP1.

Identification and binding mode of a novel Leishmania Trypanothione reductase inhibitor from high throughput screening.

Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against Leishmaniasis. This enzyme is fundamental for parasite survival in the host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize hydrogen peroxide produced by host macrophages during infection. In order to identify new lead compounds against Leishmania we developed and validated a new luminescence-based high-throughput screening (HTS) assay that allowed us to screen a library of 120,000 compounds. We identified a novel chemical class of TR inhibitors, able to kill parasites with an IC50 in the low micromolar range. The X-ray crystal structure of TR in complex with a compound from this class (compound 3) allowed the identification of its binding site in a pocket at the entrance of the NADPH binding site. Since the binding site of compound 3 identified by the X-ray structure is unique, and is not present in human homologs such as glutathione reductase (hGR), it represents a new target for drug discovery efforts.

Peptidomimetic nitrile inhibitors of malarial protease falcipain-2 with high selectivity against human cathepsins.

Falcipain-2 (FP2) is an essential enzyme in the lifecycle of malaria parasites such as Plasmodium falciparum, and its inhibition is viewed as an attractive mechanism of action for new anti-malarial agents. Selective inhibition of FP2 with respect to a family of human cysteine proteases (that include cathepsins B, K, L and S) is likely to be required for the development of agents targeting FP2. Here we describe a series of P2-modified aminonitrile based inhibitors of FP2 that provide a clear strategy toward addressing selectivity for the P. falciparum and show that it can provide potent FP2 inhibitors with strong selectivity against all four of these human cathepsin isoforms.

Discovery by organism based high-throughput screening of new multi-stage compounds affecting Schistosoma mansoni viability, egg formation and production.

Schistosomiasis, one of the most prevalent neglected parasitic diseases affecting humans and animals, is caused by the Platyhelminthes of the genus Schistosoma. Schistosomes are the only trematodes to have evolved sexual dimorphism and the constant pairing with a male is essential for the sexual maturation of the female. Pairing is required for the full development of the two major female organs, ovary and vitellarium that are involved in the production of different cell types such as oocytes and vitellocytes, which represent the core elements of the whole egg machinery. Sexually mature females can produce a large number of eggs each day. Due to the importance of egg production for both life cycle and pathogenesis, there is significant interest in the search for new strategies and compounds not only affecting parasite viability but also egg production. Here we use a recently developed high-throughput organism-based approach, based on ATP quantitation in the schistosomula larval stage of Schistosoma mansoni for the screening of a large compound library, and describe a pharmacophore-based drug selection approach and phenotypic analyses to identify novel multi-stage schistosomicidal compounds. Interestingly, worm pairs treated with seven of the eight compounds identified show a phenotype characterized by defects in eggshell assemblage within the ootype and egg formation with degenerated oocytes and vitelline cells engulfment in the uterus and/or oviduct. We describe promising new molecules that not only impair the schistosomula larval stage but also impact juvenile and adult worm viability and egg formation and production in vitro.

Identification by High-Throughput Screening of Pseudomonas Acyl-Coenzyme A Synthetase Inhibitors.

Pseudomonas infections are common among hospitalized, immunocompromised, and chronic lung disease patients. These infections are recalcitrant to common antibacterial therapies due to inherent antibiotic resistance. To meet the need of new anti- Pseudomonas drugs, a sensitive, homogenous, and robust assay was developed with the aim of identifying inhibitors of acyl-coenzyme A synthetases (ACSs) from Pseudomonas. Given the importance of fatty acids for in vivo nutrition of Pseudomonas, such inhibitors might have the potential to reduce the bacterial fitness during infection. The assay, based on a coupled reaction between the Pseudomonas spp. ACS and the firefly luciferase, allowed the identification of three classes of inhibitors by screening of a diverse compound collection. These compounds were confirmed to reversibly bind ACS with potencies in the micromolar range. Two classes were found to compete with acyl-coenzyme A, while the third one was competitive with fatty acid binding. Although these compounds inhibit the bacterial ACS in cell-free assays, they show modest or no effect on Pseudomonas growth in vitro.

The soluble Y115E-Y117E variant of human glutaminyl cyclase is a valid target for X-ray and NMR screening of inhibitors against Alzheimer disease.

Recent developments in molecular pathology and genetics have allowed the identification of human glutaminyl cyclase (hQC) among the abnormal proteins involved in many neurodegenerative disorders. Difficulties in obtaining large quantities of pure protein may limit the use of crystallographic screening for drug development on this target. Site-directed mutagenesis experiments have led to the identification of some solvent-exposed residues that are absolutely critical to achieve increased solubility and to avoid precipitation of the enzyme in inclusion bodies when expressed in Escherichia coli. The designed variant Y115E-Y117E has been found to be able to provide large amounts of monodisperse, pure hQC from an E. coli expression system. To validate the use of the artificial construct as a target for large-scale X-ray and NMR screening campaigns in the search for new inhibitors of hQC, the X-ray crystal structures of the hQC Y115E-Y117E variant and of its adduct with the inhibitor PBD-150 were determined.

Structure-activity relationship and properties optimization of a series of quinazoline-2,4-diones as inhibitors of the canonical Wnt pathway.

Wnt signaling pathway plays a critical role in numerous cellular processes, including tumor initiation, proliferation, invasion/infiltration, metastasis formation and resistance to chemotherapy. In a drug discovery project aimed at the identification of inhibitors of the canonical Wnt pathway, we selected a series of quinazoline 2,4-diones as starting point for the therapeutic treatment of glioblastoma multiforme. Despite of poor physico-chemical properties of hit compound 1, our medicinal chemistry effort allowed the discovery and characterization of lead compound 33 (SEN461), with improved ADME profile, good bioavailability and active in vitro and in vivo in glioblastoma, gastric and sarcoma tumors.

siRNA screen identifies QPCT as a druggable target for Huntington's disease.

Huntington's disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified new modifiers of mutant HTT toxicity by performing a large-scale 'druggable genome' siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen and also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone αB-crystallin and reduced the aggregation of diverse proteins. We generated new QPCT inhibitors using in silico methods followed by in vitro screening, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a new HD druggable target affecting mutant HTT aggregation and provide proof of principle for a discovery pipeline from druggable genome screen to drug development.

BACE1 inhibitors: a head group scan on a series of amides.

A series of amides bearing a variety of amidine head groups was investigated as BACE1 inhibitors with respect to inhibitory activity in a BACE1 enzyme as well as a cell-based assay. Determination of their basicity as well as their properties as substrates of P-glycoprotein revealed that a 2-amino-1,3-oxazine head group would be a suitable starting point for further development of brain penetrating compounds for potential Alzheimer's disease treatment.

Pharmacophore based receptor modeling: the case of adenosine A3 receptor antagonists. An approach to the optimization of protein models.

To design and synthesize new potent and selective antagonists of the human A(3) adenosine receptor, pharmacophoric hypotheses were generated with the software Catalyst for a comprehensive set of compounds retrieved from previous literature. Three of these pharmacophores were used to drive the optimization of a molecular model of the receptor built by homology modeling. The alignment of the ligands proposed by Catalyst was then used to manually dock a set of known A(3) antagonists into the binding site, and as a result, the model was able to explain the different binding mode of very active compounds with respect to less active ones and to reproduce, with good accuracy, free energies of binding. The docking highlighted that the nonconserved residue Tyr254 could play an important role for A(3) selectivity, suggesting that a mutagenesis study on this residue could be of interest in this respect. The reliability of the whole approach was successfully tested by rational design and synthesis of new compounds.