Modulating the selectivity of inhibitors for prolyl oligopeptidase inhibitors and fibroblast activation protein-α for different indications.

We have previously described several different chemical series of bicyclic prolyl oligopeptidase (POP) inhibitors as probes for neurodegenerative diseases that demonstrated nanomolar activity in vitro and submicromolar activity in cellulo. The more recent implication of POP in cancer, together with homologous fibroblast activation protein α (FAP), implicated in tumor growth, led us to consider developing POP/FAP dual inhibitors as a promising strategy for the development of cancer therapeutics. At this stage, we thought to evaluate the requirements for selectivity of inhibitors for POP over FAP and to evaluate molecular platforms that would enable the development of selective POP and dual POP/FAP inhibitors.

Design, synthesis and in vitro evaluation of novel SARS-CoV-2 3CL covalent inhibitors.

Severe diseases such as the ongoing COVID-19 pandemic, as well as the previous SARS and MERS outbreaks, are the result of coronavirus infections and have demonstrated the urgent need for antiviral drugs to combat these deadly viruses. Due to its essential role in viral replication and function, 3CL (main coronaviruses cysteine-protease) has been identified as a promising target for the development of antiviral drugs. Previously reported SARS-CoV 3CL non-covalent inhibitors were used as a starting point for the development of covalent inhibitors of SARS-CoV-2 3CL.

Targeting MYC: From understanding its biology to drug discovery.

The MYC oncogene is considered to be a high priority target for clinical intervention in cancer patients due to its aberrant activation in more than 50% of human cancers. Direct small molecule inhibition of MYC has traditionally been hampered by its intrinsically disordered nature and lack of both binding site and enzymatic activity. In recent years, however, a number of strategies for indirectly targeting MYC have emerged, guided by the advent of protein structural information and the growing set of computational tools that can be used to accelerate the hit to lead process in medicinal chemistry.

Design and discovery of boronic acid drugs.

Research related to boronic acids, from synthetic development to materials to drug discovery, has skyrocketed in the past 20 years. In terms of drug discovery, the incorporation of boronic acids into medicinal chemistry endeavours has seen a steady increase in recent years. In fact, the Food and Drug Administration (FDA) and Health Canada have thus far approved five boronic acid drugs, three of which were approved in the past four years, and several others are in clinical trials.

Discovery of covalent prolyl oligopeptidase boronic ester inhibitors.

Over the past decade, many drug discovery endeavors have been invested in targeting the serine proteases prolyl oligopeptidase (POP) for the treatment of Alzheimer's and Parkinson's disease and, more recently, epithelial cancers. Our research group has focused on the discovery of reversible covalent inhibitors, namely nitriles, to target the catalytic serine residue in this enzyme. While there have been many inhibitors discovered containing a nitrile to covalently bind to the catalytic serine, we have been investigating others, particularly boronic acids and boronic esters, the latter of which have been largely unexplored as covalent warheads.

Integrated Synthetic, Biophysical, and Computational Investigations of Covalent Inhibitors of Prolyl Oligopeptidase and Fibroblast Activation Protein α.

Over the past decade, there has been increasing interest in covalent inhibition as a drug design strategy. Our own interest in the development of prolyl oligopeptidase (POP) and fibroblast activation protein α (FAP) covalent inhibitors has led us to question whether these two serine proteases were equal in terms of their reactivity toward electrophilic warheads. To streamline such investigations, we exploited both computational and experimental methods to investigate the influence of different reactive groups on both potency and binding kinetics using both our own series of POP inhibitors and others' discovered hits.

Rapid measurement of inhibitor binding kinetics by isothermal titration calorimetry.

Although drug development typically focuses on binding thermodynamics, recent studies suggest that kinetic properties can strongly impact a drug candidate's efficacy. Robust techniques for measuring inhibitor association and dissociation rates are therefore essential. To address this need, we have developed a pair of complementary isothermal titration calorimetry (ITC) techniques for measuring the kinetics of enzyme inhibition.

Mitochondrial heat shock protein-90 modulates vascular smooth muscle cell survival and the vascular injury response in vivo.

The healing response of blood vessels from the vascular injury induced by therapeutic interventions is characterized by increased cellularity and tissue remodeling. Frequently, this leads to intimal hyperplasia and lumen narrowing, with significant clinical sequelae. Vascular smooth muscle cells are the primary cell type involved in this process, wherein they express a dedifferentiated phenotype that transiently resembles neoplastic transformation.

Exploiting the mitochondrial unfolded protein response for cancer therapy in mice and human cells.

Fine tuning of the protein folding environment in subcellular organelles, such as mitochondria, is important for adaptive homeostasis and may participate in human diseases, but the regulators of this process are still largely elusive. Here, we have shown that selective targeting of heat shock protein-90 (Hsp90) chaperones in mitochondria of human tumor cells triggered compensatory autophagy and an organelle unfolded protein response (UPR) centered on upregulation of CCAAT enhancer binding protein (C/EBP) transcription factors. In turn, this transcriptional UPR repressed NF-κB-dependent gene expression, enhanced tumor cell apoptosis initiated by death receptor ligation, and inhibited intracranial glioblastoma growth in mice without detectable toxicity.

Combined in silico and experimental approach for drug design: the binding mode of peptidic and non-peptidic inhibitors to hsp90 N-terminal domain.

Heat shock protein 90 (Hsp90) is a prime target for antitumor therapies. The information obtained by molecular dynamics (MD) simulations is combined with NMR data to provide a cross-validated atomic resolution model of the complementary interactions of heat shock protein 90 with a peptidic (shepherdin) and a non-peptidic (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside, AICAR) inhibitor, showing antiproliferative and proapoptotic activity in multiple tumor cell lines. This approach highlights the relevant role of imidazolic moiety in the interaction of both antagonist molecules.

Preclinical characterization of mitochondria-targeted small molecule hsp90 inhibitors, gamitrinibs, in advanced prostate cancer.

Purpose: This study aimed to characterize the preclinical activity of the first class of combinatorial, mitochondria-targeted, small molecule heat shock protein-90 (Hsp90) inhibitors, gamitrinibs, in models of hormone-refractory, drug-resistant, localized, and bone metastatic prostate cancer in vivo.

Experimental Design: Mitochondrial permeability transition, apoptosis, and changes in metabolic activity were examined by time-lapse videomicroscopy, multiparametric flow cytometry, MTT, and analysis of isolated mitochondria. Drug-resistant prostate cancer cells were generated by chronic exposure of hormone-refractory PC3 cells to the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG).

Global targeting of subcellular heat shock protein-90 networks for therapy of glioblastoma.

Drug discovery for complex and heterogeneous tumors now aims at dismantling global networks of disease maintenance, but the subcellular requirements of this approach are not understood. Here, we simultaneously targeted the multiple subcellular compartments of the molecular chaperone heat shock protein-90 (Hsp90) in a model of glioblastoma, a highly lethal human malignancy in urgent need of fresh therapeutic strategies. Treatment of cultured or patient-derived glioblastoma cells with Shepherdin, a dual peptidomimetic inhibitor of mitochondrial and cytosolic Hsp90, caused irreversible collapse of mitochondria, degradation of Hsp90 client proteins in the cytosol, and tumor cell killing by apoptosis and autophagy.

Cytoprotective mitochondrial chaperone TRAP-1 as a novel molecular target in localized and metastatic prostate cancer.

Molecular chaperones of the heat shock protein-90 (Hsp90) family promote cell survival, but the molecular requirements of this pathway in tumor progression are not understood. Here, we show that a mitochondria-localized Hsp90 chaperone, tumor necrosis factor receptor-associated protein-1 (TRAP-1), is abundantly and ubiquitously expressed in human high-grade prostatic intraepithelial neoplasia, Gleason grades 3 through 5 prostatic adenocarcinomas, and metastatic prostate cancer, but largely undetectable in normal prostate or benign prostatic hyperplasia in vivo. Prostate lesions formed in genetic models of the disease, including the transgenic adenocarcinoma of the mouse prostate and mice carrying prostate-specific deletion of the phosphatase tensin homolog tumor suppressor (Pten(pc-/-)), also exhibit high levels of TRAP-1.

Endogenous tumor suppression mediated by PTEN involves survivin gene silencing.

Endogenous tumor suppression provides a barrier against oncogenesis, but the molecular requirements of this process are not well understood. Here, we show that the dual specificity phosphatase PTEN, a gene almost universally altered in human tumors, silences the expression of survivin, an essential regulator of cell division and apoptosis in cancer. This pathway is independent of p53, involves active repression of survivin gene transcription, and is mediated by direct occupancy of the survivin promoter by FOXO1 and FOXO3a factors.

Combinatorial drug design targeting multiple cancer signaling networks controlled by mitochondrial Hsp90.

Although therapeutically targeting a single signaling pathway that drives tumor development and/or progression has been effective for a number of cancers, in many cases this approach has not been successful. Targeting networks of signaling pathways, instead of isolated pathways, may overcome this problem, which is probably due to the extreme heterogeneity of human tumors. However, the possibility that such networks may be spatially arranged in specialized subcellular compartments is not often considered in pathway-oriented drug discovery and may influence the design of new agents.

A functional Notch-survivin gene signature in basal breast cancer.

Introduction: Basal-type, or triple-negative, breast cancer (lacking estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 expression) is a high-risk disease for which no molecular therapies are currently available. We studied genetic signatures of basal breast cancer potentially suitable for therapeutic intervention.

Methods: We analyzed protein expression of the Notch-1 intracellular domain and survivin by immunohistochemistry in a series of basal breast cancer patients.

Regulation of tumor cell mitochondrial homeostasis by an organelle-specific Hsp90 chaperone network.

Molecular chaperones, especially members of the heat shock protein 90 (Hsp90) family, are thought to promote tumor cell survival, but this function is not well understood. Here, we show that mitochondria of tumor cells, but not most normal tissues, contain Hsp90 and its related molecule, TRAP-1. These chaperones interact with Cyclophilin D, an immunophilin that induces mitochondrial cell death, and antagonize its function via protein folding/refolding mechanisms.

Small-molecule targeting of heat shock protein 90 chaperone function: rational identification of a new anticancer lead.

Heat shock protein 90 (Hsp90) is a significant target in the development of rational cancer therapy due to its role at the crossroads of multiple signaling pathways associated with cell proliferation and cell viability. Here we present a combined structure- and dynamics-based computational design strategy, taking the flexibility of the receptor and of a lead peptidic antagonist into account explicitly, to identify the nonpeptidic small molecule 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) as a structurally novel inhibitor of Hsp90. The compound is selected to bind the Hsp90 N-terminal domain, mimicking the chemical and conformational properties of the recently described peptidic antagonist of the survivin-Hsp90 complex, shepherdin [Plescia et al.

Antileukemic activity of shepherdin and molecular diversity of hsp90 inhibitors.

Background: Heat shock protein 90 (Hsp90) is a molecular chaperone that is involved in signaling pathways for cell proliferation, survival, and cellular adaptation. Inhibitors of Hsp90 are being examined as cancer therapeutic agents, but the molecular mechanism of their anticancer activity is still unclear. We investigated Hsp90 as a therapeutic target for acute myeloid leukemia (AML) by use of the Hsp90 inhibitor shepherdin (a novel peptidyl antagonist of the interaction between Hsp90 and survivin, which is a regulator of cell proliferation and cell viability in cancer).

Rational design of shepherdin, a novel anticancer agent.

Anticancer agents that selectively kill tumor cells and spare normal tissues are urgently needed. Here, we engineered a cell-permeable peptidomimetic, shepherdin, modeled on the binding interface between the molecular chaperone Hsp90 and the antiapoptotic and mitotic regulator, survivin. Shepherdin makes extensive contacts with the ATP pocket of Hsp90, destabilizes its client proteins, and induces massive death of tumor cells by apoptotic and nonapoptotic mechanisms.

A survivin gene signature predicts aggressive tumor behavior.

Gene signatures that predict aggressive tumor behavior at the earliest stages of disease, ideally before overt tissue abnormalities, are urgently needed. To search for such genes, we generated a transgenic model of survivin, an essential regulator of cell division and apoptosis overexpressed in cancer. Transgenic expression of survivin in the urinary bladder did not cause histologic abnormalities of the urothelium.

Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis.

Evasion of apoptosis is a hallmark of cancer, but the molecular circuitries of this process are not understood. Here we show that survivin, a member of the inhibitor of apoptosis gene family that is overexpressed in cancer, exists in a novel mitochondrial pool in tumor cells. In response to cell death stimulation, mitochondrial survivin is rapidly discharged in the cytosol, where it prevents caspase activation and inhibits apoptosis.

IL-11 protects human microvascular endothelium from alloinjury in vivo by induction of survivin expression.

IL-11 can reduce tissue injury in animal models of inflammation but the mechanism(s) is unknown. When C.B-17 SCID/beige mice bearing human skin grafts are injected i.

Regulation of survivin function by Hsp90.

Pathways controlling cell proliferation and cell survival require flexible adaptation to environmental stresses. These mechanisms are frequently exploited in cancer, allowing tumor cells to thrive in unfavorable milieus. Here, we show that Hsp90, a molecular chaperone that is central to the cellular stress response, associates with survivin, an apoptosis inhibitor and essential regulator of mitosis.