Steven A. Rogers

A new glucocerebrosidase chaperone reduces α-synuclein and glycolipid levels in iPSC-derived dopaminergic neurons from patients with gaucher disease and parkinsonism

Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT Because GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone, which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons, indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease.

Natural product (−)-gossypol inhibits colon cancer cell growth by targeting RNA-binding protein Musashi-1

Musashi‐1 (MSI1) is an RNA‐binding protein that acts as a translation activator or repressor of target mRNAs. The best‐characterized MSI1 target is Numb mRNA, whose encoded protein negatively regulates Notch signaling. Additional MSI1 targets include the mRNAs for the tumor suppressor protein APC that regulates Wnt signaling and the cyclin‐dependent kinase inhibitor P21WAF−1. We hypothesized that increased expression of NUMB, P21 and APC, through inhibition of MSI1 RNA‐binding activity might be an effective way to simultaneously downregulate Wnt and Notch signaling, thus blocking the growth of a broad range of cancer cells. We used a fluorescence polarization assay to screen for small molecules that disrupt the binding of MSI1 to its consensus RNA binding site. One of the top hits was (−)‐gossypol (Ki = 476 ± 273 nM), a natural product from cottonseed, known to have potent anti‐tumor activity and which has recently completed Phase IIb clinical trials for prostate cancer. Surface plasmon resonance and nuclear magnetic resonance studies demonstrate a direct interaction of (−)‐gossypol with the RNA binding pocket of MSI1. We further showed that (−)‐gossypol reduces Notch/Wnt signaling in several colon cancer cell lines having high levels of MSI1, with reduced SURVIVIN expression and increased apoptosis/autophagy. Finally, we showed that orally administered (−)‐gossypol inhibits colon cancer growth in a mouse xenograft model. Our study identifies (−)‐gossypol as a potential small molecule inhibitor of MSI1‐RNA interaction, and suggests that inhibition of MSI1s RNA binding activity may be an effective anti‐cancer strategy.

Novel Cephalosporins Selectively Active on Nonreplicating Mycobacterium tuberculosis

We report two series of novel cephalosporins that are bactericidal to Mycobacterium tuberculosis alone of the pathogens tested, which only kill M. tuberculosis when its replication is halted by conditions resembling those believed to pertain in the host, and whose bactericidal activity is not dependent upon or enhanced by clavulanate, a β-lactamase inhibitor. The two classes of cephalosporins bear an ester or alternatively an oxadiazole isostere at C-2 of the cephalosporin ring system, a position that is almost exclusively a carboxylic acid in clinically used agents in the class. Representatives of the series kill M. tuberculosis within macrophages without toxicity to the macrophages or other mammalian cells.

Lifting the Mask: Identification of New Small Molecule Inhibitors of Uropathogenic Escherichia coli Group 2 Capsule Biogenesis

Uropathogenic Escherichia coli (UPEC) is the leading cause of community-acquired urinary tract infections (UTIs), with over 100 million UTIs occurring annually throughout the world. Increasing antimicrobial resistance among UPEC limits ambulatory care options, delays effective treatment, and may increase overall morbidity and mortality from complications such as urosepsis. The polysaccharide capsules of UPEC are an attractive target a therapeutic, based on their importance in defense against the host immune responses; however, the large number of antigenic types has limited their incorporation into vaccine development. The objective of this study was to identify small-molecule inhibitors of UPEC capsule biogenesis. A large-scale screening effort entailing 338,740 compounds was conducted in a cell-based, phenotypic screen for inhibition of capsule biogenesis in UPEC. The primary and concentration-response assays yielded 29 putative inhibitors of capsule biogenesis, of which 6 were selected for further studies. Secondary confirmatory assays identified two highly active agents, named DU003 and DU011, with 50% inhibitory concentrations of 1.0 µM and 0.69 µM, respectively. Confirmatory assays for capsular antigen and biochemical measurement of capsular sugars verified the inhibitory action of both compounds and demonstrated minimal toxicity and off-target effects. Serum sensitivity assays demonstrated that both compounds produced significant bacterial death upon exposure to active human serum. DU011 administration in mice provided near complete protection against a lethal systemic infection with the prototypic UPEC K1 isolate UTI89. This work has provided a conceptually new class of molecules to combat UPEC infection, and future studies will establish the molecular basis for their action along with efficacy in UTI and other UPEC infections.

Structural and Functional Evaluation of Clinically Relevant Inhibitors of Steroidogenic Cytochrome P450 17A1.

Human steroidogenic cytochrome P450 17A1 (CYP17A1) is a bifunctional enzyme that performs both hydroxylation and lyase reactions, with the latter required to generate androgens that fuel prostate cancer proliferation. The steroid abiraterone, the active form of the only CYP17A1 inhibitor approved by the Food and Drug Administration, binds the catalytic heme iron, nonselectively impeding both reactions and ultimately causing undesirable corticosteroid imbalance. Some nonsteroidal inhibitors reportedly inhibit the lyase reaction more than the preceding hydroxylase reaction, which would be clinically advantageous, but the mechanism is not understood. Thus, the nonsteroidal inhibitors seviteronel and orteronel and the steroidal inhibitors abiraterone and galeterone were compared with respect to their binding modes and hydroxylase versus lyase inhibition. Binding studies and X-ray structures of CYP17A1 with nonsteroidal inhibitors reveal coordination to the heme iron like the steroidal inhibitors. (S)-seviteronel binds similarly to both observed CYP17A1 conformations. However, (S)-orteronel and (R)-orteronel bind to distinct CYP17A1 conformations that differ in a region implicated in ligand entry/exit and the presence of a peripheral ligand. To reconcile these binding modes with enzyme function, side-by-side enzymatic analysis was undertaken and revealed that neither the nonsteroidal seviteronel nor the (S)-orteronel inhibitors demonstrated significant lyase selectivity, but the less potent (R)-orteronel was 8- to 11-fold selective for lyase inhibition. While active-site iron coordination is consistent with competitive inhibition, conformational selection for binding of some inhibitors and the differential presence of a peripheral ligand molecule suggest the possibility of CYP17A1 functional modulation by features outside the active site.

Identification of novel small molecule Beclin 1 mimetics activating autophagy

Anti-apoptotic proteins Bcl-2 and Bcl-xL could block autophagy by binding to Beclin 1 protein, an essential inducer of autophagy. Compounds mimicking Beclin 1 might be able to disrupt Bcl-xL/2-Beclin 1 interaction, free out Beclin 1, and thus trigger autophagy. In order to identify small molecule Beclin 1 mimetics, a fluorescence polarization-based high-throughput screening of 50,316 compounds was carried out with a Z’ score of 0.82 ± 0.05, and an outcome of 58 hits. After the structure analysis, three acridine analogues were unveiled and confirmed using the fluorescence polarization assay and the surface plasmon resonance assay. Moreover, a set of 17 additional acridine analogues was prepared and tested. Compound 7 showed selectivity for Bcl-xL (KD = 6.5 μM) over Bcl-2 (KD = 160 μM) protein, and potent cytotoxicity (nanomolar scale) in PC-3, PC-3a and DU145 prostate cancer cells. Furthermore, induction of autophagy was also demonstrated in PC-3 and PC-3a cells treated with some acridine compounds by LC3 conversion immunoblotting and LC3 fluorescence microscopy. These Beclin 1 mimetics will be invaluable tools for developing novel autophagy inducers, better understanding the roles of autophagy in cancer, and will contribute to cancer therapy.

Abstract 2449: Targeting an “undruggable” RNA-binding protein: Discovery of small molecule inhibitors of HuR for novel breast cancer therapy

Post-transcriptional gene regulation is essential for normal development, but when dysregulated, has many implications in disease conditions, including cancer. The RNA-binding proteins (RBPs) are critical trans factors that associate with specific cis elements present in mRNAs, thereby regulating the fate of target mRNAs. Due to the lack of well-defined binding pocket, RBPs have been considered “undruggable targets”. The RBP Hu antigen R (HuR) is overexpressed in many types of cancer, including breast cancer. Elevated cytoplasmic HuR level correlates with high-grade malignancy and serves as a prognostic factor of poor clinical outcome in breast cancer. HuR promotes tumorigenesis by interacting with cancer-associated mRNAs, those mRNAs encode proteins that are implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis. HuR also modulates the sensitivity of breast cancer cells to chemotherapy. Our hypothesis is that small molecule compounds that disrupt the HuR-mRNA interaction will block HuR function, leading to the decay and reduced translation of mRNAs of the target genes critical for breast cancer cell growth and progression. High throughput screening (HTS) was carried out in several chemical libraries (∼23,000 compounds) using fluorescence polarization (FP) assay and identified a series of initial hits with sub-micromolar inhibitory constants (Ki). Those potential disruptors were then validated by ALPHA assay (Amplified Luminescent Proximity Homogeneous Assay), confirmed by Surface Plasmon Resonance (SPR). In cell-based assays, top hit ST-3 and its optimized analogs, specifically shortened HuR target mRNAs’ half-life and decreased the level of the encoded proteins (Bcl-2, XIAP and Msi1/2). Moreover, those compounds selectively inhibited breast cancer cell proliferation but not normal cells. Knocking down HuR in breast cancer cells attenuated the activity of those HuR-mRNA disruptors. More cell-based assays are carrying out to delineate the mechanism of action. We are also testing the antitumor efficacy of those HuR inhibitors in mouse xenograft models. In conclusion, we identified potential small molecule disrupters of HuR-mRNA interaction for potential novel cancer therapy that inhibited breast cancer with HuR overexpression. Citation Format: Xiaoqing Wu, Lan Lan, Amber Smith, Rebecca Marquez, David Wilson, Steven Rogers, Philip Gao, Scott Lovell, John Karanicolas, Dan Dixon, Jeffrey Aube, Liang Xu. Targeting an “undruggable” RNA-binding protein: Discovery of small molecule inhibitors of HuR for novel breast cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2449. doi:10.1158/1538-7445.AM2015-2449

Natural product derivative Gossypolone inhibits Musashi family of RNA-binding proteins

BackgroundThe Musashi (MSI) family of RNA-binding proteins is best known for the role in post-transcriptional regulation of target mRNAs. Elevated MSI1 levels in a variety of human cancer are associated with up-regulation of Notch/Wnt signaling. MSI1 binds to and negatively regulates translation of Numb and APC (adenomatous polyposis coli), negative regulators of Notch and Wnt signaling respectively.MethodsPreviously, we have shown that the natural product (−)-gossypol as the first known small molecule inhibitor of MSI1 that down-regulates Notch/Wnt signaling and inhibits tumor xenograft growth in vivo. Using a fluorescence polarization (FP) competition assay, we identified gossypolone (Gn) with a > 20-fold increase in Ki value compared to (−)-gossypol. We validated Gn binding to MSI1 using surface plasmon resonance, nuclear magnetic resonance, and cellular thermal shift assay, and tested the effects of Gn on colon cancer cells and colon cancer DLD-1 xenografts in nude mice.ResultsIn colon cancer cells, Gn reduced Notch/Wnt signaling and induced apoptosis. Compared to (−)-gossypol, the same concentration of Gn is less active in all the cell assays tested. To increase Gn bioavailability, we used PEGylated liposomes in our in vivo studies. Gn-lip via tail vein injection inhibited the growth of human colon cancer DLD-1 xenografts in nude mice, as compared to the untreated control (P < 0.01, n = 10).ConclusionOur data suggest that PEGylation improved the bioavailability of Gn as well as achieved tumor-targeted delivery and controlled release of Gn, which enhanced its overall biocompatibility and drug efficacy in vivo. This provides proof of concept for the development of Gn-lip as a molecular therapy for colon cancer with MSI1/MSI2 overexpression.

3-Substituted biquinolinium inhibitors of AraC family transcriptional activator VirF from S. flexneri obtained through in situ chemical ionization of 3,4-disubstituted dihydroquinolines

During a structure-activity relationship optimization campaign to develop an inhibitor of AraC family transcriptional activators, we discovered an unexpected transformation of a previously reported inhibitor that occurs under the assay conditions. Once placed in the assay media, the 3, 4-disubstituted dihydroquinoline core of the active analogue rapidly undergoes a decomposition reaction to a quaternary 3-substituted biquinolinium. Further examination established an SAR for this chemotype while also demonstrating its resilience to irreversible binding of biologically relevant nucleophiles.