The disruption of protein-protein interactions with co-chaperones and client substrates as a strategy towards Hsp90 inhibition

The 90-kiloDalton (kD) heat shock protein (Hsp90) is a ubiquitous, ATP-dependent molecular chaperone whose primary function is to ensure the proper folding of several hundred client protein substrates. Because many of these clients are overexpressed or become mutated during cancer progression, Hsp90 inhibition has been pursued as a potential strategy for cancer as one can target multiple oncoproteins and signaling pathways simultaneously. The first discovered Hsp90 inhibitors, geldanamycin and radicicol, function by competitively binding to Hsp90's N-terminal binding site and inhibiting its ATPase activity. However, most of these N-terminal inhibitors exhibited detrimental activities during clinical evaluation due to induction of the pro-survival heat shock response as well as poor selectivity amongst the four isoforms. Consequently, alternative approaches to Hsp90 inhibition have been pursued and include C-terminal inhibition, isoform-selective inhibition, and the disruption of Hsp90 protein-protein interactions. Since the Hsp90 protein folding cycle requires the assembly of Hsp90 into a large heteroprotein complex, along with various co-chaperones and immunophilins, the development of small molecules that prevent assembly of the complex offers an alternative method of Hsp90 inhibition.

Novel PF74-like small molecules targeting the HIV-1 capsid protein: Balance of potency and metabolic stability

Of all known small molecules targeting human immunodeficiency virus (HIV) capsid protein (CA), PF74 represents by far the best characterized chemotype, due to its ability to confer antiviral phenotypes in both early and late phases of viral replication. However, the prohibitively low metabolic stability renders PF74 a poor antiviral lead. We report herein our medicinal chemistry efforts toward identifying novel and metabolically stable small molecules targeting the PF74 binding site. Specifically, we replaced the inter-domain-interacting, electron-rich indole ring of PF74 with less electron-rich isosteres, including imidazolidine-2,4-dione, pyrimidine-2,4-dione, and benzamide, and identified four potent antiviral compounds (

Orthogonal assays for the identification of inhibitors of the single-stranded nucleic acid binding protein YB-1

We have previously shown that high expression of the nucleic acid binding factor YB-1 is strongly associated with poor prognosis in a variety of cancer types. The 3-dimensional protein structure of YB-1 has yet to be determined and its role in transcriptional regulation remains elusive. Drug targeting of transcription factors is often thought to be difficult and there are very few published high-throughput screening approaches. YB-1 predominantly binds to single-stranded nucleic acids, adding further difficulty to drug discovery. Therefore, we have developed two novel screening assays to detect compounds that interfere with the transcriptional activation properties of YB-1, both of which may be generalizable to screen for inhibitors of other nucleic acid binding molecules. The first approach is a cell-based luciferase reporter gene assay that measures the level of activation of a fragment of the promoter by YB-1. The second approach is a novel application of the AlphaScreen system, to detect interference of YB-1 interaction with a single-stranded DNA binding site. These complementary assays examine YB-1 binding to two discrete nucleic acid sequences using two different luminescent signal outputs and were employed sequentially to screen 7360 small molecule compounds leading to the identification of three putative YB-1 inhibitors.

Lectin-Like Oxidized LDL Receptor 1 Mediates the Uptake of the C-Terminal Domain of Hsp70 (A Promising Immune Adjuvant Molecule) and Antigen Peptide Complexes.

Extracellular heat shock protein 70 (Hsp70) is an adjuvant molecule that stimulates the immune system. The C-terminal domain of Hsp70 (C70), without the ATPase domain, is sufficient for antigen cross-presentation. However, the mechanism by which the receptor mediates the uptake of C70–peptide complex remains unclear. We therefore aimed to determine the process by which the receptor mediates the uptake of antigenic peptide-bound C70. Methodology: Hsp70 and C70 individually cloned into pET28a were expressed in Escherichia coli BL21 (DE3) and were purified on Ni-NTA agarose and MonoQ HR5/5. Hsp70 and C70 were labeled with Alexa 555 and Alexa 633, respectively, to detect cellular binding. HEK293 cells stably expressing lectin-like oxidized LDL receptor-1 (LOX 1) and KG-1 human dendritic-like cells were incubated with Alexa-labeled Hsp70 and C70 individually or with C70 and antigenic complexes and were observed using fluorescence microscopy. The affinity of LOX-1 toward Hsp70 and C70 was analyzed by chip assay using surface plasmon resonance, which immobilized LOX-1 ligand recognition domain. Results: HEK293 cells stably expressing LOX-1 and KG-1 cells accepted the C70– peptide and Hsp70–peptide complexes. Anti-LOX-1-neutralizing antibody inhibited the uptake of the C70–peptide complexes by KG-1 cells. The dissociation constant (KD) of C70 toward the LOX-1 extracellular domain, measured by surface plasmon resonance, was 4.02 × 10−7 M and that of the C70–peptide complex was 6.6 × 10−8 M. C70 increased the LOX-1 affinity by forming a complex with the antigen peptide. Conclusion: Our findings suggest that LOX-1 is the primary receptor for the C70–peptide and the Hsp70–peptide complexes. C70 is a promising adjuvant molecule that is internalized via LOX-1. In addition, it is convenient to prepare C70 using an E. coli expression system and C70 is more stable than full-length Hsp70.

KvLQT1 and KCNE1 K+ Channel Gene Polymorphisms in Long QT Syndrome.

Long QT Syndrome (LQTS), a disorder of the cardiac repolarization process with prolongation of the QT interval (QTc ≥0.46 seconds), is an ion-channelopathy. Mutations in either KCNQ1 or KCNE1 genes are susceptible to LQTS. Hence, screening of KCNQ1 and KCNE1 genes is taken up to evaluate the genetic correlation of these genes in Long QT patients of Indian origin. A total of 33 Long QT Syndrome patients and 100 healthy subjects were enrolled for the present study. PCR-SSCP protocol was utilised for screening of KCNQ1 and KCNE1 genes followed by In-silico and statistical analysis. The clinical profile of the Long QT syndrome patients in our study revealed a higher percentage of females with the mean age also being higher in females when compared to males. The two variations (S546S and IVS13+36A>G) in KCNQ1 and the S38G polymorphism in KCNE1 gene were identified and their association with Long QT syndrome is being reported for the first time in Indian population. S546S is located in the KCNQ1 C terminus close to this domain and IVS13+36A>G is located in the intronic region in close proximity to the coding region for C-terminal domain; these may therefore affect the functional protein through non-assembly. S38G leads to a substitution of serine to glycine at 38th amino acid position (S38G) in the transmembrane domain of KCNE1. Our study reports compound heterozygosity/genetic compound ofS546S and IVS13+36A>G of KCNQ1 gene. Haplotype frequencies and linkage disequilibrium analysis revealed a significant association between the three biomarkers. Compound heterozygosity of the polymorphisms influence downstream signalling and KCNQ1- KCNE1 interactions.