Synthesis and Biological Activity of 3-(Heteroaryl)quinolin-2(1H)-ones Bis-Heterocycles as Potential Inhibitors of the Protein Folding Machinery Hsp90

In the context of our SAR study concerning 6BrCaQ analogues as C-terminal Hsp90 inhibitors, we designed and synthesized a novel series of 3-(heteroaryl)quinolin-2(1H), of types 3, 4, and 5, as a novel class of analogues. A Pd-catalyzed Liebeskind–Srogl cross-coupling was developed as a convenient approach for easy access to complex purine architectures. This series of analogues showed a promising biological effect against MDA-MB231 and PC-3 cancer cell lines. This study led to the identification of the best compounds, 3b (IC50 = 28 µM) and 4e, which induce a significant decrease of CDK-1 client protein and stabilize the levels of Hsp90 and Hsp70 without triggering the HSR response.

14–3-3 protein regulation of excitation–contraction coupling

14–3-3 proteins (14–3-3 s) are a family of highly conserved proteins that regulate many cellular processes in eukaryotes by interacting with a diverse array of client proteins. The 14–3-3 proteins have been implicated in several disease states and previous reviews have condensed the literature with respect to their structure, function, and the regulation of different cellular processes. This review focuses on the growing body of literature exploring the important role 14–3-3 proteins appear to play in regulating the biochemical and biophysical events associated with excitation–contraction coupling (ECC) in muscle. It presents both a timely and unique analysis that seeks to unite studies emphasizing the identification and diversity of 14–3-3 protein function and client protein interactions, as modulators of muscle contraction. It also highlights ideas within these two well-established but intersecting fields that support further investigation with respect to the mechanistic actions of 14–3-3 proteins in the modulation of force generation in muscle.

Electrostatics cause the molecular chaperone BiP to preferentially bind oligomerized states of a client protein

Hsp70-family chaperones bind short monomeric peptides with a weak characteristic affinity in the low micromolar range, but can also bind some aggregates, fibrils, and amyloids, with low nanomolar affinity. While this differential affinity enables Hsp70 to preferentially target potentially toxic aggregates, it is unknown how Hsp70s differentiate between monomeric and oligomeric states of a target protein. Here we examine the interaction of BiP (the Hsp70 paralog in the endoplasmic reticulum) with proIGF2, the pro-protein form of IGF2 that includes a long and mostly disordered E-peptide region that promotes proIGF2 oligomerization. We discover that electrostatic attraction enables the negatively charged BiP to bind positively charged E-peptide oligomers with low nanomolar affinity. We identify the specific BiP binding sites on proIGF2, and although some are positively charged, as monomers they bind BiP with characteristically low affinity in the micromolar range. We conclude that electrostatics enable BiP to preferentially recognize oligomeric states of proIGF2. Electrostatic targeting of Hsp70 to aggregates may be broadly applicable, as all the currently-documented cases in which Hsp70 binds aggregates with high-affinity involve clients that are expected to be positively charged.

3D-QSAR and Molecular Docking Approaches for the Identification of Phyto-Inhibitors of Hsp90

Inhibition of Hsp90 disrupts the Hsp90 client protein complex, resulting in its breakdown. Phytochemicals from reported anticancer plants were screened against the orthosteric site of Hsp90. The lead compounds were subjected to the Lipinski rule of five to evaluate their drug-likeness. Three-Dimensional Quantitative Structure-Activity Relationships (3D-QSAR), a mathematical model for the inhibition of Hsp90, was also derived. The lead compounds are guaiol from Cannabis sativa, actinidine from Anacadium occidentale, and choline from Tinospora cordifolia with docking scores of -11kcal/mol, -12.1kcal/mol, and -10.8kcal/mol, respectively. The 3D-QSAR model generated is robust and thoroughly validated with a correlation coefficient R of 0.94 and R2 of 0.950. Actinidine, choline and, guaiol are novel and potent inhibitors of Hsp90. They form interactions with key amino acid residues within the Hsp90 orthosteric site.

Phylogenetic evidence for asparagine to aspartic acid protein editing of N-glycosylated SARS-CoV-2 viral proteins by NGLY1 deglycosylation/deamidation suggests an unusual vaccination strategy

Many viral proteins, including multiple SARS-CoV-2 proteins, are secreted via the endoplasmic reticulum, and viral particles are assembled and exported in ER-associated replication compartments. Viral coat proteins such as the SARS-CoV-2 Spike protein are N-glycosylated at NxS/T sites as they enter the ER. N-glycosylated sites in many eukaryotic proteins are deglycosylated by the NGLY1/PNG-1 deglycosylation enzyme which also deamidates the N-glycosylated asparagine to aspartic acid, thus editing the target protein sequence. Proteomic analysis of mammalian cell lines has revealed deamidation of many host N-glycosylated asparagines to aspartic acid by NGLY1/PNG-1 on peptides that are presented by mammalian HLA for immune surveillance. The key client protein for NGLY1/PNG-1 deglycosylation and N to D protein editing was revealed by genetic analysis of C. elegans proteasome regulation to be the intact endoplasmic reticulum-transiting SKN-1A transcription factor. Strikingly, an analysis of cancer cell genetic dependencies for growth revealed that the mammalian orthologue of SKN-1A, NRF1 (also called NFE2L1) is required by a highly correlated set of cell lines as NGLY1/PNG-1, supporting that NGLY1/PNG-1 and NRF1 act in the same pathway. NGLY1/PNG-1 edits N-glycosylated asparagines on the intact SKN-1 protein as it is retrieved by ERAD from the ER to in turn activate the transcription of target proteasomal genes. The normal requirement for NGLY1/PNG-1 editing of SKN-1A can be bypassed by a genomic substituion of N to D in four NxS/T N-glycosylation motifs of SKN-1A. Thus NGLY1/PNG-1-mediated N to D protein editing is more than a degradation step for the key client protein for proteasomal homeostasis in C. elegans or tumor growth in particular mammalian cell lines, SKN-1A/NRF1. In addition, such N to D substitutions in NxS/T N-glycosylation motifs occur in evolution: N to D substitutions are observed in phylogenetic comparisons of SKN-1A between nematode species that diverged hundreds of millions of years ago or of the vertebrate NRF1 between disparate vertebrates. Genomic N to D mutations bypass the many steps in N-glycosylation in the ER and deglycosylation-based editing of N to D, perhaps based on differences in the competency of divergent species for various N-glycosylation or deglycosylation steps.We surveyed the N-glycosylation sites in coronavirus proteins for such phylogenetic evidence for N to D protein editing in viral life cycles, and found evidence for preferential N to D residue substitutions in NxS/T N-glycosylation sites in comparisons of the genome sequences of hundreds of coronaviruses. This suggests that viruses use NGLY1/PNG-1 in some hosts, for example humans, to edit particular N-glycosylated residues to aspartic acid, but that in other hosts, often in bats, an N to D substitution mutation in the virus genome is selected. Single nucleotide mutations in Asp or Asn codons can produce viruses with N to D or D to N substitutions that might be selected in different animal hosts from the population of viral variants produced in any previous host. NGLY1/PNG-1 has been implicated in viral immunity in mammalian cell culture, favoring this hypothesis.Because of the phylogenetic evidence that the NGLY1/PNG-1 editing of protein sequences has functional importance for SKN-1A/NRF1 and viruses, and because most immunization protocols do not address the probable editing and functional importance of N-glycosylated aspargines to aspartic acid in normal viral infections, we suggest that immunization with viral proteins engineered to substitute D at genomically encoded NxS/T sites of N-glycosylated viral proteins that show a high frequency of N to D substitution in viral phylogeny may enhance immunological response to peptide antigens. Such genomically-edited peptides would not require ER-localization for N-glycosylation or other cell compartment localization for NGLY1/PNG-1 N to D protein editing. In addition, such N to D edited protein vaccines could be produced in bacteria since N-glycosylation and deglycosylation which do not occur in bacteria would no longer be required to immunize with a D-substituted peptide. Bacterially-expressed vaccines would be much lower cost and with fewer failure modes than attenuated viral vaccines or recombinant animal viruses produced in chicken eggs, mammalian tissue culture cells, or delivered by mRNA vectors to the patient directly. Because N to D edited peptides are clearly produced by NGLY1/PNG-1, and may be and presented by mammalian HLA, such peptides may more robustly activate T-cell killing or B-cell maturation to mediate more robust viral immunity.

Redefining Molecular Chaperones as Chaotropes

Molecular chaperones are the key instruments of bacterial protein homeostasis. Chaperones not only facilitate folding of client proteins, but also transport them, prevent their aggregation, dissolve aggregates and resolve misfolded states. Despite this seemingly large variety, single chaperones can perform several of these functions even on multiple different clients, thus suggesting a single biophysical mechanism underlying. Numerous recently elucidated structures of bacterial chaperone–client complexes show that dynamic interactions between chaperones and their client proteins stabilize conformationally flexible non-native client states, which results in client protein denaturation. Based on these findings, we propose chaotropicity as a suitable biophysical concept to rationalize the generic activity of chaperones. We discuss the consequences of applying this concept in the context of ATP-dependent and -independent chaperones and their functional regulation.

HSP-90/kinase complexes are stabilized by the large PPIase FKB-6

Protein kinases are important regulators in cellular signal transduction. As one major type of Hsp90 client, protein kinases rely on the ATP-dependent molecular chaperone Hsp90, which maintains their structure and supports their activation. Depending on client type, Hsp90 interacts with different cofactors. Here we report that besides the kinase-specific cofactor Cdc37 large PPIases of the Fkbp-type strongly bind to kinase•Hsp90•Cdc37 complexes. We evaluate the nucleotide regulation of these assemblies and identify prominent interaction sites in this quaternary complex. The synergistic interaction between the participating proteins and the conserved nature of the interaction suggests functions of the large PPIases Fkbp51/Fkbp52 and their nematode homolog FKB-6 as contributing factors to the kinase cycle of the Hsp90 machinery.

Snapshots of actin and tubulin folding inside the TRiC chaperonin

The integrity of a cell’s proteome depends on correct folding of polypeptides by chaperonins. The TCP-1 ring chaperonin (TRiC) acts as obligate folder for >10% of cytosolic proteins, including cytoskeletal proteins actin and tubulin. While its architecture and how it recognises folding substrates is emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and co-chaperone (PhLP2A) at different folding stages, for structure determination by cryogenic electron microscopy. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions towards the central space to achieve their folding. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Furthermore, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging atomistic model of client protein folding through TRiC.

Novel Small Molecule Hsp90/Cdc37 Interface Inhibitors Indirectly Target K-Ras-Signaling

The ATP-competitive inhibitors of Hsp90 have been tested predominantly in kinase addicted cancers; however, they have had limited success. A mechanistic connection between Hsp90 and oncogenic K-Ras is not known. Here, we show that K-Ras selectivity is enabled by the loss of the K-Ras membrane nanocluster modulator galectin-3 downstream of the Hsp90 client HIF-1α. This mechanism suggests a higher drug sensitivity in the context of KRAS mutant, HIF-1α-high and/or Gal3-high cancer cells, such as those found, in particular, in pancreatic adenocarcinoma. The low toxicity of conglobatin further indicates a beneficial on-target toxicity profile for Hsp90/Cdc37 interface inhibitors. We therefore computationally screened >7 M compounds, and identified four novel small molecules with activities of 4 μM–44 μM in vitro. All of the compounds were K-Ras selective, and potently decreased the Hsp90 client protein levels without inducing the heat shock response. Moreover, they all inhibited the 2D proliferation of breast, pancreatic, and lung cancer cell lines. The most active compounds from each scaffold, furthermore, significantly blocked 3D spheroids and the growth of K-Ras-dependent microtumors. We foresee new opportunities for improved Hsp90/Cdc37 interface inhibitors in cancer and other aging-associated diseases.