The androgen receptor is a major driver of prostate cancer and inhibition of its transcriptional activity using competitive antagonists, such as enzalutamide remains a frontline therapy for prostate cancer management. However, the majority of patients eventually develop drug resistance. We propose that targeting the androgen receptor for degradation via Proteolysis Targeting Chimeras (PROTACs) will be a better therapeutic strategy for targeting androgen receptor signaling in prostate cancer cells. Here we perform a head-to-head comparison between a currently approved androgen receptor antagonist enzalutamide, and its PROTAC derivative, ARCC-4, across different cellular models of prostate cancer drug resistance. ARCC-4 is a low-nanomolar androgen receptor degrader able to degrade about 95% of cellular androgen receptors. ARCC-4 inhibits prostate tumor cell proliferation, degrades clinically relevant androgen receptor point mutants and unlike enzalutamide, retains antiproliferative effect in a high androgen environment. Thus, ARCC-4 exemplifies how protein degradation can address the drug resistance hurdles of enzalutamide.
Of late, targeted protein degradation (TPD) has surfaced as a novel and innovative chemical tool and therapeutic modality. By co-opting protein degradation pathways, TPD facilitates complete removal of the protein molecules from within or outside the cell. While the pioneering Proteolysis-Targeting Chimera (PROTAC) technology and molecular glues hijack the ubiquitin-proteasome system, newer modalities co-opt autophagy or the endo-lysosomal pathway. Using this mechanism, TPD is posited to largely expand the druggable space far beyond small-molecule inhibitors. In this review, we discuss the major advances in TPD, highlight our current understanding, and explore outstanding questions in the field.
Over 300 BRAF missense mutations have been identified in patients, yet currently approved drugs target V600 mutants alone. Moreover, acquired resistance inevitably emerges, primarily due to RAF lesions that prevent inhibition of BRAF V600 with current treatments. Therefore, there is a need for new therapies that target other mechanisms of activated BRAF. In this study, we use the Proteolysis Targeting Chimera (PROTAC) technology, which promotes ubiquitination and degradation of neo-substrates, to address the limitations of BRAF inhibitor-based therapies. Using vemurafenib-based PROTACs, we achieve low nanomolar degradation of all classes of BRAF mutants, but spare degradation of WT RAF family members. Our lead PROTAC outperforms vemurafenib in inhibiting cancer cell growth and shows in vivo efficacy in a Class 2 BRAF xenograft model. Mechanistic studies reveal that BRAFWT is spared due to weak ternary complex formation in cells owing to its quiescent inactivated conformation, and activation of BRAFWT sensitizes it to degradation. This study highlights the degree of selectivity achievable with degradation-based approaches by targeting mutant BRAF-driven cancers while sparing BRAFWT, providing an anti-tumor drug modality that expands the therapeutic window.
Christianson syndrome (CS), an X-linked neurological disorder characterized by postnatal attenuation of brain growth (postnatal microcephaly), is caused by mutations in SLC9A6, the gene encoding endosomal Na+/H+ exchanger 6 (NHE6). To hasten treatment development, we established induced pluripotent stem cell (iPSC) lines from patients with CS representing a mutational spectrum, as well as biologically related and isogenic control lines. We demonstrated that pathogenic mutations lead to loss of protein function by a variety of mechanisms: The majority of mutations caused loss of mRNA due to nonsense-mediated mRNA decay; however, a recurrent, missense mutation (the G383D mutation) had both loss-of-function and dominant-negative activities. Regardless of mutation, all patient-derived neurons demonstrated reduced neurite growth and arborization, likely underlying diminished postnatal brain growth in patients. Phenotype rescue strategies showed mutation-specific responses: A gene transfer strategy was effective in nonsense mutations, but not in the G383D mutation, wherein residual protein appeared to interfere with rescue. In contrast, application of exogenous trophic factors (BDNF or IGF-1) rescued arborization phenotypes across all mutations. These results may guide treatment development in CS, including gene therapy strategies wherein our data suggest that response to treatment may be dictated by the class of mutation.
Polynucleotide phosphorylase (PNPase), a 3-to-5 phosphorolytic exoribonuclease, is thought to be the primary enzyme responsible for turnover of Bacillus subtilis mRNA. The role of PNPase in B. subtilis mRNA decay has been analyzed previously by comparison of mRNA profiles in a wild-type strain versus a strain that is deleted for pnpA, the gene encoding PNPase. Bacterial mRNA turnover is an essential process that replenishes the ribonucleotide pool and plays a role in regulation of gene expression. We have been studying 3Ј-exonuclease activity in Bacillus subtilis, the Gram-positive model organism. Although at least four 3Ј-exonucleases are known to exist in B. subtilis (1), the phosphorolytic enzyme, polynucleotide phosphorylase (PNPase, 3 encoded by the pnpA gene), has long been thought to play the major role in mRNA turnover in this organism (2, 3). The B. subtilis pnpA gene was originally identified in a transposon insertion screen of competence-deficient mutants, and was also shown to be required for cold adaptation (4). A deletion strain lacking most of the pnpA coding sequence (the ⌬pnpA strain) was constructed, and additional phenotypes were observed: growth as non-motile chains, tetracycline sensitivity (5), and accumulation of mRNA decay intermediates (1, 6). More recently, reliance of global mRNA turnover on PNPase was examined in an RNA sequencing analysis of the B. subtilis transcriptome in wild-type and ⌬pnpA strains, and this demonstrated the broad impact of PNPase on mRNA turnover (7). Many of the above mentioned studies were performed before the discovery of RNase Y, a B. subtilis endoribonuclease that appears to function in initiation of decay similarly to the more well known RNase E of Escherichia coli (8,9). Endonucleolytic cleavage in the body of a message by RNase E in E. coli or RNase Y in B. subtilis generates mRNA fragments that are susceptible to further steps in mRNA turnover. In addition, just as RNase E of E. coli coordinates a complex of several proteins known as the "degradosome," so, too, there is evidence that RNase Y of B. subtilis is the organizing protein for a complex that includes, at least, PNPase, CshA (an RNA helicase), and the glycolytic enzymes, enolase and phosphofructokinase (8,10,11). A PNPase-RNase Y interaction was first indicated by bacterial-2-hybrid (B2H) assay and in vivo protein crosslinking experiments (8). This interaction has more recently been confirmed by surface plasmon resonance analysis, which showed a strong PNPase-RNase Y interaction with a K d of 5 nM (12). Structural mapping of regions of PNPase that interact with an RNase E peptide has been done for the E. coli enzyme (13). Structural studies have not been reported for the B. subtilis PNPaseRNase Y interaction.The existence of a PNPase-RNase Y complex raises the question of whether this interaction is relevant to the ability of PNPase to rapidly degrade upstream decay intermediates that are created by RNase Y cleavage. In addition, prior results of experiments in the strain that contained no PNPase...
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