Chemical Translational Biology: Redefining Druggability of Protein-Protein Interactions.

Chemical biologists use chemical tools to answer biological questions. The translational application of these principles has led to an explosion in the discovery and druggability of new protein targets, including protein-protein interactions (PPIs). Proteins tend to interact with other macromolecules using relatively large and featureless binding surfaces, which has hampered traditional drug discovery efforts, particularly for interactions with weaker affinity. In this article, I discuss several emerging strategies for targeting PPIs, including computational and structural methods and novel screening approaches. In particular, I focus on hijacking intrinsic protein allosteric pathways for the discovery and design of small-molecule and peptide ligands.

H4K20me0 recognition by BRCA1-BARD1 directs homologous recombination to sister chromatids.

Genotoxic DNA double-strand breaks (DSBs) can be repaired by error-free homologous recombination (HR) or mutagenic non-homologous end-joining1. HR supresses tumorigenesis1, but is restricted to the S and G2 phases of the cell cycle when a sister chromatid is present2. Breast cancer type 1 susceptibility protein (BRCA1) promotes HR by antagonizing the anti-resection factor TP53-binding protein 1(53BP1) (refs. 2-5), but it remains unknown how BRCA1 function is limited to the S and G2 phases. We show that BRCA1 recruitment requires recognition of histone H4 unmethylated at lysine 20 (H4K20me0), linking DSB repair pathway choice directly to sister chromatid availability. We identify the ankyrin repeat domain of BRCA1-associated RING domain protein 1 (BARD1)-the obligate BRCA1 binding partner3-as a reader of H4K20me0 present on new histones in post-replicative chromatin6. BARD1 ankyrin repeat domain mutations disabling H4K20me0 recognition abrogate accumulation of BRCA1 at DSBs, causing aberrant build-up of 53BP1, and allowing anti-resection activity to prevail in S and G2. Consequently, BARD1 recognition of H4K20me0 is required for HR and resistance to poly (ADP-ribose) polymerase inhibitors. Collectively, this reveals that BRCA1-BARD1 monitors the replicative state of the genome to oppose 53BP1 function, routing only DSBs within sister chromatids to HR.

Heat Shock Protein 70 (Hsp70) Suppresses RIP1-Dependent Apoptotic and Necroptotic Cascades.

Hsp70 is a molecular chaperone that binds to "client" proteins and protects them from protein degradation. Hsp70 is essential for the survival of many cancer cells, but it is not yet clear which of its clients are involved. Using structurally distinct chemical inhibitors, we found that many of the well-known clients of the related chaperone, Hsp90, are not strikingly responsive to Hsp70 inhibition. Rather, Hsp70 appeared to be important for the stability of the RIP1 (RIPK1) regulators: cIAP1/2 (BIRC1 and BIRC3), XIAP, and cFLIPS/L (CFLAR). These results suggest that Hsp70 limits apoptosis and necroptosis pathways downstream of RIP1. Consistent with this model, MDA-MB-231 breast cancer cells treated with Hsp70 inhibitors underwent apoptosis, while cotreatment with z-VAD.fmk switched the cell death pathway to necroptosis. In addition, cell death in response to Hsp70 inhibitors was strongly suppressed by RIP1 knockdown or inhibitors. Thus, these data indicate that Hsp70 plays a previously unrecognized and important role in suppressing RIP1 activity.Implications: These findings clarify the role of Hsp70 in prosurvival signaling and suggest IAPs as potential new biomarkers for Hsp70 inhibition. Mol Cancer Res; 16(1); 58-68. ©2017 AACR.

X-linked inhibitor of apoptosis protein (XIAP) is a client of heat shock protein 70 (Hsp70) and a biomarker of its inhibition.

Heat shock protein 70 (Hsp70) and Hsp90 are molecular chaperones that play essential roles in tumor growth by stabilizing pro-survival client proteins. However, although the development of Hsp90 inhibitors has benefited from the identification of clients, such as Raf-1 proto-oncogene, Ser/Thr kinase (RAF1), that are particularly dependent on this chaperone, no equivalent clients for Hsp70 have been reported. Using chemical probes and MDA-MB-231 breast cancer cells, we found here that the inhibitors of apoptosis proteins, including c-IAP1 and X-linked inhibitor of apoptosis protein (XIAP), are obligate Hsp70 clients that are rapidly (within ∼3-12 h) lost after inhibition of Hsp70 but not of Hsp90. Mutagenesis and pulldown experiments revealed multiple Hsp70-binding sites on XIAP, suggesting that it is a direct, physical Hsp70 client. Interestingly, this interaction was unusually tight (∼260 nm) for an Hsp70-client interaction and involved non-canonical regions of the chaperone. Finally, we also found that Hsp70 inhibitor treatments caused loss of c-IAP1 and XIAP in multiple cancer cell lines and in tumor xenografts, but not in healthy cells. These results are expected to significantly accelerate Hsp70 drug discovery by providing XIAP as a pharmacodynamic biomarker. More broadly, our findings further suggest that Hsp70 and Hsp90 have partially non-overlapping sets of obligate protein clients in cancer cells.

Direct and Propagated Effects of Small Molecules on Protein-Protein Interaction Networks.

Networks of protein-protein interactions (PPIs) link all aspects of cellular biology. Dysfunction in the assembly or dynamics of PPI networks is a hallmark of human disease, and as such, there is growing interest in the discovery of small molecules that either promote or inhibit PPIs. PPIs were once considered undruggable because of their relatively large buried surface areas and difficult topologies. Despite these challenges, recent advances in chemical screening methodologies, combined with improvements in structural and computational biology have made some of these targets more tractable. In this review, we highlight developments that have opened the door to potent chemical modulators. We focus on how allostery is being used to produce surprisingly robust changes in PPIs, even for the most challenging targets. We also discuss how interfering with one PPI can propagate changes through the broader web of interactions. Through this analysis, it is becoming clear that a combination of direct and propagated effects on PPI networks is ultimately how small molecules re-shape biology.

Inhibitors of difficult protein-protein interactions identified by high-throughput screening of multiprotein complexes.

Protein-protein interactions (PPIs) are important in all aspects of cellular function, and there is interest in finding inhibitors of these contacts. However, PPIs with weak affinities and/or large interfaces have traditionally been more resistant to the discovery of inhibitors, partly because it is more challenging to develop high-throughput screening (HTS) methods that permit direct measurements of these physical interactions. Here, we explored whether the functional consequences of a weak PPI might be used as a surrogate for binding. As a model, we used the bacterial ATPase DnaK and its partners DnaJ and GrpE. Both DnaJ and GrpE bind DnaK and catalytically accelerate its ATP cycling, so we used stimulated nucleotide turnover to indirectly report on these PPIs. In pilot screens, we identified compounds that block activation of DnaK by either DnaJ or GrpE. Interestingly, at least one of these molecules blocked binding of DnaK to DnaJ, while another compound disrupted allostery between DnaK and GrpE without altering the physical interaction. These findings suggest that the activity of a reconstituted multiprotein complex might be used in some cases to identify allosteric inhibitors of challenging PPIs.

Sekikaic acid and lobaric acid target a dynamic interface of the coactivator CBP/p300.

Capturing a coactivator, naturally: the natural products sekikaic acid and lobaric acid, isolated after a high-throughput screen of a structurally diverse extract collection, effectively target the dynamic binding interfaces of the GACKIX domain of the coactivator CBP/p300. These molecules are the most effective inhibitors of the GACKIX domain yet described and are uniquely selective for this domain.

Specificity determinants of allosteric modulation in the neuronal nicotinic acetylcholine receptor: a fine line between inhibition and potentiation.

We are interested in the allosteric modulation of neuronal nicotinic acetylcholine receptors (nAChRs). We have postulated that the anthelmintic morantel (Mor) positively modulates (potentiates) rat α3ß2 receptors through a site located at the ß(+)/α(-) interface that is homologous to the canonical agonist site (J Neurosci 29:8734-8742, 2009). On this basis, we aimed to determine the site specificity by studying differences in modulation between α3ß2 and α4ß2 receptors. We also compared modulation by Mor with that of the related compound oxantel (Oxa). Whereas Mor and Oxa each potentiated α3ß2 receptors 2-fold at saturating acetylcholine (ACh) concentrations, Mor had no effect on α4ß2 receptors, and Oxa inhibited ACh-evoked responses. The inhibition was noncompetitive, but not due to open channel block. Furthermore, the nature and extent of modulation did not depend on subunit stoichiometry. We studied six positions at the α(-) interface that differ between α3 and α4. Two positions (α3Ile57 and α3Thr115) help mediate the effects of the modulators but do not seem to contribute to specificity. Mutations in two others (α3Leu107 and α3Ile117) yielded receptors with appreciable α4-character; that is, Mor potentiation was reduced compared with wild-type α3ß2 control and Oxa inhibition was evident. A fifth position (α3Glu113) was unique in that it discriminated between the two compounds, showing no change in Mor potentiation from control but substantial Oxa inhibition. Our work has implications for rational drug design for nicotinic receptors and sheds light on mechanisms of allosteric modulation in nAChRs, especially the subtle differences between potentiation and inhibition.