The p62 protein functions as a scaffold in signaling pathways that lead to activation of NF-B and is an important regulator of osteoclastogenesis. Mutations affecting the receptor activator of NF-B signaling axis can result in human skeletal disorders, including those identified in the C-terminal ubiquitin-associated (UBA) domain of p62 in patients with Paget disease of bone. These observations suggest that the disease may involve a common mechanism related to alterations in the ubiquitin-binding properties of p62. The structural basis for ubiquitin recognition by the UBA domain of p62 has been investigated using NMR and reveals a novel binding mechanism involving a slow exchange structural reorganization of the UBA domain to a "bound" noncanonical UBA conformation that is not significantly populated in the absence of ubiquitin. The repacking of the three-helix bundle generates a binding surface localized around the conserved Xaa-Gly-Phe-Xaa loop that appears to optimize both hydrophobic and electrostatic surface complementarity with ubiquitin. NMR titration analysis shows that the p62-UBA binds to Lys 48 -linked di-ubiquitin with ϳ4-fold lower affinity than to mono-ubiquitin, suggesting preferential binding of the p62-UBA to single ubiquitin units, consistent with the apparent in vivo preference of the p62 protein for Lys 63 -linked polyubiquitin chains (which adopt a more open and extended structure). The conformational switch observed on binding may represent a novel mechanism that underlies specificity in regulating signalinduced protein recognition events.The p62 protein (encoded by the sequestosome 1 (SQSTM1) gene) functions as a scaffold in signaling pathways downstream of the interleukin-1, tumor necrosis factor-␣, nerve growth factor, and receptor activator of NF-B 3 receptors, which ultimately lead to activation of the NF-B transcription factor, with receptor activator of NF-B signaling being a critical determinant in the regulation of osteoclast formation (1-3). Mice that are deficient in p62 show no obvious skeletal phenotype under normal conditions but exhibit defective osteoclastogenesis when challenged with bone-resorbing factors (4). Moreover, mutations affecting p62 are a common cause of Paget disease of bone (PDB), a condition associated with increased osteoclast and osteoblast activity (5-8). PDB is characterized by excessive bone turnover leading to bone expansion, structural weakness, deformity, and pain (9, 10).The p62 protein has a domain structure consistent with its participation in multiple signaling complexes, although p62 also appears to be multifunctional, not least in controlling protein recruitment to endosomes (3) and proteasomal proteolysis (11). Within the p62 sequence, an N-terminal PB1 domain has been identified that binds atypical protein kinase C. In addition, a ZZ motif is evident, a binding site for the RING finger protein TRAF6, and two PEST sequences that lie adjacent to the C-terminal ubiquitin-associated (UBA) domain (Fig. 1), a motif that occurs in enzymes of the ubiq...

Aggregation is a common problem affecting biopharmaceutical development that can have a significant effect on the quality of the product, as well as the safety to patients, particularly because of the increased risk of immune reactions. Here, we describe a new high-throughput screening algorithm developed to classify antibody molecules based on their propensity to aggregate. The tool, constructed and validated on experimental aggregation data for over 500 antibodies, is able to discern molecules with a high aggregation propensity as defined by experimental criteria relevant to bioprocessing and manufacturing of these molecules. Furthermore, we show how this tool can be combined with other computational approaches during early drug development to select molecules with reduced risk of aggregation and optimal developability properties.

Ubiquitin-associated (UBA) domain mutations of SQSTM1 are an important cause of Paget's disease of bone (PDB), which is a human skeletal disorder characterized by abnormal bone turnover. We previously showed that, when introduced into the full-length SQSTM1 protein, the disease-causing P392L, M404V, G411S, and G425R missense mutations and the E396X truncating mutation (representative of all of the SQSTM1 truncating mutations) cause a generalized loss of monoubiquitin binding and impaired K48-linked polyubiquitin binding at physiological temperature. Here, we show that the remaining three known PDB missense mutations, P387L, S399P, and M404T, have similar deleterious effects on monoubiquitin binding and K48-linked polyubiquitin binding by SQSTM1. The P387L mutation affects an apparently unstructured region at the N terminus of the UBA domain, some five residues from the start of the first helix, which is dispensable for polyubiquitin binding by the isolated UBA domain. Our findings support the proposal that the disease mechanism in PDB with SQSTM1 mutations involves a common loss of ubiquitin binding function of SQSTM1 and implicate a sequence extrinsic to the compact globular region of the UBA domain as a critical determinant of ubiquitin recognition by the full-length SQSTM1 protein.

In drug development, the “onus” of the low R&D efficiency has been put traditionally onto the drug discovery process (i.e., finding the right target or “binding” functionality). Here, we show that manufacturing is not only a central component of product success, but also that, by integrating manufacturing and discovery activities in a “holistic” interpretation of QbD methodologies, we could expect to increase the efficiency of the drug discovery process as a whole. In this new context, early risk assessment, using developability methodologies and computational methods in particular, can assist in reducing risks during development in a cost-effective way. We define specific areas of risk and how they can impact product quality in a broad sense, including essential aspects such as product efficacy and patient safety. Emerging industry practices around developability are introduced, including some specific examples of applications to biotherapeutics. Furthermore, we suggest some potential workflows to illustrate how developability strategies can be introduced in practical terms during early drug development in order to mitigate risks, reduce drug attrition and ultimately increase the robustness of the biopharmaceutical supply chain. Finally, we also discuss how the implementation of such methodologies could accelerate the access of new therapeutic treatments to patients in the clinic.

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