No abstract

Aspartic acid, glutamic acid and histidine are ionizable residues occupying various protein environments and perform many different functions in structures. Their roles are tied to their acid/base equilibria, solvent exposure, and backbone conformations. We propose that the number of unique environments for ASP, GLU and HIS is quite limited. We generated maps of these residue's environments using a hydropathic scoring function to record the type and magnitude of interactions for each residue in a 2703-protein structural dataset. These maps are backbone-dependent and suggest the existence of new structural motifs for each residue type. Additionally, we developed an algorithm for tuning these maps to any pH, a potentially useful element for protein design and structure building. Here, we elucidate the complex interplay between secondary structure, relative solvent accessibility, and residue ionization states: the degree of protonation for ionizable residues increases with solvent accessibility, which in turn is notably dependent on backbone structure.

Background Deregulated translation initiation is implicated extensively in cancer initiation and progression. It is actively pursued as a viable target that circumvents the dependency on oncogenic signaling, a significant factor in current strategies. Eukaryotic translation initiation factor (eIF) 4A plays an essential role in translation initiation by unwinding the secondary structure of messenger RNA (mRNA) upstream of the start codon, enabling active ribosomal recruitment on the downstream genes. Several natural product molecules with similar scaffolds, such as Rocaglamide A (RocA), targeting eIF4A have been reported in the last decade. However, their clinical utilization is still elusive due to several pharmacological limitations. In this study we identified new eIF4A1 inhibitors and their possible mechanisms. Methods In this report, we conducted a pharmacophore-based virtual screen of RocA complexed with eIF4A and a polypurine RNA strand for novel eIF4A inhibitors from commercially available compounds in the MolPort Database. We performed target-based screening and optimization of active pharmacophores. We assessed the effects of novel compounds on biochemical and cell-based assays for efficacy and mechanistic evaluation. Results We validated three new potent eIF4A inhibitors, RBF197, RBF 203, and RBF 208, which decreased diffuse large B-cell lymphoma (DLBCL) cell viability. Biochemical and cellular studies, molecular docking, and functional assays revealed that thosenovel compounds clamp eIF4A into mRNA in an ATP-independent manner. Moreover, we found that RBF197 and RBF208 significantly depressed eIF4A-dependent oncogene expression as well as the colony formation capacity of DLBCL. Interestingly, exposure of these compounds to non-malignant cells had only minimal impact on their growth and viability. Conclusions Identified compounds suggest a new strategy for designing novel eIF4A inhibitors.

Graphical abstract

The aliphatic hydrophobic amino acid residues—alanine, isoleucine, leucine, proline and valine—are among the most common found in proteins. Their structural role in proteins is seemingly obvious: engage in hydrophobic interactions to stabilize secondary, and to a lesser extent, tertiary and quaternary structure. However, favorable hydrophobic interactions involving the sidechains of these residue types are generally less significant than the unfavorable set arising from interactions with polar atoms. Importantly, the constellation of interactions between residue sidechains and their environments can be recorded as three-dimensional maps that, in turn, can be clustered. The clustered average map sets compose a library of interaction profiles encoding interaction strengths, interaction types and the optimal 3D position for the interacting partners. This library is backbone angle-dependent and suggests solvent and lipid accessibility for each unique interaction profile. In this work, in addition to analysis of soluble proteins, a large set of membrane proteins that contained optimized artificial lipids were evaluated by parsing the structures into three distinct components: soluble extramembrane domain, lipid facing transmembrane domain, core transmembrane domain. The aliphatic residues were extracted from each of these sets and passed through our calculation protocol. Notable observations include: the roles of aliphatic residues in soluble proteins and in the membrane protein’s soluble domains are nearly identical, although the latter are slightly more solvent accessible; by comparing maps calculated with sidechain-lipid interactions to maps ignoring those interactions, the potential extent of residue-lipid and residue-interactions can be assessed and likely exploited in structure prediction and modeling; amongst these residue types, the levels of lipid engagement show isoleucine as the most engaged, while the other residues are largely interacting with neighboring helical residues.

Human sirtuins play important roles in various cellular events including DNA repair, gene silencing, mitochondrial biogenesis, insulin secretion and apoptosis. They regulate a wide array of protein and enzyme targets through their NAD+‐dependent deacetylase activities. Sirtuins are also thought to mediate the beneficial effects of low‐calorie intake to extend longevity in diverse organisms from yeast to mammals. Small molecules mimicking calorie restriction to stimulate sirtuin activity are attractive therapeutics against age‐related disorders such as cardiovascular diseases, diabetes and neurodegeneration. Little is known about one of the mitochondrial sirtuins, SIRT5. SIRT5 has emerged as a critical player in maintaining cardiac health and neuronal viability upon stress and functions as a tumour suppressor in a context‐specific manner. Much has been debated about whether SIRT5 has evolved away from being a deacetylase because of its weak catalytic activity, especially in the in vitro testing. We have, for the first time, identified a SIRT5‐selective allosteric activator, nicotinamide riboside (NR). It can increase SIRT5 catalytic efficiency with different synthetic peptide substrates. The mechanism of action was further explored using a combination of molecular biology and biochemical strategies. Based on the existing structural biology information, the NR binding site was also mapped out. These activators are powerful chemical probes for the elucidation of cellular regulations and biological functions of SIRT5. The knowledge gained in this study can be used to guide the design and synthesis of more potent, isotype‐selective SIRT5 activators and to develop them into therapeutics for metabolic disorders and age‐related diseases.

Ubiquitin-mediated events are emerging as the gatekeeper in cell proliferation, and the recognition of deubiquitinating enzymes (DUBs) as the critical regulators of these processes is rapidly growing. Several studies in the last decade confirmed that the USP11 (Ubiquitin-Specific proteases 11) had promoted tumor growth and metastasis. Indeed, pan-cancer data analysis reveals enhanced expression of USP11 in numerous malignancies, including lymphoma. Our lab has established that USP11 deubiquitinates and stabilizes the translation initiation factor eIF4B (Eukaryotic Initiation Factor 4B) to promote eIF4B-dependent oncogenic translation and to facilitate the proliferation of diffuse large B-cell lymphoma (DLBCL). Brody and colleagues screened 2000 FDA- approved compounds and reported that the anti-neoplastic agent mitoxantrone is an effective non-specific inhibitor of USP11, which was later observed to target USP15. Thus, catalytically targeting USP11 provides substantial challenges as it encompasses high homology with the functional orthologs USP4 and USP15. Addressing this major limitation, Spiliotopoulos et al. identified a unique USP11-specific allosteric site without impeding USP15 and USP4 activities. Utilizing this platform, we performed a virtual screening of more than 10 million compounds from publicly available databases and identified 307 potential unique hits. The chemical library of these hits was assessed using an in-house optimized in-cell high throughput screening assay. The primary screening revealed that 16 of the hit compounds with EC50 less than 10μM. The two most potent, structurally different, USP11 inhibitors, RBF4 and RBF11, were further evaluated. In-vitro deubiquitinase activity assay of USP11 showed minimal inhibition on treatment with compounds (as we are targeting an allosteric site), which indicates that RBF4 and 11 do not bind to the catalytic domain. Thermal shift assays revealed that RBF4 preferentially stabilizes USP11 compared to other functional paralogues USP4 and USP15, which validates the selective USP11-RBF4 interaction. Further, treatment with RBF4 and 11 hampered DLBCL proliferation in a dose-dependent manner. Significantly, treatment with selected compounds depleted USP11-dependent oncogenic expression and, thus, the colony-forming capacity of DLBCL. These results show that these novel compounds can serve as an ideal tool to increase our understanding of USP11 biology and may emerge as an example of potent and selective USP11 inhibitors. Currently, we are evaluating their anti-cancer potency in pre-clinical models. Citation Format: Forum Kayastha, Bandish Kapadia, Noah B. Herrington, Anirban Roychowdhury, Glen E. Kellogg, Ronald B. Gartenhaus. Discovery, development and characterization of potent and selective USP11 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB023.