Elucidating the correct binding mode of drug-like compounds is crucial to reveal the molecular determinants underlying the recognition by the target protein and to estimate the binding affinity, thus guiding ensuing hit-to-lead optimization. However, identifying the near-native binding pose in docking experiments remains a major hurdle. Assuming that the hydropathic complementarity principle is the major driving force in ligand-protein recognition, the suitability of simple hydropathicity-based scoring functions to reveal the near-native pose was examined. A benchmarking dataset of 1000 ligand-protein complexes purposely designed to encompass bioactive and decoy poses was used to assess the performance of 3D hydropathicity atomic descriptors derived from empirical and quantum mechanical models. This strategy led to a predictive accuracy of ca. 90% when the non-negligible influence of the conformational stress is also considered. The results reveal the need to leverage an understanding of nonpolar/polar chemical features for the successful discrimination of the near-native pose.

Canonically known both for structural contributions to lipid bilayers and roles in cell signaling, the sphingolipids comprise a dynamic, multifaceted class of molecules which are studied to understand cell biology and pathophysiology. All sphingolipids are downstream products of the rate-limiting and initiating enzyme in the de novo sphingolipid synthesis pathway, serine palmitoyltransferase (SPT). SPT activity is strictly regulated. This regulation is accomplished through the ORMDLs, transmembrane polypeptides embedded in the lipid bilayer of the endoplasmic reticulum, which are the regulatory subunits of the SPT complex. Recently the specific mechanism of ORMDL's regulation of SPT was established: ceramide, a downstream product of the de novo biosynthetic pathway, binds directly to a binding site of ORMDL to induce an inhibitory conformational change. Here, we validate a computational docking approach to interrogate the binding efficiency of a range of sphingolipids in the ceramide binding site. We demonstrate that docking poses predicted by this in silico approach reflect experimental data on the efficiency of sphingolipid species to accomplish ORMDL-dependent inhibition of SPT. We propose that this docking analysis will be a valuable complement to experimental tests of compounds that bind to this site to regulate sphingolipid biosynthesis.

The amino acid sequence of the tryptophan-rich sensory proteins (TSPO) is substantially conserved throughout all kingdoms of life. Human mitochondrial TSPO1 (HsTSPO1) binds to porphyrins and steroids, although its interactions with these molecules remains unknown. HsTSPO1 is associated with numerous physiological and pathological disorders, but the underlying molecular mechanisms are unknown. Here, we disclose the finding of human mitochondrial TSPO as a cholesterol-dependent protoporphyrin IX oxygenase. The results of our biochemical characterization are consistent with structural data and evolutionary analysis. The dependence of HsTSPO1 activity on cholesterol may be the result of the coevolution of this membrane protein with the membrane system. Our study provides a molecular foundation for comprehending the various roles played by mitochondrial TSPO in normal physiological and pathological situations.

Deubiquitinases (DUBs) have long been viewed through the narrow lens of enzymatic catalysis, but emerging evidence reveals their non-catalytic domains as master regulators of oncogenic signaling. USP11, a structurally modular DUB, exemplifies this duality: beyond its canonical role in DNA repair, USP11 scaffolds key translational effectors such as eIF4B, sustaining the expression of pro-survival oncogenes in aggressive lymphomas. Here, we unveil RBF4 and RBF11, first-in-class, non-catalytic USP11 inhibitors discovered through pharmacophore-guided virtual screening anchored on the UBL domain interface. These small molecules selectively bind USP11 without disrupting its catalytic activity yet interrupt critical interactions essential for eIF4B stabilization and oncogenic translation. Mechanistically, USP11 inhibition collapses MYC-driven translational networks, destabilizes DNA repair factors, rewires calcium homeostasis, and induces a post-transcriptional apoptotic program while sparing non-malignant cells. RBF4, chemically identical to the FDA-approved anti-arrhythmic agent Dronedarone, exhibits potent antitumor efficacy in orthotopic EμMyc lymphoma models, suppressing tumor growth, metastatic spread, and ascites formation with no overt toxicity. Transcriptomic analyses reveal broad rewiring of EMT, immune, and metabolic programs, underscoring USP11’s role as a nodal regulator of tumor cell identity and plasticity. These findings establish the UBL domain of USP11 as a druggable scaffold, redefining DUBs not merely as enzymes but as structural signaling platforms. RBF4 emerges as a clinically actionable prototype for dismantling USP11-driven oncogenic circuits, illuminating a new therapeutic axis in lymphoid malignancies and beyond. One Sentence Summary Potent, selective USP11 inhibitors exhibit anti-tumor activity.

Triple-negative breast cancer (TNBC) is associated with a generally poor prognosis due to its highly aggressive and metastatic nature, lack of targetable receptors, as well as the frequent development of resistance to chemotherapy. We previously reported that AU1, a small molecule developed as an inhibitor of BPTF (bromodomain PHD finger-containing transcription factor), was capable of sensitizing preclinical models of TNBC to chemotherapy in part via the promotion of autophagy. In studies reported here, we identify an additional property of this compound, specifically that sensitization is associated with the inhibition of the P-glycoprotein (P-gp) efflux pump. In silico molecular docking studies indicate that AU1 binds to active regions of the efflux pump in a manner consistent with the inhibition of the pump function. This work identifies a novel chemical structure that can influence multidrug efflux, an established mechanism of drug resistance in TNBC, that has not yet been successfully addressed by clinical efforts.

A method is described to deconstruct the network of hydropathic interactions within and between a protein’s sidechain and its environment into residue-based three-dimensional maps. These maps encode favorable and unfavorable hydrophobic and polar interactions, in terms of spatial positions for optimal interactions, relative interaction strength, as well as character. In addition, these maps are backbone angle-dependent. After map calculation and clustering, a finite number of unique residue sidechain interaction maps exist for each backbone conformation, with the number related to the residue’s size and interaction complexity. Structures for soluble proteins (~749,000 residues) and membrane proteins (~387,000 residues) were analyzed, with the latter group being subdivided into three subsets related to the residue’s position in the membrane protein: soluble domain, core-facing transmembrane domain, and lipid-facing transmembrane domain. This work suggests that maps representing residue types and their backbone conformation can be reassembled to optimize the medium-to-high resolution details of a protein structure. In particular, the information encoded in maps constructed from the lipid-facing transmembrane residues appears to paint a clear picture of the protein–lipid interactions that are difficult to obtain experimentally.

The Department of Medicinal Chemistry, together with the Institute for Structural Biology, Drug Discovery and Development, at Virginia Commonwealth University (VCU) has evolved, organically with quite a bit of bootstrapping, into a unique drug discovery ecosystem in response to the environment and culture of the university and the wider research enterprise. Each faculty member that joined the department and/or institute added a layer of expertise, technology and most importantly, innovation, that fertilized numerous collaborations within the University and with outside partners. Despite moderate institutional support with respect to a typical drug discovery enterprise, the VCU drug discovery ecosystem has built and maintained an impressive array of facilities and instrumentation for drug synthesis, drug characterization, biomolecular structural analysis and biophysical analysis, and pharmacological studies. Altogether, this ecosystem has had major impacts on numerous therapeutic areas, such as neurology, psychiatry, drugs of abuse, cancer, sickle cell disease, coagulopathy, inflammation, aging disorders and others. Novel tools and strategies for drug discovery, design and development have been developed at VCU in the last five decades; e.g., fundamental rational structure-activity relationship (SAR)-based drug design, structure-based drug design, orthosteric and allosteric drug design, design of multi-functional agents towards polypharmacy outcomes, principles on designing glycosaminoglycans as drugs, and computational tools and algorithms for quantitative SAR (QSAR) and understanding the roles of water and the hydrophobic effect.

Sphingolipids are a diverse class of lipids with essential functions as determinants of membrane physical properties and as intra- and intercellular signaling agents. Disruption of the normal biochemical processes that establish the levels of individual sphingolipids is associated with a variety of human diseases including cancer, cardiovascular disease, metabolic disease, skin diseases, and lysosomal storage diseases. A unique aspect of this metabolic network is that there is a single enzymatic step that initiates the biosynthetic pathway for all sphingolipids. This step is catalyzed by the enzyme serine palmitoyltranserase (SPT). Under most circumstances SPT condenses serine and the 16-carbon acyl-CoA, palmitoyl-CoA to produce the precursor of all sphingolipids. SPT, a four-subunit protein complex, is subject to classic feedback regulation: when cellular sphingolipids are elevated, SPT activity is inhibited. Ceramide is the sphingolipid sensed by this system and it regulates SPT by directly binding to the complex. The ceramide binding site in the SPT complex, and how ceramide binding results in SPT inhibition, has now been determined in vertebrates, plants, and yeast using molecular modeling and cryo-electron microscopy. Here we discuss the similarities and differences revealed by these resolved structures and the surprising result that ceramide binds at almost identical positions in the SPT complex of these divergent organisms, but accomplishes SPT regulation in very different ways.

EDITORIAL article Front. Mol. Biosci., 15 September 2023Sec. Protein Biochemistry for Basic and Applied Sciences Volume 10 - 2023 | https://doi.org/10.3389/fmolb.2023.1289441

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.

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.

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.

A long-lasting goal of computational biochemists, medicinal chemists, and structural biologists has been the development of tools capable of deciphering the molecule-molecule interaction code that produces a rich variety of complex biomolecular assemblies comprised of the many different simple and biological molecules of life: water, small metabolites, cofactors, substrates, proteins, DNAs, and RNAs. Software applications that can mimic the interactions amongst all of these species, taking account of the laws of thermodynamics, would help gain information for understanding qualitatively and quantitatively key determinants contributing to the energetics of the bimolecular recognition process. This, in turn, would allow the design of novel compounds that might bind at the intermolecular interface by either preventing or reinforcing the recognition. HINT, hydropathic interaction, was a model and software code developed from a deceptively simple idea of Donald Abraham with the close collaboration with Glen Kellogg at Virginia Commonwealth University. HINT is based on a function that scores atom-atom interaction using LogP, the partition coefficient of any molecule between two phases; here, the solvents are water that mimics the cytoplasm milieu and octanol that mimics the protein internal hydropathic environment. This review summarizes the results of the extensive and successful collaboration between Abraham and Kellogg at VCU and the group at the University of Parma for testing HINT in a variety of different biomolecular interactions, from proteins with ligands to proteins with DNA.

Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, serves as a cofactor for scores of B6-dependent (PLP-dependent) enzymes involved in many cellular processes. One such B6 enzyme is dopa decarboxylase (DDC), which is required for the biosynthesis of key neurotransmitters, e.g., dopamine and serotonin. PLP-dependent enzymes are biosynthesized as apo-B6 enzymes and then converted to the catalytically active holo-B6 enzymes by Schiff base formation between the aldehyde of PLP and an active site lysine of the protein. In eukaryotes, PLP is made available to the B6 enzymes through the activity of the B6-salvage enzymes, pyridoxine 5′-phosphate oxidase (PNPO) and pyridoxal kinase (PLK). To minimize toxicity, the cell keeps the content of free PLP (unbound) very low through dephosphorylation and PLP feedback inhibition of PNPO and PLK. This has led to a proposed mechanism of complex formation between the B6-salvage enzymes and apo-B6 enzymes prior to the transfer of PLP, although such complexes are yet to be characterized at the atomic level, presumably due to their transient nature. A computational study, for the first time, was used to predict a likely PNPO and DDC complex, which suggested contact between the allosteric PLP tight-binding site on PNPO and the active site of DDC. Using isothermal calorimetry and/or surface plasmon resonance, we also show that PNPO binds both apoDDC and holoDDC with dissociation constants of 0.93 ± 0.07 μM and 2.59 ± 0.11 μM, respectively. Finally, in the presence of apoDDC, the tightly bound PLP on PNPO is transferred to apoDDC, resulting in the formation of about 35% holoDDC.

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.

Evaluation of the intramolecular stability of proteins plays a key role in the comprehension of their biological behavior and mechanism of action. Small structural alterations such as mutations induced by single nucleotide polymorphism can impact biological activity and pharmacological modulation. Covid-19 mutations, that affect viral replication and the susceptibility to antibody neutralization, and the action of antiviral drugs, are just one example. In this work, the intramolecular stability of mutated proteins, like Spike glycoprotein and its complexes with the human target, is evaluated through hydropathic intramolecular energy scoring originally conceived by Abraham and Kellogg based on the “Extension of the fragment method to calculate amino acid zwitterion and side-chain partition coefficients” by Abraham and Leo in Proteins : Struct. Funct. Genet. 1987, 2:130-52. HINT is proposed as a fast and reliable tool for the stability evaluation of any mutated system. This work has been written in honor of Prof. Donald J. Abraham (1936-2021).

Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and important regulator of the DNA damage response (DDR). One critical ATM target is the structural subunit A (PR65-S401) of protein phosphatase 2A (PP2A), known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylating many proteins during the recovery from the DDR. We generated mouse embryonic fibroblasts expressing PR65-WT, -S401A (cannot be phosphorylated), and -S401D (phospho-mimetic) transgenes. Significantly, S401 mutants exhibited extensive chromosomal aberrations, impaired DNA double-strand break (DSB) repair and underwent increased mitotic catastrophe after radiation. Both S401A and the S401D cells showed impaired DSB repair (nonhomologous end joining and homologous recombination repair) and exhibited delayed DNA damage recovery, which was reflected in reduced radiation survival. Furthermore, S401D cells displayed increased ERK and AKT signaling resulting in enhanced growth rate further underscoring the multiple roles ATM-PP2A signaling plays in regulating prosurvival responses. Time-lapse video and cellular localization experiments showed that PR65 was exported to the cytoplasm after radiation by CRM1, a nuclear export protein, in line with the very rapid pleiotropic effects observed. A putative nuclear export sequence (NES) close to S401 was identified and when mutated resulted in aberrant PR65 shuttling. Our study demonstrates that the phosphorylation of a single, critical PR65 amino acid (S401) by ATM fundamentally controls the DDR, and balances DSB repair quality, cell survival and growth by spatiotemporal PR65 nuclear-cytoplasmic shuttling mediated by the nuclear export receptor CRM1.

Three-dimensional (3D) maps of the hydropathic environments of protein amino acid residues are information-rich descriptors of preferred conformations, interaction types and energetics, and solvent accessibility. The interactions made by each residue are the primary factor for rotamer selection and the secondary, tertiary, and even quaternary protein structure. Our evolving basis set of environmental data for each residue type can be used to understand the protein structure. This work focuses on the aromatic residues phenylalanine, tyrosine, and tryptophan and their structural roles. We calculated and analyzed side chain-to-environment 3D maps for over 70,000 residues of these three types that reveal, with respect to hydrophobic and polar interactions, the environment around each. After binning with backbone ϕ/ψ and side chain χ1, we clustered each bin by 3D similarities between map-map pairs. For each of the three residue types, four bins were examined in detail: one in the β-pleat, two in the right-hand α-helix, and one in the left-hand α-helix regions of the Ramachandran plot. For high degrees of side chain burial, encapsulation of the side chain by hydrophobic interactions is ubiquitous. The more solvent-exposed side chains are more likely to be involved in polar interactions with their environments. Evidence for π-π interactions was observed in about half of the residues surveyed [phenylalanine (PHE): 53.3%, tyrosine (TYR): 34.1%, and tryptophan (TRP): 55.7%], but on an energy basis, this contributed to only ∼4% of the total. Evidence for π-cation interactions was observed in 14.1% of PHE, 8.3% of TYR, and 26.8% of TRP residues, but on an energy basis, this contributed to only ∼1%. This recognition of even these subtle interactions in the 3D hydropathic environment maps is key support for our interaction homology paradigm of protein structure elucidation and possibly prediction.

Atomic-resolution protein structural models are prerequisites for many downstream activities like structurefunction studies or structure-based drug discovery. Unfortunately, this data is often unavailable for some of the most interesting and therapeutically important proteins. Thus, computational tools for building native-like structural models from less-than-ideal experimental data are needed. To this end, interaction homology exploits the character, strength and loci of the sets of interactions that define a structure. Each residue type has its own limited set of backbone angle-dependent interaction motifs, as defined by their environments. In this work, we characterize the interactions of serine, cysteine and S-bridged cysteine in terms of 3D hydropathic environment maps. As a result, we explore several intriguing questions. Are the environments different between the isosteric serine and cysteine residues? Do some environments promote the formation of cystine S-S bonds? With the increasing availability of structural data for water-insoluble membrane proteins, are there environmental differences for these residues between soluble and membrane proteins? The environments surrounding serine and cysteine residues are dramatically different: serine residues are about 50% solvent exposed, while cysteines are only 10% exposed; the latter are more involved in hydrophobic interactions although there are backbone angledependent differences. Our analysis suggests that one driving force for -S-S- bond formation is a rather substantial increase in burial and hydrophobic interactions in cystines. Serine and cysteine become less and more, respectively, solvent-exposed in membrane proteins. 3D hydropathic environment maps are an evolving structure analysis tool showing promise as elements in a new protein structure prediction paradigm.