Development of Allosteric Small Molecule APOBEC3B Inhibitors from In Silico Screening.

APOBEC3B cytosine deaminase contributes to the mutational burdens of tumors, resulting in tumor progression and therapy resistance. Small molecule APOBEC3B inhibitors have potential to slow or mitigate these detrimental outcomes. Through molecular dynamics (MD) simulations and computational solvent mapping analysis, we identified a novel putative allosteric pocket on the C-terminal domain of APOBEC3B (A3Bctd), and virtually screened the ChemBridge Diversity Set (N~110,000) against both the active and potential allosteric sites. Selected high-scoring compounds were subsequently purchased, characterized for purity and composition, and tested in biochemical assays, which yielded 13 hit compounds. Orthogonal NMR assays verified binding to the target protein. Initial selectivity studies suggest these compounds preferentially target A3Bctd over related deaminase APOBEC3A (A3A), and MD simulations indicate this selectivity may be due to the steric repulsion from H56 that is unique to A3A. Taken together, our studies represent the first virtual screening effort against A3Bctd that has yielded candidate inhibitors suitable for further development.

Binding and Activation of LRP1-Dependent Cell Signaling in Schwann Cells Using a Peptide Derived from the Hemopexin Domain of MMP-9.

Schwann cells (SCs) undergo phenotypic transformation and then orchestrate nerve repair following a peripheral nervous system injury. The low-density lipoprotein receptor-related protein-1 (LRP1) is significantly upregulated in SCs in response to acute injury, activating cJun and promoting SC survival. Matrix-metalloproteinase-9 (MMP-9) is an LRP1 ligand that binds LRP1 through its hemopexin domain (PEX) and activates SC survival signaling and migration. To identify novel peptide mimetics within the hemopexin domain of MMP-9, we examined the crystal structure of PEX, synthesized four peptides, and examined their potential to bind and activate LRP1. We demonstrate that a 22 amino acid peptide, peptide 2, was the only peptide that activated Akt and ERK1/2 signaling in SCs, similar to a glutathione s-transferase (GST)-fused holoprotein, GST-PEX. Intraneural injection of peptide 2, but not vehicle, into crush-injured sciatic nerves activated cJun greater than 2.5-fold in wild-type mice, supporting that peptide 2 can activate the SC repair signaling in vivo. Peptide 2 also bound to Fc-fusion proteins containing the ligand-binding motifs of LRP1, clusters of complement-like repeats (CCRII and CCRIV). Pulldown and computational studies of alanine mutants of peptide 2 showed that positively charged lysine and arginine amino acids within the peptide are critical for stability and binding to CCRII. Collectively, these studies demonstrate that a novel peptide derived from PEX can serve as an LRP1 agonist and possesses qualities previously associated with LRP1 binding and SC signaling in vitro and in vivo.

Inhibition of PRMT5 by market drugs as a novel cancer therapeutic avenue.

Market drugs, such as Food and Drug Administration (FDA) or European Medicines Agency (EMA)-approved drugs for specific indications provide opportunities for repurposing for newer therapeutics. This potentially saves resources invested in clinical trials that verify drug safety and tolerance in humans prior to alternative indication approval. Protein arginine methyltransferase 5 (PRMT5) overexpression has been linked to promoting the tumor phenotype in several cancers, including pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), and breast cancer (BC), making PRMT5 an important target for cancer therapy. Previously, we showed that PRMT5-mediated methylation of the nuclear factor (NF)-κB, partially contributes to its constitutive activation observed in cancers. In this study, we utilized an AlphaLISA-based high-throughput screening method adapted in our lab, and identified one FDA-approved drug, Candesartan cilexetil (Can, used in hypertension treatment) and one EMA-approved drug, Cloperastine hydrochloride (Clo, used in cough treatment) that had significant PRMT5-inhibitory activity, and their anti-tumor properties were validated using cancer phenotypic assays in vitro. Furthermore, PRMT5 selective inhibition of methyltransferase activity was confirmed by reduction of both NF-κB methylation and its subsequent activation upon drug treatment. Using in silico prediction, we identified critical residues on PRMT5 targeted by these drugs that may interfere with its enzymatic activity. Finally, Clo and Can treatment have exhibited marked reduction in tumor growth in vivo. Overall, we provide basis for pursuing repurposing Clo and Can as anti-PRMT5 cancer therapies. Our study offers potential safe and fast repurposing of previously unknown PRMT5 inhibitors into clinical practice.

Selectivity and Ranking of Tight-Binding JAK-STAT Inhibitors Using Markovian Milestoning with Voronoi Tessellations.

Janus kinases (JAK), a group of proteins in the nonreceptor tyrosine kinase (NRTKs) family, play a crucial role in growth, survival, and angiogenesis. They are activated by cytokines through the Janus kinase-signal transducer and activator of a transcription (JAK-STAT) signaling pathway. JAK-STAT signaling pathways have significant roles in the regulation of cell division, apoptosis, and immunity. Identification of the V617F mutation in the Janus homology 2 (JH2) domain of JAK2 leading to myeloproliferative disorders has stimulated great interest in the drug discovery community to develop JAK2-specific inhibitors. However, such inhibitors should be selective toward JAK2 over other JAKs and display an extended residence time. Recently, novel JAK2/STAT5 axis inhibitors (N-(1H-pyrazol-3-yl)pyrimidin-2-amino derivatives) have displayed extended residence times (hours or longer) on target and adequate selectivity excluding JAK3. To facilitate a deeper understanding of the kinase-inhibitor interactions and advance the development of such inhibitors, we utilize a multiscale Markovian milestoning with Voronoi tessellations (MMVT) approach within the Simulation-Enabled Estimation of Kinetic Rates v.2 (SEEKR2) program to rank order these inhibitors based on their kinetic properties and further explain the selectivity of JAK2 inhibitors over JAK3. Our approach investigates the kinetic and thermodynamic properties of JAK-inhibitor complexes in a user-friendly, fast, efficient, and accurate manner compared to other brute force and hybrid-enhanced sampling approaches.

APOBEC3A Catalytic Inactivity Mutation Induces Tertiary Structure Destabilization.

APOBEC3A (A3A)-catalyzed DNA cytosine deamination is implicated in virus and cancer mutagenesis, and A3A is a target for small molecule drug discovery. The catalytic glutamic acid (E72) is frequently mutated in biochemical studies to characterize deamination-dependent versus deamination-independent A3A functions. Additionally, catalytically active A3A is toxic in bacterial expression systems, which adversely affects yield during recombinant A3A expression. Here, we demonstrate that mutating the catalytic glutamic acid to an isosteric glutamine (E72Q) significantly decreases the thermal stability of the protein, compared to the alanine-inactivating mutation (E72A). Differential scanning fluorimetry and mass spectrometry reveal that A3A E72Q is less thermally stable than A3A E72A or wild-type A3A. Strikingly, A3A E72Q is partially denatured at 37 °C and binds single-stranded DNA with significantly poorer affinity compared to A3A E72A. This study constitutes an important cautionary note on A3A protein design and informs that A3A E72A is the preferred catalytic inactivation mutation for most applications.

Glycoproteomic landscape and structural dynamics of TIM family immune checkpoints enabled by mucinase SmE.

Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of biological functions. In particular, the T cell immunoglobulin and mucin-domain containing family of proteins (TIM-1, -3, -4) decorate immune cells and act as key checkpoint inhibitors in cancer. However, their dense O-glycosylation remains enigmatic both in terms of glycoproteomic landscape and structural dynamics, primarily due to the challenges associated with studying mucin domains. Here, we present a mucinase (SmE) and demonstrate its ability to selectively cleave along the mucin glycoprotein backbone, similar to others of its kind. Unlike other mucinases, though, SmE harbors the unique ability to cleave at residues bearing extremely complex glycans which enabled improved mass spectrometric analysis of several mucins, including the entire TIM family. With this information in-hand, we performed molecular dynamics (MD) simulations of TIM-3 and -4 to demonstrate how glycosylation affects structural features of these proteins. Overall, we present a powerful workflow to better understand the detailed molecular structures of the mucinome.

Discovery of compounds that reactivate p53 mutants in vitro and in vivo.

The tumor suppressor p53 is the most frequently mutated protein in human cancer. The majority of these mutations are missense mutations in the DNA binding domain of p53. Restoring p53 tumor suppressor function could have a major impact on the therapy for a wide range of cancers. Here we report a virtual screening approach that identified several small molecules with p53 reactivation activities. The UCI-LC0023 compound series was studied in detail and was shown to bind p53, induce a conformational change in mutant p53, restore the ability of p53 hotspot mutants to associate with chromatin, reestablish sequence-specific DNA binding of a p53 mutant in a reconstituted in vitro system, induce p53-dependent transcription programs, and prevent progression of tumors carrying mutant p53, but not p53null or p53WT alleles. Our study demonstrates feasibility of a computation-guided approach to identify small molecule corrector drugs for p53 hotspot mutations.

Development of Dimethylisoxazole-Attached Imidazo[1,2-a]pyridines as Potent and Selective CBP/P300 Inhibitors.

The use of epigenetic bromodomain inhibitors as anticancer therapeutics has transitioned from targeting bromodomain extraterminal domain (BET) proteins into targeting non-BET bromodomains. The two most relevant non-BET bromodomain oncology targets are cyclic AMP response element-binding protein (CBP) and E1A binding protein P300 (EP300). To explore the growing CBP/EP300 interest, we developed a highly efficient two-step synthetic route for dimethylisoxazole-attached imidazo[1,2-a]pyridine scaffold-containing inhibitors. Our efficient two-step reactions enabled high-throughput synthesis of compounds designed by molecular modeling, which together with structure-activity relationship (SAR) studies facilitated an overarching understanding of selective targeting of CBP/EP300 over non-BET bromodomains. This led to the identification of a new potent and selective CBP/EP300 bromodomain inhibitor, UMB298 (compound 23, CBP IC50 72 nM and bromodomain 4, BRD4 IC50 5193 nM). The SAR we established is in good agreement with literature-reported CBP inhibitors, such as CBP30, and demonstrates the advantage of utilizing our two-step approach for inhibitor development of other bromodomains.

Structural Characterization of a Minimal Antibody against Human APOBEC3B.

APOBEC3B (A3B) is one of seven human APOBEC3 DNA cytosine deaminases that restrict viral infections as part of the overall innate immune response, but it also plays a major role in tumor evolution by mutating genomic DNA. Given the importance of A3B as a restriction factor of viral infections and as a driver of multiple human cancers, selective antibodies against A3B are highly desirable for its specific detection in various research and possibly diagnostic applications. Here, we describe a high-affinity minimal antibody, designated 5G7, obtained via a phage display screening against the C-terminal catalytic domain (ctd) of A3B. 5G7 also binds APOBEC3A that is highly homologous to A3Bctd but does not bind the catalytic domain of APOBEC3G, another Z1-type deaminase domain. The crystal structure of 5G7 shows a canonical arrangement of the heavy and light chain variable domains, with their complementarity-determining region (CDR) loops lining an antigen-binding cleft that accommodates a pair of α-helices. To understand the mechanism of A3Bctd recognition by 5G7, we used the crystal structures of A3Bctd and 5G7 as templates and computationally predicted the A3B-5G7 complex structure. Stable binding poses obtained by the simulation were further tested by site-directed mutagenesis and in vitro binding analyses. These studies mapped the epitope for 5G7 to a portion of C-terminal α6 helix of A3Bctd, with Arg374 playing an essential role. The same region of A3Bctd was used previously as a peptide antigen for generating a rabbit monoclonal antibody (mAb 5210-87-13), suggesting that this region is particularly immunogenic and that these antibodies from very different origins may share similar binding modes. Our studies provide a platform for the development of selective antibodies against A3B and other APOBEC3 family enzymes.

Disease-related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveal a path to selective inhibitors.

Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the class IB PI3K catalytic subunit (p110γ) playing key roles in immune signalling. p110γ is a key factor in inflammatory diseases and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the C-terminus can inactivate or activate enzyme activity. Screening of inhibitors using HDX-MS revealed that activation loop-binding inhibitors induce allosteric conformational changes that mimic those in the R1021C mutant. Structural analysis of advanced PI3K inhibitors in clinical development revealed novel binding pockets that can be exploited for further therapeutic development. Overall, this work provides unique insights into regulatory mechanisms that control PI3Kγ kinase activity and shows a framework for the design of PI3K isoform and mutant selective inhibitors.

A potential interaction between the SARS-CoV-2 spike protein and nicotinic acetylcholine receptors.

Changeux et al. (Changeux et al. C. R. Biol. 343:33-39.) recently suggested that the SARS-CoV-2 spike protein may interact with nicotinic acetylcholine receptors (nAChRs) and that such interactions may be involved in pathology and infectivity. This hypothesis is based on the fact that the SARS-CoV-2 spike protein contains a sequence motif similar to known nAChR antagonists. Here, we use molecular simulations of validated atomically detailed structures of nAChRs and of the spike to investigate the possible binding of the Y674-R685 region of the spike to nAChRs. We examine the binding of the Y674-R685 loop to three nAChRs, namely the human α4ß2 and α7 subtypes and the muscle-like αßγδ receptor from Tetronarce californica. Our results predict that Y674-R685 has affinity for nAChRs. The region of the spike responsible for binding contains a PRRA motif, a four-residue insertion not found in other SARS-like coronaviruses. The conformational behavior of the bound Y674-R685 is highly dependent on the receptor subtype; it adopts extended conformations in the α4ß2 and α7 complexes but is more compact when bound to the muscle-like receptor. In the α4ß2 and αßγδ complexes, the interaction of Y674-R685 with the receptors forces the loop C region to adopt an open conformation, similar to other known nAChR antagonists. In contrast, in the α7 complex, Y674-R685 penetrates deeply into the binding pocket in which it forms interactions with the residues lining the aromatic box, namely with TrpB, TyrC1, and TyrC2. Estimates of binding energy suggest that Y674-R685 forms stable complexes with all three nAChR subtypes. Analyses of simulations of the glycosylated spike show that the Y674-R685 region is accessible for binding. We suggest a potential binding orientation of the spike protein with nAChRs, in which they are in a nonparallel arrangement to one another.

An integrated view of p53 dynamics, function, and reactivation.

The tumor suppressor p53 plays a vital role in responding to cell stressors such as DNA damage, hypoxia, and tumor formation by inducing cell-cycle arrest, senescence, or apoptosis. Expression level alterations and mutational frequency implicates p53 in most human cancers. In this review, we show how both computational and experimental methods have been used to provide an integrated view of p53 dynamics, function, and reactivation potential. We argue that p53 serves as an exceptional case study for developing methods in modeling intrinsically disordered proteins. We describe how these methods can be leveraged to improve p53 reactivation molecule design and other novel therapeutic modalities, such as PROteolysis TARgeting Chimeras (PROTACs).

OCRE Domains of Splicing Factors RBM5 and RBM10: Tyrosine Ring-Flip Frequencies Determined by Integrated Use of 1 H NMR Spectroscopy and Molecular Dynamics Simulations.

The 55-residue OCRE domains of the splicing factors RBM5 and RBM10 contain 15 tyrosines in compact, globular folds. At 25 °C, all 15 tyrosines show symmetric 1 H NMR spectra, with averaged signals for the pairs of δ- and ϵ-ring hydrogens. At 4 °C, two tyrosines were identified as showing 1 H NMR line-broadening due to lowered frequency of the ring-flipping. For the other 13 tyrosine rings, it was not evident, from the 1 H NMR data alone, whether they were either all flipping at high frequencies, or whether slowed flipping went undetected due to small chemical-shift differences between pairs of exchanging ring hydrogen atoms. Here, we integrate 1 H NMR spectroscopy and molecular dynamics (MD) simulations to determine the tyrosine ring-flip frequencies. In the RBM10-OCRE domain, we found that, for 11 of the 15 tyrosines, these frequencies are in the range 2.0×106 to 1.3×108  s-1 , and we established an upper limit of <1.0×106  s-1 for the remaining four residues. The experimental data and the MD simulation are mutually supportive, and their combined use extends the analysis of aromatic ring-flip events beyond the limitations of routine 1 H NMR line-shape analysis into the nanosecond frequency range.

Active site plasticity and possible modes of chemical inhibition of the human DNA deaminase APOBEC3B.

The single-stranded DNA cytosine deaminase APOBEC3B (A3B) functions in innate immunity against viruses, but it is also strongly implicated in eliciting mutations in cancer genomes. Because of the critical role of A3B in promoting virus and tumor evolution, small molecule inhibitors are desirable. However, there is no reported structure for any of the APOBEC3-family enzymes in complex with a small molecule bound in the active site, which hampers the development of small molecules targeting A3B. Here we report high-resolution structures of an active A3B catalytic domain chimera with loop 7 residues exchanged with those from the corresponding region of APOBEC3G (A3G). The structures reveal novel open conformations lacking the catalytically essential zinc ion, with the highly conserved active site residues extensively rearranged. These inactive conformations are stabilized by 2-pyrimidone or an iodide ion bound in the active site. Molecular dynamics simulations corroborate the remarkable plasticity of the engineered active site and identify key interactions that stabilize the native A3B active site. These data provide insights into A3B active site dynamics and suggest possible modes of its inhibition by small molecules, which would aid in rational design of selective A3B inhibitors for constraining virus and tumor evolution.

Markov state models and NMR uncover an overlooked allosteric loop in p53.

The tumor suppressor p53 is the most frequently mutated gene in human cancer, and thus reactivation of mutated p53 is a promising avenue for cancer therapy. Analysis of wildtype p53 and the Y220C cancer mutant long-timescale molecular dynamics simulations with Markov state models and validation by NMR relaxation studies has uncovered the involvement of loop L6 in the slowest motions of the protein. Due to its distant location from the DNA-binding surface, the conformational dynamics of this loop has so far remained largely unexplored. We observe mutation-induced stabilization of alternate L6 conformations, distinct from all experimentally-determined structures, in which the loop is both extended and located further away from the DNA-interacting surface. Additionally, the effect of the L6-adjacent Y220C mutation on the conformational landscape of the functionally-important loop L1 suggests an allosteric role to this dynamic loop and the inactivation mechanism of the mutation. Finally, the simulations reveal a novel Y220C cryptic pocket that can be targeted for p53 rescue efforts. Our approach exemplifies the power of the MSM methodology for uncovering intrinsic dynamic and kinetic differences among distinct protein ensembles, such as for the investigation of mutation effects on protein function.

Structural basis for ligand modulation of the CCR2 conformational landscape.

CC chemokine receptor 2 (CCR2) is a part of the chemokine receptor family, an important class of therapeutic targets. These class A G-protein coupled receptors (GPCRs) are involved in mammalian signaling pathways and control cell migration toward endogenous CC chemokine ligands, named for the adjacent cysteine motif on their N terminus. Chemokine receptors and their associated ligands are involved in a wide range of diseases and thus have become important drug targets. CCR2, in particular, promotes the metastasis of cancer cells and is also implicated in autoimmunity-driven type-1 diabetes, diabetic nephropathy, multiple sclerosis, asthma, atherosclerosis, neuropathic pain, and rheumatoid arthritis. Although promising, CCR2 antagonists have been largely unsuccessful to date. Here, we investigate the effect of an orthosteric and an allosteric antagonist on CCR2 dynamics by coupling long-timescale molecular dynamics simulations with Markov-state model theory. We find that the antagonists shift CCR2 into several stable inactive conformations that are distinct from the crystal structure conformation and disrupt a continuous internal water and sodium ion pathway, preventing transitions to an active-like state. Several metastable conformations present a cryptic drug-binding pocket near the allosteric site that may be amenable to targeting with small molecules. Without antagonists, the apo dynamics reveal intermediate conformations along the activation pathway that provide insight into the basal dynamics of CCR2 and may also be useful for future drug design.

Mechanisms for Benzene Dissociation through the Excited State of T4 Lysozyme L99A Mutant.

The atomic-level mechanisms that coordinate ligand release from protein pockets are only known for a handful of proteins. Here, we report results from accelerated molecular dynamics simulations for benzene dissociation from the buried cavity of the T4 lysozyme Leu99Ala mutant (L99A). In these simulations, benzene is released through a previously characterized, sparsely populated room-temperature excited state of the mutant, explaining the coincidence for experimentally measured benzene off rate and apo protein slow-timescale NMR relaxation rates between ground and excited states. The path observed for benzene egress is a multistep ligand migration from the buried cavity to ultimate release through an opening between the F/G-, H-, and I-helices and requires a number of cooperative multiresidue and secondary-structure rearrangements within the C-terminal domain of L99A. These rearrangements are identical to those observed along the ground state to excited state transitions characterized by molecular dynamic simulations run on the Anton supercomputer. Analyses of the molecular properties of the residues lining the egress path suggest that protein surface electrostatic potential may play a role in the release mechanism. Simulations of wild-type T4 lysozyme also reveal that benzene-egress-associated dynamics in the L99A mutant are potentially exaggerations of the substrate-processivity-related dynamics of the wild type.

Determinants of Oligonucleotide Selectivity of APOBEC3B.

APOBEC3B (A3B) is a prominent source of mutation in many cancers. To date, it has been difficult to capture the native protein-DNA interactions that confer A3B's substrate specificity by crystallography due to the highly dynamic nature of wild-type A3B active site. We use computational tools to restore a recent crystal structure of a DNA-bound A3B C-terminal domain mutant construct to its wild type sequence, and run molecular dynamics simulations to study its substrate recognition mechanisms. Analysis of these simulations reveal dynamics of the native A3Bctd-oligonucleotide interactions, including the experimentally inaccessible loop 1-oligonucleotide interactions. A second series of simulations in which the target cytosine nucleotide was computationally mutated from a deoxyribose to a ribose show a change in sugar ring pucker, leading to a rearrangement of the binding site and revealing a potential intermediate in the binding pathway. Finally, apo simulations of A3B, starting from the DNA-bound open state, experience a rapid and consistent closure of the binding site, reaching conformations incompatible with substrate binding. This study reveals a more realistic and dynamic view of the wild type A3B binding site and provides novel insights for structure-guided design efforts for A3B.

Dynamics and Molecular Mechanisms of p53 Transcriptional Activation.

The "guardian of the genome", p53, functions as a tumor suppressor that responds to cell stressors such as DNA damage, hypoxia, and tumor formation by inducing cell-cycle arrest, senescence, or apoptosis. Mutation of p53 disrupts its tumor suppressor function, leading to various types of human cancers. One particular mutant, R175H, is a structural mutant that inactivates the DNA damage response pathway and acquires oncogenic functions that promotes both cancer and drug resistance. Our current work aims to understand how p53 wild-type function is disrupted due to the R175H mutation. We use a series of atomistic integrative models built previously from crystal structures of the full-length p53 tetramer bound to DNA and model the R175H mutant using in silico site-directed mutagenesis. Explicitly solvated all-atom molecular dynamics (MD) simulations on wild-type and the R175H mutant p53 reveal insights into how wild-type p53 searches and recognizes DNA, and how this mechanism is disrupted as a result of the R175H mutation. Specifically, our work reveals the optimal quaternary DNA binding mode of the DNA binding domain and shows how this binding mode is altered via symmetry loss as a result of the R175H mutation, indicating a recognition mechanism that is reminiscent of the asymmetry seen in wild type p53 binding to nonspecific genomic elements. Altogether our work sheds new light into the hitherto unseen molecular mechanisms governing transcription factor, DNA recognition.

APOBEC3H Subcellular Localization Determinants Define Zipcode for Targeting HIV-1 for Restriction.

APOBEC enzymes are DNA cytosine deaminases that normally serve as virus restriction factors, but several members, including APOBEC3H, also contribute to cancer mutagenesis. Despite their importance in multiple fields, little is known about cellular processes that regulate these DNA mutating enzymes. We show that APOBEC3H exists in two distinct subcellular compartments, cytoplasm and nucleolus, and that the structural determinants for each mechanism are genetically separable. First, native and fluorescently tagged APOBEC3Hs localize to these two compartments in multiple cell types. Second, a series of genetic, pharmacologic, and cell biological studies demonstrate active cytoplasmic and nucleolar retention mechanisms, whereas nuclear import and export occur through passive diffusion. Third, APOBEC3H cytoplasmic retention determinants relocalize APOBEC3A from a passive cell-wide state to the cytosol and, additionally, endow potent HIV-1 restriction activity. These results indicate that APOBEC3H has a structural zipcode for subcellular localization and selecting viral substrates for restriction.