Design, Synthesis, and Structure-Activity Relationships of Novel Peptide Derivatives of the Severe Acute Respiratory Syndrome-Coronavirus-2 Spike-Protein that Potently Inhibit Nicotinic Acetylcholine Receptors.

The spike-protein of SARS-CoV-2 has a distinctive amino-acid sequence (682RRARS686) that forms a cleavage site for the enzyme furin. Strikingly, the structure of the spike-protein loop containing the furin cleavage site bears substantial similarity to neurotoxin peptides found in the venoms of certain snakes and marine cone snails. Leveraging this relationship, we designed and synthesized disulfide-constrained peptides with amino-acid sequences corresponding to the furin cleavage-sites of wild-type (B.1 variant) SARS-CoV-2 or the Alpha, Delta, and Omicron variants. Remarkably, some of these peptides potently inhibited α7 and α9α10 nicotinic acetylcholine receptors (nAChR) with nM affinity and showed SARS-CoV-2 variant and nAChR subtype-dependent potencies. Nuclear magnetic resonance spectroscopy and molecular dynamics were used to rationalize structure-activity relationships between peptides and their cognate receptors. These findings delineate nAChR subtypes that can serve as high-affinity spike-protein targets in tissues central to COVID-19 pathophysiology and identify ligands and target receptors to inform the development of novel SARS-CoV-2 therapeutics.

A Targetable Self-association Surface of the Huntingtin exon1 Helical Tetramer Required for Assembly of Amyloid Pre-nucleation Oligomers.

Polyglutamine (polyQ) sequences undergo repeat-length dependent formation of disease-associated, amyloid-like cross-ß core structures with kinetics and aggregate morphologies often influenced by the flanking sequences. In Huntington's disease (HD), the httNT segment on the polyQ's N-terminal flank enhances aggregation rates by changing amyloid nucleation from a classical homogeneous mechanism to a two-step process requiring an ɑ-helix-rich oligomeric intermediate. A folded, helix-rich httNT tetrameric structure suggested to be this critical intermediate was recently reported. Here we employ single alanine replacements along the httNT sequence to assess this proposed structure and refine the mechanistic model. We find that Ala replacement of hydrophobic residues within simple httNT peptides greatly suppresses helicity, supporting the tetramer model. These same helix-disruptive replacements in the httNT segment of an exon-1 analog greatly reduce aggregation kinetics, suggesting that an ɑ-helix rich multimer - either the tetramer or a larger multimer - plays an on-pathway role in nucleation. Surprisingly, several other Ala replacements actually enhance helicity and/or amyloid aggregation. The spatial localization of these residues on the tetramer surface suggests a self-association interface responsible for formation of the octomers and higher-order multimers most likely required for polyQ amyloid nucleation. Multimer docking of the tetramer, using the protein-protein docking algorithm ClusPro, predicts this symmetric surface to be a viable tetramer dimerization interface. Intriguingly, octomer formation brings the emerging polyQ chains into closer proximity at this tetramer-tetramer interface. Further supporting the potential importance of tetramer super-assembly, computational docking with a known exon-1 aggregation inhibitor predicts ligand contacts with residues at this interface.

A peptidomimetic modulator of the CaV2.2 N-type calcium channel for chronic pain.

Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Cav2.2 (e.g., with Prialt/Ziconotide) or indirect modulation [e.g., with gabapentinoids such as Gabapentin (GBP)] mitigates chronic pain but is encumbered by side effects and abuse liability. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Cav2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A1R2 dipeptide in CBD3 as the anchoring Cav2.2 motif and designed pharmacophore models to screen 27 million compounds on the open-access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons. Nine small molecules were tested electrophysiologically, while one (CBD3063) was also evaluated biochemically and behaviorally. CBD3063 uncoupled Cav2.2 from CRMP2, reduced membrane Cav2.2 expression and Ca2+ currents, decreased neurotransmission, reduced fiber photometry-based calcium responses in response to mechanical stimulation, and reversed neuropathic and inflammatory pain across sexes in two different species without changes in sensory, sedative, depressive, and cognitive behaviors. CBD3063 is a selective, first-in-class, CRMP2-based peptidomimetic small molecule, which allosterically regulates Cav2.2 to achieve analgesia and pain relief without negative side effect profiles. In summary, CBD3063 could potentially be a more effective alternative to GBP for pain relief.

Characterization of allosteric modulators that disrupt androgen receptor co-activator protein-protein interactions to alter transactivation-Drug leads for metastatic castration resistant prostate cancer.

Three series of compounds were prioritized from a high content screening campaign that identified molecules that blocked dihydrotestosterone (DHT) induced formation of Androgen Receptor (AR) protein-protein interactions (PPIs) with the Transcriptional Intermediary Factor 2 (TIF2) coactivator and also disrupted preformed AR-TIF2 PPI complexes; the hydrobenzo-oxazepins (S1), thiadiazol-5-piperidine-carboxamides (S2), and phenyl-methyl-indoles (S3). Compounds from these series inhibited AR PPIs with TIF2 and SRC-1, another p160 coactivator, in mammalian 2-hybrid assays and blocked transcriptional activation in reporter assays driven by full length AR or AR-V7 splice variants. Compounds inhibited the growth of five prostate cancer cell lines, with many exhibiting differential cytotoxicity towards AR positive cell lines. Representative compounds from the 3 series substantially reduced both endogenous and DHT-enhanced expression and secretion of the prostate specific antigen (PSA) cancer biomarker in the C4-2 castration resistant prostate cancer (CRPC) cell line. The comparatively weak activities of series compounds in the H3-DHT and/or TIF2 box 3 LXXLL-peptide binding assays to the recombinant ligand binding domain of AR suggest that direct antagonism at the orthosteric ligand binding site or AF-2 surface respectively are unlikely mechanisms of action. Cellular enhanced thermal stability assays (CETSA) indicated that compounds engaged AR and reduced the maximum efficacy and right shifted the EC50 of DHT-enhanced AR thermal stabilization consistent with the effects of negative allosteric modulators. Molecular docking of potent representative hits from each series to AR structures suggest that S1-1 and S2-6 engage a novel binding pocket (BP-1) adjacent to the orthosteric ligand binding site, while S3-11 occupies the AR binding function 3 (BF-3) allosteric pocket. Hit binding poses indicate spaces and residues adjacent to the BP-1 and BF-3 pockets that will be exploited in future medicinal chemistry optimization studies. Small molecule allosteric modulators that prevent/disrupt AR PPIs with coactivators like TIF2 to alter transcriptional activation in the presence of orthosteric agonists might evade the resistance mechanisms to existing prostate cancer drugs and provide novel starting points for medicinal chemistry lead optimization and future development into therapies for metastatic CRPC.

Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein.

The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIF pocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small-molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1.

Discovery and Biological Characterization of PRMT5:MEP50 Protein-Protein Interaction Inhibitors.

Protein arginine methyltransferase 5 (PRMT5) is a master epigenetic regulator and an extensively validated therapeutic target in multiple cancers. Notably, PRMT5 is the only PRMT that requires an obligate cofactor, methylosome protein 50 (MEP50), to function. We developed compound 17, a novel small-molecule PRMT5:MEP50 protein-protein interaction (PPI) inhibitor, after initial virtual screen hit identification and analogue refinement. Molecular docking indicated that compound 17 targets PRMT5:MEP50 PPI by displacing the MEP50 W54 burial into a hydrophobic pocket of the PRMT5 TIM barrel. In vitro analysis indicates IC50 < 500 nM for prostate and lung cancer cells with selective, specific inhibition of PRMT5:MEP50 substrate methylation and target gene expression, and RNA-seq analysis suggests that compound 17 may dysregulate TGF-ß signaling. Compound 17 provides a proof of concept in targeting PRMT5:MEP50 PPI, as opposed to catalytic targeting, as a novel mechanism of action and supports further preclinical development of inhibitors in this class.

More than a marker: potential pathogenic functions of MAP2.

Microtubule-associated protein 2 (MAP2) is the predominant cytoskeletal regulator within neuronal dendrites, abundant and specific enough to serve as a robust somatodendritic marker. It influences microtubule dynamics and microtubule/actin interactions to control neurite outgrowth and synaptic functions, similarly to the closely related MAP Tau. Though pathology of Tau has been well appreciated in the context of neurodegenerative disorders, the consequences of pathologically dysregulated MAP2 have been little explored, despite alterations in its immunoreactivity, expression, splicing and/or stability being observed in a variety of neurodegenerative and neuropsychiatric disorders including Huntington's disease, prion disease, schizophrenia, autism, major depression and bipolar disorder. Here we review the understood structure and functions of MAP2, including in neurite outgrowth, synaptic plasticity, and regulation of protein folding/transport. We also describe known and potential mechanisms by which MAP2 can be regulated via post-translational modification. Then, we assess existing evidence of its dysregulation in various brain disorders, including from immunohistochemical and (phospho) proteomic data. We propose pathways by which MAP2 pathology could contribute to endophenotypes which characterize these disorders, giving rise to the concept of a "MAP2opathy"-a series of disorders characterized by alterations in MAP2 function.

A dimer-monomer switch controls CHIP-dependent substrate ubiquitylation and processing.

The high substrate selectivity of the ubiquitin/proteasome system is mediated by a large group of E3 ubiquitin ligases. The ubiquitin ligase CHIP regulates the degradation of chaperone-controlled and chaperone-independent proteins. To understand how CHIP mediates substrate selection and processing, we performed a structure-function analysis of CHIP and addressed its physiological role in Caenorhabditis elegans and human cells. The conserved function of CHIP in chaperone-assisted degradation requires dimer formation to mediate proteotoxic stress resistance and to prevent protein aggregation. The CHIP monomer, however, promotes the turnover of the membrane-bound insulin receptor and longevity. The dimer-monomer transition is regulated by CHIP autoubiquitylation and chaperone binding, which provides a feedback loop that controls CHIP activity in response to cellular stress. Because CHIP also binds other E3 ligases, such as Parkin, the molecular switch mechanism described here could be a general concept for the regulation of substrate selectivity and ubiquitylation by combining different E3s.

A heterotypic assembly mechanism regulates CHIP E3 ligase activity.

CHIP (C-terminus of Hsc70-interacting protein) and its worm ortholog CHN-1 are E3 ubiquitin ligases that link the chaperone system with the ubiquitin-proteasome system (UPS). CHN-1 can cooperate with UFD-2, another E3 ligase, to accelerate ubiquitin chain formation; however, the basis for the high processivity of this E3s set has remained obscure. Here, we studied the molecular mechanism and function of the CHN-1-UFD-2 complex in Caenorhabditis elegans. Our data show that UFD-2 binding promotes the cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. However, HSP70/HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding, thereby promoting a shift to the autoinhibited CHN-1 state by acting on a conserved residue in its U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitylate and regulate S-adenosylhomocysteinase (AHCY-1), a key enzyme in the S-adenosylmethionine (SAM) regeneration cycle, which is essential for SAM-dependent methylation. Our results define the molecular mechanism underlying the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitylation.

A specific inhibitor of ALDH1A3 regulates retinoic acid biosynthesis in glioma stem cells.

Elevated aldehyde dehydrogenase (ALDH) activity correlates with poor outcome for many solid tumors as ALDHs may regulate cell proliferation and chemoresistance of cancer stem cells (CSCs). Accordingly, potent, and selective inhibitors of key ALDH enzymes may represent a novel CSC-directed treatment paradigm for ALDH+ cancer types. Of the many ALDH isoforms, we and others have implicated the elevated expression of ALDH1A3 in mesenchymal glioma stem cells (MES GSCs) as a target for the development of novel therapeutics. To this end, our structure of human ALDH1A3 combined with in silico modeling identifies a selective, active-site inhibitor of ALDH1A3. The lead compound, MCI-INI-3, is a selective competitive inhibitor of human ALDH1A3 and shows poor inhibitory effect on the structurally related isoform ALDH1A1. Mass spectrometry-based cellular thermal shift analysis reveals that ALDH1A3 is the primary binding protein for MCI-INI-3 in MES GSC lysates. The inhibitory effect of MCI-INI-3 on retinoic acid biosynthesis is comparable with that of ALDH1A3 knockout, suggesting that effective inhibition of ALDH1A3 is achieved with MCI-INI-3. Further development is warranted to characterize the role of ALDH1A3 and retinoic acid biosynthesis in glioma stem cell growth and differentiation.

Novel FOXM1 inhibitor identified via gene network analysis induces autophagic FOXM1 degradation to overcome chemoresistance of human cancer cells.

FOXM1 transcription factor is an oncogene and a master regulator of chemoresistance in multiple cancers. Pharmacological inhibition of FOXM1 is a promising approach but has proven to be challenging. We performed a network-centric transcriptomic analysis to identify a novel compound STL427944 that selectively suppresses FOXM1 by inducing the relocalization of nuclear FOXM1 protein to the cytoplasm and promoting its subsequent degradation by autophagosomes. Human cancer cells treated with STL427944 exhibit increased sensitivity to cytotoxic effects of conventional chemotherapeutic treatments (platinum-based agents, 5-fluorouracil, and taxanes). RNA-seq analysis of STL427944-induced gene expression changes revealed prominent suppression of gene signatures characteristic for FOXM1 and its downstream targets but no significant changes in other important regulatory pathways, thereby suggesting high selectivity of STL427944 toward the FOXM1 pathway. Collectively, the novel autophagy-dependent mode of FOXM1 suppression by STL427944 validates a unique pathway to overcome tumor chemoresistance and improve the efficacy of treatment with conventional cancer drugs.

Tripeptide IRW Upregulates NAMPT Protein Levels in Cells and Obese C57BL/6J Mice.

Nicotinamide adenine dinucleotide (NAD+) plays a vital role in cellular processes that govern human health and disease. Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in NAD+ biosynthesis. Thus, boosting NAD+ level via an increase in NAMPT levels is an attractive approach for countering the effects of aging and metabolic disease. This study aimed to establish IRW (Ile-Arg-Trp), a small tripeptide derived from ovotransferrin, as a booster of NAMPT levels. Treatment of muscle (L6) cells with IRW increased intracellular NAMPT protein levels (2.2-fold, p < 0.05) and boosted NAD+ (p < 0.01). Both immunoprecipitation and recombinant NAMPT assays indicated the possible NAMPT-activating ability of IRW (p < 0.01). Similarly, IRW increased NAMPT mRNA and protein levels in the liver (2.6-fold, p < 0.01) and muscle tissues (2.3-fold, p < 0.05) of C57BL/6J mice fed with a high-fat diet (HFD). A significantly increased level of circulating NAD+ was also observed following IRW treatment (4.7 fold, p < 0.0001). Dosing of Drosophila melanogaster with IRW elevated both D-NAAM (fly NAMPT) and NAD+ in vivo (p < 0.05). However, IRW treatment did not boost NAMPT levels in SIRT1 KO cells, indicating a possible SIRT1 dependency for the pharmacological effect. Overall, these data indicate that IRW is a novel small peptide booster of the NAMPT pool.

On the molecular mechanism of SARS-CoV-2 retention in the upper respiratory tract.

Cell surface receptor engagement is a critical aspect of viral infection. At low pH, binding of SARS-CoV and its ACE2 receptor has a tight interaction that catalyzes the fusion of the spike and endosomal membranes followed by genome release. Largely overlooked has been the role of neutral pH in the respiratory tract, where we find that SARS-CoV stabilizes a transition state that enhances the off-rate from its receptor. An alternative pH-switch is found in CoV-2-like coronaviruses of tropical pangolins, but with a reversed phenotype where the tight interaction with ACE2 is at neutral pH. We show that a single point mutation in pangolin-CoV, unique to CoV-2, that deletes the last His residue in their receptor binding domain perpetuates this tight interaction independent of pH. This tight bond, not present in previous respiratory syndromes, implies that CoV-2 stays bound to the highly expressed ACE2 receptors in the nasal cavity about 100 times longer than CoV. This finding supports the unfamiliar pathology of CoV-2, observed virus retention in upper respiratory tract 1 , longer incubation times and extended periods of shedding. Implications to combat pandemics that, like SARS-CoV-2, export evolutionarily successful strains via higher transmission rates due to retention in nasal epithelium and their evolutionary origin are discussed.

De novo emergence of adaptive membrane proteins from thymine-rich genomic sequences.

Recent evidence demonstrates that novel protein-coding genes can arise de novo from non-genic loci. This evolutionary innovation is thought to be facilitated by the pervasive translation of non-genic transcripts, which exposes a reservoir of variable polypeptides to natural selection. Here, we systematically characterize how these de novo emerging coding sequences impact fitness in budding yeast. Disruption of emerging sequences is generally inconsequential for fitness in the laboratory and in natural populations. Overexpression of emerging sequences, however, is enriched in adaptive fitness effects compared to overexpression of established genes. We find that adaptive emerging sequences tend to encode putative transmembrane domains, and that thymine-rich intergenic regions harbor a widespread potential to produce transmembrane domains. These findings, together with in-depth examination of the de novo emerging YBR196C-A locus, suggest a novel evolutionary model whereby adaptive transmembrane polypeptides emerge de novo from thymine-rich non-genic regions and subsequently accumulate changes molded by natural selection.

Cross-docking benchmark for automated pose and ranking prediction of ligand binding.

Significant efforts have been devoted in the last decade to improving molecular docking techniques to predict both accurate binding poses and ranking affinities. Some shortcomings in the field are the limited number of standard methods for measuring docking success and the availability of widely accepted standard data sets for use as benchmarks in comparing different docking algorithms throughout the field. In order to address these issues, we have created a Cross-Docking Benchmark server. The server is a versatile cross-docking data set containing 4,399 protein-ligand complexes across 95 protein targets intended to serve as benchmark set and gold standard for state-of-the-art pose and ranking prediction in easy, medium, hard, or very hard docking targets. The benchmark along with a customizable cross-docking data set generation tool is available at http://disco.csb.pitt.edu. We further demonstrate the potential uses of the server in questions outside of basic benchmarking such as the selection of the ideal docking reference structure.

Discovery of Non-peptide Small Molecule Allosteric Modulators of the Src-family Kinase, Hck.

The eight mammalian Src-family tyrosine kinases are dynamic, multi-domain structures, which adopt distinct "open" and "closed" conformations. In the closed conformation, the regulatory SH3 and SH2 domains pack against the back of the kinase domain, providing allosteric control of kinase activity. Small molecule ligands that engage the regulatory SH3-SH2 region have the potential to modulate Src-family kinase activity for therapeutic advantage. Here we describe an HTS-compatible fluorescence polarization assay to identify small molecules that interact with the unique-SH3-SH2-linker (U32L) region of Hck, a Src-family member expressed exclusively in cells of myeloid lineage. Hck has significant potential as a drug target in acute myeloid leukemia, an aggressive form of cancer with substantial unmet clinical need. The assay combines recombinant Hck U32L protein with a fluorescent probe peptide that binds to the SH3 domain in U32L, resulting in an increased FP signal. Library compounds that interact with the U32L protein and interfere with probe binding reduce the FP signal, scoring as hits. Automated 384-well high-throughput screening of 60,000 compounds yielded Z'-factor coefficients > 0.7 across nearly 200 assay plates, and identified a series of hit compounds with a shared pyrimidine diamine substructure. Surface plasmon resonance assays confirmed direct binding of hit compounds to the Hck U32L target protein as well as near-full-length Hck. Binding was not observed with the individual SH3 and SH2 domains, demonstrating that these compounds recognize a specific three-dimensional conformation of the regulatory regions. This conclusion is supported by computational docking studies, which predict ligand contacts with a pocket formed by the juxtaposition of the SH3 domain, the SH3-SH2 domain connector, and the SH2-kinase linker. Each of the four validated hits stimulated recombinant, near-full-length Hck activity in vitro, providing evidence for allosteric effects on the kinase domain. These results provide a path to discovery and development of chemical scaffolds to target the regulatory regions of Hck and other Src family kinases as a new approach to pharmacological kinase control.

Defining the Kv2.1-syntaxin molecular interaction identifies a first-in-class small molecule neuroprotectant.

The neuronal cell death-promoting loss of cytoplasmic K+ following injury is mediated by an increase in Kv2.1 potassium channels in the plasma membrane. This phenomenon relies on Kv2.1 binding to syntaxin 1A via 9 amino acids within the channel intrinsically disordered C terminus. Preventing this interaction with a cell and blood-brain barrier-permeant peptide is neuroprotective in an in vivo stroke model. Here a rational approach was applied to define the key molecular interactions between syntaxin and Kv2.1, some of which are shared with mammalian uncoordinated-18 (munc18). Armed with this information, we found a small molecule Kv2.1-syntaxin-binding inhibitor (cpd5) that improves cortical neuron survival by suppressing SNARE-dependent enhancement of Kv2.1-mediated currents following excitotoxic injury. We validated that cpd5 selectively displaces Kv2.1-syntaxin-binding peptides from syntaxin and, at higher concentrations, munc18, but without affecting either synaptic or neuronal intrinsic properties in brain tissue slices at neuroprotective concentrations. Collectively, our findings provide insight into the role of syntaxin in neuronal cell death and validate an important target for neuroprotection.