The interaction of the HIV-1 transactivator protein Tat with its transactivation response (TAR) RNA is an essential step in viral replication and therefore an attractive target for developing antivirals with new mechanisms of action. Numerous compounds that bind to the 3-nt bulge responsible for binding Tat have been identified in the past, but none of these molecules had sufficient potency to warrant pharmaceutical development. We have discovered conformationally-constrained cyclic peptide mimetics of Tat that are specific nM inhibitors of the Tat-TAR interaction by using a structure-based approach. The lead peptides are nearly as active as the antiviral drug nevirapine against a variety of clinical isolates in human lymphocytes. The NMR structure of a peptide-RNA complex reveals that these molecules interfere with the recruitment to TAR of both Tat and the essential cellular cofactor transcription elongation factor-b (P-TEFb) by binding simultaneously at the RNA bulge and apical loop, forming an unusually deep pocket. This structure illustrates additional principles in RNA recognition: RNA-binding molecules can achieve specificity by interacting simultaneously with multiple secondary structure elements and by inducing the formation of deep binding pockets in their targets. It also provides insight into the P-TEFb binding site and a rational basis for optimizing the promising antiviral activity observed for these cyclic peptides.NMR ͉ transcription elongation factor-b ͉ antiviral ͉ Tat-TAR interaction ͉ RNA recognition T ranscription of the HIV-1 RNA in infected cells is strongly activated by the complex between the viral Tat protein and its cognate transactivation response (TAR) RNA, a 59-nt RNA found at the 5Ј end of all nascent viral transcripts (Fig. 1A). Tat and its cellular cofactor, the transcription elongation factor-b (P-TEFb), are recruited to the elongating RNA polymerase II (RNAP II) through interactions with TAR and are required for reactivation of the integrated proviral genome in latently infected cells (1). The cooperative binding of Tat and P-TEFb to TAR activates the CDK9 kinase of P-TEFb that phosphorylates RNAP II and the repressive NELF factors, leading to greatly enhanced RNAP II processivity (1-3).The Tat-TAR complex is an attractive target for developing new antivirals because the interaction between Tat and TAR is unique to the virus, whereas P-TEFb is used widely for transcription of most host genes. Furthermore, TAR is extremely conserved among viral isolates and P-TEFb plays a key role in promoting infectivity through the Tat-TAR complex and in the emergence from latency. These considerations have led to the synthesis and evaluation of numerous small-molecule and peptidic inhibitors of the Tat-TAR interaction during the last 15 years (4-7). However, none of these molecules had sufficient potency or selectivity to progress into preclinical studies and, indeed, inhibiting the Tat-TAR interaction has proven challenging. First, there is little precedent for the pharmacological disruption...

The amine-catalyzed enantioselective Michael addition of aldehydes to nitro alkenes (Scheme 1) is known to be acid-catalyzed (Fig. 1). A mechanistic investigation of this reaction, catalyzed by diphenylprolinol trimethylsilyl ether is described. Of the 13 acids tested, 4-NO 2 ÀC 6 H 4 OH turned out to be the most effective additive, with which the amount of catalyst could be reduced to 1 mol-% (Tables 2 -5). Fast formation of an amino-nitro-cyclobutane 12 was discovered by in situ NMR analysis of a reaction mixture. Enamines, preformed from the prolinol ether and aldehydes (benzene/molecular sieves), and nitroolefins underwent a stoichiometric reaction to give single all-trans-isomers of cyclobutanes (Fig. 3) in a [2 þ 2] cycloaddition. This reaction was shown, in one case, to be acid-catalyzed (Fig. 4) and, in another case, to be thermally reversible (Fig. 5). Treatment of benzene solutions of the isolated aminonitro-cyclobutanes with H 2 O led to mixtures of 4-nitro aldehydes (the products 7 of overall Michael addition) and enamines 13 derived thereof (Figs. 6 -9). From the results obtained with specific examples, the following tentative, general conclusions are drawn for the mechanism of the reaction (Schemes 2 and 3): enamine and cyclobutane formation are fast, as compared to product formation; the zwitterionic primary product 5 of C,C-bond formation is in equilibrium with the product of its collapse (the cyclobutane) and with its precursors (enamine and nitro alkene); when protonated at its nitronate anion moiety the zwitterion gives rise to an iminium ion 6, which is hydrolyzed to the desired nitro aldehyde 7 or deprotonated to an enamine 13. While the enantioselectivity of the reaction is generally very high (> 97% ee), the diastereoselectivity depends upon the conditions, under which the reaction is carried out (Fig. 10 and Tables 1 -5). Various acid-catalyzed steps have been identified. The cyclobutanes 12 may be considered an off-cycle reservoir of catalyst, and the zwitterions 5 the key players of the process (bottom part of Scheme 2 and Scheme 3).

The HIV-1 TAR RNA represents a well-known paradigm to study the role of dynamics and conformational change in RNA function. This regulatory RNA changes conformation in response to binding of Tat protein and of a variety of peptidic and small molecule ligands, indicating that its conformational flexibility and intrinsic dynamics play important roles in molecular recognition. We have used 13C NMR relaxation experiments to examine changes in the motional landscape of HIV-1 TAR in the presence of three ligands of different affinity and specificity. The ligands are argininamide, a linear peptide mimic of the Tat basic domain and a cyclic peptide that potently inhibits Tat-dependent activation of transcription. All three molecules induce the same motional characteristics within the three nucleotides bulge that represents the Tat-binding site. However, the cyclic peptide has a unique motional signature in the apical loop, which represents a binding site for the essential host co-factor cyclin T1. These results suggest that all peptidic mimics of Tat induce the same dynamics in TAR within this protein binding site. However, the new cyclic peptide mimic of Tat represents a new class of ligands with a unique effect on the dynamics and the structure of the apical loop.

The pharmacological disruption of the interaction between the HIV Tat protein and its cognate transactivation response RNA (TAR) would generate novel anti-viral drugs with a low susceptibility to drug resistance, but efforts to discover ligands with sufficient potency to warrant pharmaceutical development have been unsuccessful. We have previously described a family of structurally constrained β-hairpin peptides that potently inhibits viral growth in HIV-infected cells. The nuclear magnetic resonance (NMR) structure of an inhibitory complex revealed that the peptide makes intimate contacts with the 3-nt bulge and the upper helix of the RNA hairpin, but that a single residue contacts the apical loop where recruitment of the essential cellular co-factor cyclin T1 occurs. Attempting to extend the peptide to form more interactions with the RNA loop, we examined a library of longer peptides and achieved >6-fold improvement in affinity. The structure of TAR bound to one of the extended peptides reveals that the peptide slides down the major groove of the RNA, relative to our design, in order to maintain critical interactions with TAR. These conserved contacts involve three amino acid side chains and identify critical interaction points required for potent and specific binding to TAR RNA. They constitute a template of essential interactions required for inhibition of this RNA.

Graphical AbstractHighlights d HDAC4 is an active enzyme d HDAC4 deacetylates myosin heavy chain, PGC-1a, and Hsc70 d HDAC4 inhibition increases myosin heavy chain and PGC-1a protein levels d Maintaining acetylation of myosin heavy chain and Hsc70 prevents atrophy SUMMARY HDAC4, a class IIa histone deacetylase, is upregulated in skeletal muscle in response to denervationinduced atrophy. When HDAC4 is deleted postnatally, mice are partially protected from denervation. Despite the name ''histone'' deacetylase, HDAC4 demonstrably deacetylates cytosolic and non-histone nuclear proteins. We developed potent and selective class IIa HDAC inhibitors. Using these tools and genetic knockdown, we identified three previously unidentified substrates of HDAC4: myosin heavy chain, peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1a), and heat shock cognate 71 kDa protein (Hsc70). HDAC4 inhibition almost completely prevented denervation-induced loss of myosin heavy chain isoforms and blocked the action of their E3 ligase, MuRF1. PGC-1a directly interacts with class IIa HDACs; selective inhibitors increased PGC-1a protein in muscles. Hsc70 deacetylation by HDAC4 affects its chaperone activity. Through these endogenous HDAC4 substrates, we identified several muscle metabolic pathways that are regulated by class IIa HDACs, opening up new therapeutic options to treat skeletal muscle disorders and potentially other disease where these specific pathways are affected. Cell Reports 29, 749-763, October 15, 2019 ª 2019 The Author(s). 749 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).(D) Densitometry of Immunoreactive bands in (C). The ratio of MyHC IIb to sarcomeric actin was determined. Relative mean fold change (versus CON of HDAC4 iRKO mice) ± SEM was calculated and plotted. #### p < 0.0001 comparing control legs (CONs) to denervated legs (DENs) of WT mice. ***p < 0.01 comparing linked groups using two-way ANOVA and Sidak's post hoc test. (E) MYH gene expression in GAS muscles from CONs and DENs of female WT and HDAC4 iRKO mice. Geometric mean of TBP and VPS26A levels was used to normalize RNA amount added. The relative mean fold change (versus CON of flox À/À Cremice) ± SEM is plotted.(C) Venn diagram of genes regulated in DENs of HDAC4 iRKO mice or WT mice treated with NVS-HD1, both compared to WT mice treated with VEH. (D) Gene expression changes in GAS muscles from CONs and DENs of 8-month-old mice treated with NVS-HD1 5-days post denervation. Geometric mean of TBP, B2M, and VPS26A levels was used to normalize RNA amount added. Relative mean fold change (versus CONs of VEH-treated mice) ± SEM is plotted.

The terminal homologation by CH(2) insertion into the peptides mentioned in the title is described. This involves replacement of the N-terminal amino acid residue by a β(2) - and of the C-terminal amino acid residue by a β(3) -homo-amino acid moiety (β(2) hXaa and β(3) hXaa, resp.; Fig. 1). In this way, the structure of the peptide chain from the N-terminal to the C-terminal stereogenic center is identical, and the modified peptide is protected against cleavage by exopeptidases (Figs. 2 and 3). Neurotensin (NT; 1) and its C-terminal fragment NT(8-13) are ligands of the G-protein-coupled receptors (GPCR) NT1, NT2, NT3, and NT analogs are promising tools to be used in cancer diagnostics and therapy. The affinities of homologated NT analogs, 2b-2e, for NT1 and NT2 receptors were determined by using cell homogenates and tumor tissues (Table 1); in the latter experiments, the affinities for the NT1 receptor are more or less the same as those of NT (0.5-1.3 vs. 0.6 nM). At the same time, one of the homologated NT analogs, 2c, survives in human plasma for 7 days at 37° (Fig. 6). An NMR analysis of NT(8-13) (Tables 2 and 4, and Fig. 8) reveals that this N-terminal NT fragment folds to a turn in CD(3) OH. - In the case of the human analgesic opiorphin (3a), a pentapeptide, and of the HIV-derived B27-KK10 (4a), a decapeptide, terminal homologation (→3b and 4b, resp.) led to a 7- and 70-fold half-life increase in plasma (Fig. 9). With N-terminally homologated NPY, 5c, we were not able to determine serum stability; the peptide consisting of 36 amino acid residues is subject to cleavage by endopetidases. Three of the homologated compounds, 2b, 2c, and 5c, were shown to be agonists (Fig. 7 and 11). A comparison of terminal homologation with other stability-increasing terminal modifications of peptides is performed (Fig. 5), and possible applications of the neurotensin analogs, described herein, are discussed.

Facioscapulohumeral muscular dystrophy (FSHD) is among the most prevalent of the adult-onset muscular dystrophies. FSHD causes a loss of muscle mass and function, resulting in severe debilitation and reduction in quality of life. Currently, only the symptoms of FSHD can be treated, and such treatments have minimal benefit. The available options are not curative, and none of the treatments address the underlying cause of FSHD. The genetic, epigenetic, and molecular mechanisms triggering FSHD are now quite well-understood, and it has been shown that expression of the transcriptional regulator () is necessary for disease onset and is largely thought to be the causative factor in FSHD. Therefore, we sought to identify compounds suppressing expression in a phenotypic screen using FSHD patient-derived muscle cells, a zinc finger and SCAN domain-containing 4 (ZSCAN4)-based reporter gene assay for measuring DUX4 activity, and ∼3,000 small molecules. This effort identified molecules that reduce gene expression and hence DUX4 activity. Among those, β-adrenergic receptor agonists and phosphodiesterase inhibitors, both leading to increased cellular cAMP, effectively decreased expression by>75% in cells from individuals with FSHD. Of note, we found that cAMP production reduces expression through a protein kinase A-dependent mode of action in FSHD patient myotubes. These findings increase our understanding of how expression is regulated in FSHD and point to potential areas of therapeutic intervention.

Abstract:We report structural studies in aqueous solution on backbone cyclic peptides that possess potent antimicrobial activity specifically against Pseudomonas sp. The peptides target the -barrel outer membrane protein LptD, which plays an essential role in lipopolysaccharide transport to the outer membrane. The peptide L27-11 contains a 12-residue loop (T(1)W(2)L(3)K(4)K(5)R(6)R(7)W(8)K(9)K(10)A(11)K(12)) linked to a DPro-LPro template. Two related peptides were also studied, one with various Lys to ornithine or diaminobutyric acid substitutions as well as a DLys(6) (called LB-01), and another containing the same loop sequence, but linked to an LPro-DPro template (called LB-02). NMR studies and MD simulations show that L27-11 and LB-01 adopt -hairpin structures in solution. In contrast, LB-02 is more flexible and importantly, adopts a wide variety of different backbone conformations, but not -hairpin conformations. L27-11 and LB-01 show antimicrobial activity in the nanomolar range against Pseudomonas aeruginosa, whereas LB-02 is essentially inactive. Thus the -hairpin structure of the peptide is important for antimicrobial activity. An alanine scan of L27-11 revealed that tryptophan side chains (W(2)/W(8)) displayed on opposite faces of the -hairpin represent key groups contributing to antimicrobial activity. conformations. L27-11 and LB-01 both show antimicrobial activity in the nanomolar range against P. aeruginosa, whereas LB-02 is essentially inactive. Thus the ß-hairpin structure of the peptides appears to be very important for their antimicrobial activity.We surmise that a ß-hairpin-shaped peptide might be especially well suited to bind to proteins rich in ß-structure such as LptD in a membrane environment.