Essential structural requirements for specific recognition of HIV TAR RNA by peptide mimetics of Tat protein

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 RN

Essential structural requirements for specific recognition of HIV TAR RNA by peptide mimetics of Tat protein

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

Recombinant Expression, Purification, and Characterization of an HIV-1 Tat-Human Cyclin T1 Chimera

Efficient transcription of the human immunodeficiency virus type 1 (HIV-1) requires the interaction of the viral protein Tat with the trans-activation response (TAR) stem-loop of the long-terminal repeat (LTR) portion of nascent viral RNA. The production of viable transcripts is enhanced dramatically by the interaction of HIV-1 Tat with the host protein human Cyclin T1. Interaction with hCycT1 remodels Tat protein contributing a single cysteine residue that is critical to the formation of the second of two zinc fingers (Zn2). Here we suggest that it is the presence of this critical cysteine residue and not the presence of arginine residues from human Cyclin T1 that imparts high affinity and specificity to the interaction with HIV-1 TAR RNA. Crucial structural features of this interaction remain unresolved by NMR or existing crystal structures. Specifically, the structure of the Tat activation domain (AD), and Tat interaction with hCycT1 while bound to HIV-1 TAR RNA remain elusive. Much of the difficulty in obtaining structural data is a result of the notoriously difficult expression of native HIV-1 Tat caused in large part by the high cysteine count, and poor solubility of the Tat protein. This work presents a protocol for the expression and purification of a high affinity recombinant chimeric protein which includes the full 101 amino acid Tat protein fused to an essential minimal portion of CycT1m) necessary for TAR binding in sufficient purity and concentration for structural study by nuclear magnetic resonance (NMR).The elucidation of this critical region has the potential for profound impact in the structural based drug design of HIV-1 therapeutics

From cell penetrating peptides to peptoids and polyamines as novel artificial molecular transporters

In recent years, RNA interference has gained a lot of importance as a tool for posttranscriptional silencing of genes due to its high specificity, efficiency and ease of application. In mammalian cells RNAi is triggered by the application of 21 bp short dsRNAs, so-called short interfering RNAs (siRNAs). Numerous studies indicate the great potential of RNAi in the therapy of viral infections and inherited diseases. Cell-penetrating peptides can be used to apply siRNAs to primary cells, non-dividing cells or fully grown organisms, that are diffcult to transfect. These short positively charged peptides are internalized by cells. They can be detected in the endosomes, lysosomes but also in the cytosol. If attached to CPPs, large cargo molecules can be taken up with a high efficiency that surpasses that of most conventional transfection methods. In the work presented here, peptide-coupled siRNAs (pepsiRNAs) have been developed as a novel tool for transient RNAi in mammalian cells. The peptides were attached to the siRNA by a disulfide bond, that is cleaved under the reducing conditions of the cytosol and thus releases the siRNA cargo. PepsiRNAs are readily taken up by many cell types that are difficult to address by conventional transfection methods. An siRNA-induced downregulation of the targeted genes was observed at concentrations between 10 and 100 nM. SiRNAs with a 5´-thiol modification upon their sense-strand were generated by in vitro transcription, for which a thiol-modified nucleotide was synthesized via an optimized route. The cell-penetrating peptides Penetratin and Tat were recombinantly expressed as fusion-proteins with glutathione-S-transferase (GST) and purified with a modified procedure to overcome the strong membrane interaction of the GST-tagged CPPs. Recombinant TEV protease was expressed to cleave the CPPs from the fusion tag, and the cleavage activity was assessed by comparison with commercially obtained TEV protease. Thus, alternative routes to the building blocks for pepsiRNAs have been provided to scale up the amount of pepsiRNAs. Finally, small molecules with cell-penetrating properties have been developed as a future replacement of the peptide moiety. Fluorescently labeled peptoids of differing length with amine-functionalized side chains have been shown to enter different mammalian cells lines at concentrations in the lower micromolar range by an endocytosis dependent mechanism. Cationic molecules as small as spermine attached to fluorescein are taken up by an endocytosis-related mechanism. By the same approach porphyrins were delivered into the interior of the cells, where they exhibited a cytotoxic effect upon illumination, so that spermine-coupled porphyrins may be developed into a novel drug for photodynamic therapy.Entwicklung neuer Delivery-Strategien für siRNAs - Von zellpenetrierenden Peptiden zu Peptoiden und Polyaminen als neuartige molekulare Transporter In den vergangenen Jahren, hat die RNA Interferenz (RNAi) aufgrund ihrer Spezifität, Effizienz und einfachen Anwendbarkeit eine große Bedeutung als Technik für das posttranskriptionale Gene-Silencing gewonnen. In Säugerzellen wird RNAi durch die Gabe von 21 bp kurzen doppelsträngigen RNAs, sogenannten short interfering RNAs (siRNAs), ausgelöst. Zahlreiche Studien belegen das große Potential dieser Methode für die Therapie viraler Infektionen und Erbkrankheiten. Um siRNAs auch in schlecht transfizierbare Systeme wie primäre und nicht-teilende Zellen bzw. ausgewachsene Organismen einzubringen, können zellpenetrierende Peptide (CPPs) verwendet werden. Diese kurzen positiv geladenen Peptide werden von Zellen aufgenommen, wo sie in Endosomen, Phagosomen, aber auch im Zytosol detektierbar sind. Verknüpft mit CPPs werden große Moleküle mit hoher Effizienz von Zellen aufgenommen, die die der meisten konventionellen Transfektionsmethoden übertrifft. In dieser Arbeit wurden peptidgekuppelte siRNAs (pepsiRNAs) als neue Methode zur transienten RNAi in Säugerzellen entwickelt. Hierzu wurden die Peptide über eine Disulfidbrücke mit den siRNAs verknüpft, unter den reduzierenden Bedingungen des Zytosols gespalten wird und , so dass die siRNA im Innern der Zelle freigesetzt wird. PepsiRNAs werden von einer Reihe von Zelltypen aufgenommen, die mit konventionellen Techniken nur schlecht behandelbar sind. Eine deutliche Herunterregulation der Ziel-Genprodukte wurde in einem Konzentrationsbereich von 10 -100 nM beobachtet. SiRNAs mit einer 5´-Thiolmodifikation am sense-Strang wurden durch in vitro Transkription gewonnen, für die ein thiolmodifizierten Nucleotid synthetisiert und dessen Synthese optimiert wurde. Die zellpenetrierenden Peptide Penetratin und Tat wurden rekombinant als Fusionproteine mit Gluatathion-S-Transferase (GST) exprimiert und mit modifizierten Verfahren gereinigt, um die starken Wechselwirkungen der GST-CPPs mit den Zellmembranen zu überwinden. Um die CPPs vom GST-Fusionstag zu trennen, wurde rekombinante TEV-Protease exprimiert und die Aktivität mit der von kommerziell erhältlicher TEV-Protease verglichen. Somit stehen nun Alternativen zur Festphasensynthese zur Verfügung, um die Bestandteile der pepsiRNAs in größeren Mengen herzustellen. Schließlich wurden kleine Moleküle mit zellpenetrierenden Eigenschaften als Ersatz für die Peptideinheit entwickelt. Es konnte gezeigt werden, dass fluoreszenzmarkierte Peptoide verschiedener Länge mit aminofunktionalisierten Seitenketten in Konzentrationen von einigen 10 µM von verschiedenen Säugerzelllinien durch einen endozytoseabhängigen Mechanismus aufgenommen werden. Selbst kleine positiv geladene Moleküle wie fluoresceinmarkiertes Spermin werden über einen endozytoseartigen Mechanismus aufgenommen. Auf diese Weise konnten auch Porphyrine ins Innere der Zellen gebracht werden, wo sie bei Belichtung ihre zytotoxische Wirkung entfalten, so dass sie als neuartiger Wirkstoff für die Photodynamische Therapie weiterentwickelt werden können

Study of macromolecular interactions using computational solvent mapping

The term "binding hot spots" refers to regions of a protein surface with large contributions to the binding free energy. Computational solvent mapping serves as an analog to the major experimental techniques developed for the identification of such hot spots using X-ray and nuclear magnetic resonance (NMR) methods. Applications of the fast Fourier-transform-based mapping algorithm FTMap show that similar binding hot spots also occur in DNA molecules and interact with small molecules that bind to DNA with high affinity. Solvent mapping results on B-DNA, with or without Hoogsteen (HG) base pairing, have revealed the significance of "HG breathing" on the reactivity of DNA with formaldehyde. Extending the method to RNA molecules, I applied the FTMap algorithm to flexible structures of HIV-1 transactivation response element (TAR) RNA and Tau exon 10 RNA. Results show that despite the extremely flexible nature of these small RNA molecules, nucleic acid bases that interact with ligands consistently have high hit rates, and thus binding sites can be successfully identified. Based on this experience as well as the prior work on DNA, I extended the FTMap algorithm to mapping nucleic acids and implemented it in an automated online server available to the research community. FTSite, a related server for finding binding sites of proteins, was also extended to develop PeptiMap, an accurate and robust protocol that can determine peptide binding sites on proteins. Analyses of structural ensembles of ligand-free proteins using solvent mapping have shown that such ensembles contain pre-existing binding hot spots, and that such hot spots can be identified without any a priori knowledge of the ligand-bound structure. Furthermore, the structures in the ensemble having the highest binding-site hit rate are closest to the ligand-bound structure, and a higher hit rate implies improved structural similarity between the unbound protein and its bound state, resulting in high correlation coefficient between the two measures. These advances should greatly enhance researchers' ability to identify functionally important interactions among biomolecules in silico

Affinity maturation and characterization of novel binders to the HIV-1 TAR element based on the U1A RNA recognition motif

2018 Fall.Includes bibliographical references.The increased understanding of the importance of RNA, both as a carrier of information and as a functional molecule, has led to a greater demand for the ability to target specific RNAs, but the limited chemical diversity of RNA makes this challenging. This thesis documents the use of yeast display to perform affinity maturation for the ability of a protein to bind the TAR element of HIV-1, which is a desirable therapeutic target due to its prominent role in the HIV-1 infection cycle. To accomplish this, we used a "semi-design" strategy—repurposing a natural RNA bind- ing protein to bind a different target—by creating a library based on important binding regions (especially the β2β3 loop) of the U1A RRM. Following selection for TAR binding, a strong consensus sequence in the β2β3 loop emerged. The affinity of certain library members for TAR was measured by ELISA and SPR, and it was determined that the best binder (TBP 6.7) had remarkable affinity (KD = ~500 pM). This TAR binding protein also proved capable of disrupting the Tat–TAR interaction (necessary for HIV-1 replication) both in vitro and in the context of extracellular transcription. Through collaboration, we were able to obtain a co-crystal structure of TBP 6.7 and TAR. This crystal structure showed that the overall structure of TBP 6.7 was largely unchanged from that of U1A, thereby validating our semi-design strategy. We also found that the β2β3 loop played a disproportionately large role in the binding interaction (~2⁄3 of the buried surface area). The importance of this region inspired the creation and characterization of peptide derivatives of the TBP 6.7 β2β3 loop. These β2β3 loop derived peptides maintain affinity for TAR RNA (KD = ~1.8 μM), and can disrupt Tat/TAR-dependent transcription. Ultimately, the project has yielded the most avid known binders of TAR RNA, a potential novel platform of TAR binding peptides, and a crystal structure which will hopefully inform future targeting efforts

Isolation And Analysis Of Peptides Binding To Helix 69 Of E. Coli 23 S Rrna From M13 Phage Display

Peptides binding to helix 69 of domain IV or residues 1906-1924, of 23 S rRNA of E.coli were being selected from a heptapeptide phage library. An experimental system including biotin labeling of RNA and then affinity selection through four rounds was being followed. After sequencing phage clones of the fourth round, two peptide sequences dominated the phage pool, STYTSVS and NQVANHQ. The later sequence was a unique sequence since this sequence contained an abundance of amino acid residues that are also present in the ribosome recycling factor, RRF, and known to make contacts with H69. Phage-display methodology demonstrated the rapid feasibility of identification, and isolation of small peptides that bind to 23 S rRNA in an effort to discover new RNA-binding motifs that have potential therapeutic applications. For evaluating the preliminary binding affinity of these peptides with H69, fluorescence assays were applied. For this assay, the fluorescence intensity of the NQVANHQ Tentagel beads was observed to be higher than STYTSVS Tentagel beads indicating that peptide NQVANHQ is having higher affinity for H69 as compared to STYTSVS peptide. But the higher binding affinity of the NQVANHQ peptide was further validated with more sensitive method of electrospray ionization (ESI) mass spectroscopy. The apparent dissociation constant (Kd) obtained for H69 and NQVANHQ-NH2 peptide was in the low micromolar range (11 µM). This value is comparable to that of aminoglycoside antibiotics binding to the A-site RNA (1 to 10 µM). The ESI-MS experiments with H69 variant UUU RNA and peptide NQVANHQ-NH2 gave the relative dissociation constant (Kd) at 1:1 stoichiometry as 19 µM. The higher value of Kd for this complex revealed that the presence of all three pseudouridine residues positively contributed towards binding of this peptide to H69. Consecutively, to learn about the role of individual pseudouridines at position 1911 and 1915 towards binding of the peptide, the ESI-MS experiments were performed with two H69 variants,UYY and YUY. The apparent dissociation constants (Kd) for the 1:1 complex for these two RNAs decreased by 2.5-fold showing that peptide binding site is located at or near the loop region containing the pseudouridines at positions 1911 and 1915. In addition, the effect of pH on the complex formation of H69 and UUU RNA with NQVANHQ-NH2 peptide was studied at two different pH values of 7.0 and 5.2. There was three-fold decrease of the apparent dissociation constant for the 1:1 complex of RNA and the peptide indicating that either protonation of the RNA or the peptide structure influenced this change in binding of the two species. The specificity of the peptide for H69 was tested with related RNA such as human H69 and unrelated RNAs such as helix 31 and A-site rRNA. The peptide showed three-fold lower affinity than the target H69 RNA for all these RNAs suggesting that the peptide has features for developing it as a lead compound for novel antimicrobial

Engineering and evolving helical proteins that improve in vivo stability and inhibit entry of Enfuvirtide-resistant HIV-1

2019 Spring.Includes bibliographical references.Methods for the stabilization of well-defined helical peptide drugs and basic research tools have received considerable attention in the last decade. Enfuvirtide is a 36-residue chemically synthesized helical peptide that targets the viral gp41 protein and inhibits viral membrane fusion. Enfuvirtide-resistant HIV, however, has been prolific, and this peptide therapy requires daily injection due to proteolytic degradation. In this dissertation I have developed a method for stabilizing helical peptide therapeutics termed helix-grafted display proteins. These consist of the HIV-1 gp41 C-peptide helix grafted onto Pleckstrin Homology domains. Some of these earlier protein biologics inhibit HIV-1 entry with modest and variable potencies (IC50 190 nM - >1 μM). After optimization of the scaffold and the helix, our designer peptide therapeutic potently inhibited HIV-1 entry in a live-virus assay (IC50 1.9-12.4 nM). Sequence optimization of solvent-exposed helical residues using yeast display as a screening method led to improved biologics with enhanced protein expression in Escherichia coli (E. coli, a common bio-expression host), with no appreciable change in viral membrane fusion suppression. Optimized proteins suppress the viral entry of a clinically-relevant double mutant of HIV-1 that is gp41 C-peptide sensitive and Enfuvirtide-resistant. Protein fusions engineered for serum-stability also potently inhibit HIV-1 entry