The multifaceted roles of mass spectrometric analysis in nucleic acids drug discovery and development.

The deceptively simple concepts of mass determination and fragment analysis are the basis for the application of mass spectrometry (MS) to a boundless range of analytes, including fundamental components and polymeric forms of nucleic acids (NAs). This platform affords the intrinsic ability to observe first-hand the effects of NA-active drugs on the chemical structure, composition, and conformation of their targets, which might affect their ability to interact with cognate NAs, proteins, and other biomolecules present in a natural environment. The possibility of interfacing with high-performance separation techniques represents a multiplying factor that extends these capabilities to cover complex sample mixtures obtained from organisms that were exposed to NA-active drugs. This report provides a brief overview of these capabilities in the context of the analysis of the products of NA-drug activity and NA therapeutics. The selected examples offer proof-of-principle of the applicability of this platform to all phases of the journey undertaken by any successful NA drug from laboratory to bedside, and provide the rationale for its rapid expansion outside traditional laboratory settings in support to ever growing manufacturing operations.

B-CePs as cross-linking probes for the investigation of RNA higher-order structure.

Elucidating the structure of RNA and RNA ensembles is essential to understand biological functions. In this work, we explored the previously uncharted reactivity of bis-chloropiperidines (B-CePs) towards RNA. We characterized at the molecular level the different adducts induced by the fast reacting compound B-CeP 1 with RNA. Following an approach based on solution thermal melting coupled with ESI mass spectrometry (STHEM-ESI), we proved the ability of B-CePs to induce inter-molecular cross-links between guanines in double stranded RNA. These results open the possibility of using B-CePs as structural probes for investigating higher-order structures, such as the kissing loop complex established by the dimerization initiation site (DIS) of the HIV-1 genome. We confirmed the potential of B-CePs to reveal the identity of RNA structures involved in long-range interactions, expecting to benefit the characterization of samples that are not readily amenable to traditional high-resolution techniques, and thus promoting the elucidation of pertinent RNA systems associated with old and new diseases.

In Vitro Evaluation of Bis-3-Chloropiperidines as RNA Modulators Targeting TAR and TAR-Protein Interaction.

After a long limbo, RNA has gained its credibility as a druggable target, fully earning its deserved role in the next generation of pharmaceutical R&D. We have recently probed the trans-activation response (TAR) element, an RNA stem-bulge-loop domain of the HIV-1 genome with bis-3-chloropiperidines (B-CePs), and revealed the compounds unique behavior in stabilizing TAR structure, thus impairing in vitro the chaperone activity of the HIV-1 nucleocapsid (NC) protein. Seeking to elucidate the determinants of B-CePs inhibition, we have further characterized here their effects on the target TAR and its NC recognition, while developing quantitative analytical approaches for the study of multicomponent RNA-based interactions.

Bis-3-Chloropiperidines Targeting TAR RNA as A Novel Strategy to Impair the HIV-1 Nucleocapsid Protein.

Specific RNA sequences regulate functions essential to life. The Trans-Activation Response element (TAR) is an RNA stem-bulge-loop structure involved in several steps of HIV-1 replication. In this work, we show how RNA targeting can inhibit HIV-1 nucleocapsid (NC), a highly conserved protein known to catalyze nucleic acid melting and strand transfers during reverse transcription. Our RNA targeting strategy consists of the employment of bis-3-chloropiperidines (B-CePs) to impair RNA melting through bifunctional alkylation. Specific interactions between B-CePs and TAR RNA were analytically investigated by gel electrophoresis and mass spectrometry, allowing the elucidation of B-CePs' recognition of TAR, and highlighting an RNA-directed mechanism of protein inhibition. We propose that B-CePs can freeze TAR tridimensional conformation, impairing NC-induced dynamics and finally inhibiting its functions in vitro.

Structure of an engineered intein reveals thiazoline ring and provides mechanistic insight.

We have engineered an intein which spontaneously and reversibly forms a thiazoline ring at the native N-terminal Lys-Cys splice junction. We identified conditions to stablize the thiazoline ring and provided the first crystallographic evidence, at 1.54 Å resolution, for its existence at an intein active site. The finding bolsters evidence for a tetrahedral oxythiazolidine splicing intermediate. In addition, the pivotal mutation maps to a highly conserved B-block threonine, which is now seen to play a causative role not only in ground-state destabilization of the scissile N-terminal peptide bond, but also in steering the tetrahedral intermediate toward thioester formation, giving new insight into the splicing mechanism. We demonstrated the stability of the thiazoline ring at neutral pH as well as sensitivity to hydrolytic ring opening under acidic conditions. A pH cycling strategy to control N-terminal cleavage is proposed, which may be of interest for biotechnological applications requiring a splicing activity switch, such as for protein recovery in bioprocessing.

Non-Natural Linker Configuration in 2,6-Dipeptidyl-Anthraquinones Enhances the Inhibition of TAR RNA Binding/Annealing Activities by HIV-1 NC and Tat Proteins.

The HIV-1 nucleocapsid (NC) protein represents an excellent molecular target for the development of anti-retrovirals by virtue of its well-characterized chaperone activities, which play pivotal roles in essential steps of the viral life cycle. Our ongoing search for candidates able to impair NC binding/annealing activities led to the identification of peptidyl-anthraquinones as a promising class of nucleic acid ligands. Seeking to elucidate the inhibition determinants and increase the potency of this class of compounds, we have now explored the effects of chirality in the linker connecting the planar nucleus to the basic side chains. We show here that the non-natural linker configuration imparted unexpected TAR RNA targeting properties to the 2,6-peptidyl-anthraquinones and significantly enhanced their potency. Even if the new compounds were able to interact directly with the NC protein, they manifested a consistently higher affinity for the TAR RNA substrate and their TAR-binding properties mirrored their ability to interfere with NC-TAR interactions. Based on these findings, we propose that the viral Tat protein, sharing the same RNA substrate but acting in distinct phases of the viral life cycle, constitutes an additional druggable target for this class of peptidyl-anthraquinones. The inhibition of Tat-TAR interaction for the test compounds correlated again with their TAR-binding properties, while simultaneously failing to demonstrate any direct Tat-binding capabilities. These considerations highlighted the importance of TAR RNA in the elucidation of their inhibition mechanism, rather than direct protein inhibition. We have therefore identified anti-TAR compounds with dual in vitro inhibitory activity on different viral proteins, demonstrating that it is possible to develop multitarget compounds capable of interfering with processes mediated by the interactions of this essential RNA domain of HIV-1 genome with NC and Tat proteins.

Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription.

Folding of the LTR promoter into dynamic G-quadruplex conformations has been shown to suppress its transcriptional activity in HIV-1. Here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing G-quadruplex structures. The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex structures present in the LTR promoter. Nucleolin recognized with high affinity and specificity the majority, but not all the possible G-quadruplexes folded by this sequence. In addition, it displayed greater binding preference towards DNA than RNA G-quadruplexes, thus indicating two levels of selectivity based on the sequence and nature of the target. The interaction translated into stabilization of the LTR G-quadruplexes and increased promoter silencing activity; in contrast, disruption of nucleolin binding in cells by both siRNAs and a nucleolin binding aptamer greatly increased LTR promoter activity. These data indicate that nucleolin possesses a specific and regulated activity toward the HIV-1 LTR promoter, which is mediated by G-quadruplexes. These observations provide new essential insights into viral transcription and a possible low mutagenic target for antiretroviral therapy.

Mechanisms of HIV-1 Nucleocapsid Protein Inhibition by Lysyl-Peptidyl-Anthraquinone Conjugates.

The Nucleocapsid protein NCp7 (NC) is a nucleic acid chaperone responsible for essential steps of the HIV-1 life cycle and an attractive candidate for drug development. NC destabilizes nucleic acid structures and promotes the formation of annealed substrates for HIV-1 reverse transcription elongation. Short helical nucleic acid segments bordered by bulges and loops, such as the Trans-Activation Response element (TAR) of HIV-1 and its complementary sequence (cTAR), are nucleation elements for helix destabilization by NC and also preferred recognition sites for threading intercalators. Inspired by these observations, we have recently demonstrated that 2,6-disubstituted peptidyl-anthraquinone-conjugates inhibit the chaperone activities of recombinant NC in vitro, and that inhibition correlates with the stabilization of TAR and cTAR stem-loop structures. We describe here enhanced NC inhibitory activity by novel conjugates that exhibit longer peptidyl chains ending with a conserved N-terminal lysine. Their efficient inhibition of TAR/cTAR annealing mediated by NC originates from the combination of at least three different mechanisms, namely, their stabilizing effects on nucleic acids dynamics by threading intercalation, their ability to target TAR RNA substrate leading to a direct competition with the protein for the same binding sites on TAR, and, finally, their effective binding to the NC protein. Our results suggest that these molecules may represent the stepping-stone for the future development of NC-inhibitors capable of targeting the protein itself and its recognition site in RNA.

H/ACA snRNP-dependent ribosome biogenesis regulates translation of polyglutamine proteins.

Stem cells in many systems, including Drosophila germline stem cells (GSCs), increase ribosome biogenesis and translation during terminal differentiation. Here, we show that the H/ACA small nuclear ribonucleoprotein (snRNP) complex that promotes pseudouridylation of ribosomal RNA (rRNA) and ribosome biogenesis is required for oocyte specification. Reducing ribosome levels during differentiation decreased the translation of a subset of messenger RNAs that are enriched for CAG trinucleotide repeats and encode polyglutamine-containing proteins, including differentiation factors such as RNA-binding Fox protein 1. Moreover, ribosomes were enriched at CAG repeats within transcripts during oogenesis. Increasing target of rapamycin (TOR) activity to elevate ribosome levels in H/ACA snRNP complex-depleted germlines suppressed the GSC differentiation defects, whereas germlines treated with the TOR inhibitor rapamycin had reduced levels of polyglutamine-containing proteins. Thus, ribosome biogenesis and ribosome levels can control stem cell differentiation via selective translation of CAG repeat-containing transcripts.

Methylation of rRNA as a host defense against rampant group II intron retrotransposition.

BACKGROUND: Group II introns are mobile retroelements, capable of invading new sites in DNA. They are self-splicing ribozymes that complex with an intron-encoded protein to form a ribonucleoprotein that targets DNA after splicing. These molecules can invade DNA site-specifically, through a process known as retrohoming, or can invade ectopic sites through retrotransposition. Retrotransposition, in particular, can be strongly influenced by both environmental and cellular factors. RESULTS: To investigate host factors that influence retrotransposition, we performed random insertional mutagenesis using the ISS1 transposon to generate a library of over 1000 mutants in Lactococcus lactis, the native host of the Ll.LtrB group II intron. By screening this library, we identified 92 mutants with increased retrotransposition frequencies (RTP-ups). We found that mutations in amino acid transport and metabolism tended to have increased retrotransposition frequencies. We further explored a subset of these RTP-up mutants, the most striking of which is a mutant in the ribosomal RNA methyltransferase rlmH, which exhibited a reproducible 20-fold increase in retrotransposition frequency. In vitro and in vivo experiments revealed that ribosomes in the rlmH mutant were defective in the m3Ψ modification and exhibited reduced binding to the intron RNA. CONCLUSIONS: Taken together, our results reinforce the importance of the native host organism in regulating group II intron retrotransposition. In particular, the evidence from the rlmH mutant suggests a role for ribosome modification in limiting rampant retrotransposition.

Kinetic study of beta-amyloid residue accessibility using reductive alkylation and mass spectrometry.

Beta-amyloid peptide (Abeta) is the major protein constituent found in senile plaques in Alzheimer's disease (AD). It is believed that Abeta plays a role in neurodegeneration associated with AD and that its toxicity is related to its structure or aggregation state. In this study, an approach based on chemical modification of primary amines and mass spectrometric (MS) detection was used to identify residues on Abeta peptide that were exposed or buried upon changes in peptide structure associated with aggregation. Results indicate that the N terminus was the most accessible primary amine in the fibril, followed by lysine 28, then lysine 16. A kinetic analysis of the data was then performed to quantify differences in accessibility between these modification sites. We estimated apparent equilibrium unfolding constants for each modified site of the peptide, and determined that the unfolding constant for the N terminus was approximately 100 times greater than that for K28, which was about six times greater than that for K16. Understanding Abeta peptide structure at the residue level is a first step in designing novel therapies for prevention of Abeta structural transitions and/or cell interactions associated with neurotoxicity in Alzheimer's disease.

Identification of novel 2-benzoxazolinone derivatives with specific inhibitory activity against the HIV-1 nucleocapsid protein.

In this report, we present a new benzoxazole derivative endowed with inhibitory activity against the HIV-1 nucleocapsid protein (NC). NC is a 55-residue basic protein with nucleic acid chaperone properties, which has emerged as a novel and potential pharmacological target against HIV-1. In the pursuit of novel NC-inhibitor chemotypes, we performed virtual screening and in vitro biological evaluation of a large library of chemical entities. We found that compounds sharing a benzoxazolinone moiety displayed putative inhibitory properties, which we further investigated by considering a series of chemical analogues. This approach provided valuable information on the structure-activity relationships of these compounds and, in the process, demonstrated that their anti-NC activity could be finely tuned by the addition of specific substituents to the initial benzoxazolinone scaffold. This study represents the starting point for the possible development of a new class of antiretroviral agents targeting the HIV-1 NC protein.

Direct and Topoisomerase†II Mediated DNA Damage by Bis-3-chloropiperidines: The Importance of Being an Earnest G.

Bis-3-chloropiperidines are a new class of DNA-active compounds capable of alkylating nucleobases and inducing strand cleavage. In this study, we investigated the reactivity of these mustard-based agents with both single- and double-stranded DNA constructs. Polyacrylamide gel electrophoresis (PAGE) and electrospray ionization mass spectrometry (ESI-MS) were used to obtain valuable insight into their mechanism at the molecular level and to investigate their time- and concentration-dependent activity. The results revealed the preferential formation of mono- and bifunctional adducts at nucleophilic guanine sites. In a stepwise fashion, alkylation was followed by depurination and subsequent strand scission at the ensuing apurinic site. We demonstrated that the covalent modifications introduced by this new class of compounds can inhibit the activity of essential DNA-processing proteins, such as topoisomerase IIα, thereby suggesting that bis-3-chloropiperidines may have excellent anticancer potential.

Synthesis and in Vitro Screening of New Series of 2,6-Dipeptidyl-anthraquinones: Influence of Side Chain Length on HIV-1 Nucleocapsid Inhibitors.

2,6-Dipeptidyl-anthraquinones are a promising class of nucleic acid-binding compounds that act as NC inhibitors in vitro. We designed, synthesized, and tested new series of 2,6-disubstituted-anthraquinones, which are able to bind viral nucleic acid substrates of NC. We demonstrate here that these novel derivatives interact preferentially with noncanonical structures of TAR and cTAR, stabilize their dynamics, and interfere with NC chaperone activity.

Decreased asialotransferrin in cerebrospinal fluid of patients with childhood-onset ataxia and central nervous system hypomyelination/vanishing white matter disease.

BACKGROUND: A biomarker for the diagnosis of childhood-onset ataxia and central nervous system hypomyelination (CACH)/vanishing white matter disease (VWM) would have clinical utility and pathophysiologic significance. METHODS: We used 2-dimensional gel electrophoresis/mass spectrometry to compare the cerebrospinal fluid proteome of patients with mutation-confirmed CACH/VWM with that of unaffected controls. We characterized selected spots by in-gel digestion, matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry, and nanospray Fourier transform mass spectrometry. RESULTS: A specific transferrin spot pattern was detected in the CSF samples of the CACH/VWM group (n = 7), distinguishing them from the control group (n = 23) and revealing that patients with CACH/VWM have a deficiency of the asialo form of transferrin usually present in healthy cerebrospinal fluid. The glycopeptide structure, determined from isolated transferrin spots by use of in-gel digestion and extraction, was found to be consistent with earlier reports. CONCLUSIONS: The transferrin isoform abnormality in the cerebrospinal fluid of patients with CACH/VWM appears unique and is a potential clinical diagnostic biomarker. The rapid, efficient diagnosis of this disorder would have a significant impact on clinical studies exploring new strategies for the management and treatment of this disease.