Selective small-molecule inhibition of an RNA structural element.

Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.

Factor XIIa as a Novel Target for Thrombosis: Target Engagement Requirement and Efficacy in a Rabbit Model of Microembolic Signals.

Coagulation Factor XII (FXII) plays a critical role in thrombosis. What is unclear is the level of enzyme occupancy of FXIIa that is needed for efficacy and the impact of FXIIa inhibition on cerebral embolism. A selective activated FXII (FXIIa) inhibitor, recombinant human albumin-tagged mutant Infestin-4 (rHA-Mut-inf), was generated to address these questions. rHA-Mut-inf displayed potency comparable to the original wild-type HA-Infestin-4 (human FXIIa inhibition constant = 0.07 and 0.12 nM, respectively), with markedly improved selectivity against Factor Xa (FXa) and plasmin. rHA-Mut-inf binds FXIIa, but not FXII zymogen, and competitively inhibits FXIIa protease activity. Its mode of action is hence akin to typical small-molecule inhibitors. Plasma shift and aPTT studies with rHA-Mut-inf demonstrated that calculated enzyme occupancy for FXIIa in achieving a putative aPTT doubling target in human, nonhuman primate, and rabbit is more than 99.0%. The effects of rHA-Mut-inf in carotid arterial thrombosis and microembolic signal (MES) in middle cerebral artery were assessed simultaneously in rabbits. Dose-dependent inhibition was observed for both arterial thrombosis and MES. The ED50 of thrombus formation was 0.17 mg/kg i.v. rHA-Mut-inf for the integrated blood flow and 0.16 mg/kg for thrombus weight; the ED50 for MES was 0.06 mg/kg. Ex vivo aPTT tracked with efficacy. In summary, our findings demonstrated that very high enzyme occupancy will be required for FXIIa active site inhibitors, highlighting the high potency and exquisite selectivity necessary for achieving efficacy in humans. Our MES studies suggest that targeting FXIIa may offer a promising strategy for stroke prevention associated with thromboembolic events.

Atomic resolution mechanistic studies of ribocil: A highly selective unnatural ligand mimic of the E. coli FMN riboswitch.

Bacterial riboswitches are non-coding RNA structural elements that direct gene expression in numerous metabolic pathways. The key regulatory roles of riboswitches, and the urgent need for new classes of antibiotics to treat multi-drug resistant bacteria, has led to efforts to develop small-molecules that mimic natural riboswitch ligands to inhibit metabolic pathways and bacterial growth. Recently, we reported the results of a phenotypic screen targeting the riboflavin biosynthesis pathway in the Gram-negative bacteria Escherichia coli that led to the identification of ribocil, a small molecule inhibitor of the flavin mononucleotide (FMN) riboswitch controlling expression of this biosynthetic pathway. Although ribocil is structurally distinct from FMN, ribocil functions as a potent and highly selective synthetic mimic of the natural ligand to repress riboswitch-mediated ribB gene expression and inhibit bacterial growth both in vitro and in vivo. Herein, we expand our analysis of ribocil; including mode of binding in the FMN binding pocket of the riboswitch, mechanisms of resistance and structure-activity relationship guided efforts to generate more potent analogs.

A hybrid NMR/SAXS-based approach for discriminating oligomeric protein interfaces using Rosetta.

Oligomeric proteins are important targets for structure determination in solution. While in most cases the fold of individual subunits can be determined experimentally, or predicted by homology-based methods, protein-protein interfaces are challenging to determine de novo using conventional NMR structure determination protocols. Here we focus on a member of the bet-V1 superfamily, Aha1 from Colwellia psychrerythraea. This family displays a broad range of crystallographic interfaces none of which can be reconciled with the NMR and SAXS data collected for Aha1. Unlike conventional methods relying on a dense network of experimental restraints, the sparse data are used to limit conformational search during optimization of a physically realistic energy function. This work highlights a new approach for studying minor conformational changes due to structural plasticity within a single dimeric interface in solution.

Structures of apo- and ssDNA-bound YdbC from Lactococcus lactis uncover the function of protein domain family DUF2128 and expand the single-stranded DNA-binding domain proteome.

Single-stranded DNA (ssDNA) binding proteins are important in basal metabolic pathways for gene transcription, recombination, DNA repair and replication in all domains of life. Their main cellular role is to stabilize melted duplex DNA and protect genomic DNA from degradation. We have uncovered the molecular function of protein domain family domain of unknown function DUF2128 (PF09901) as a novel ssDNA binding domain. This bacterial domain strongly associates into a dimer and presents a highly positively charged surface that is consistent with its function in non-specific ssDNA binding. Lactococcus lactis YdbC is a representative of DUF2128. The solution NMR structures of the 20 kDa apo-YdbC dimer and YdbC:dT(19)G(1) complex were determined. The ssDNA-binding energetics to YdbC were characterized by isothermal titration calorimetry. YdbC shows comparable nanomolar affinities for pyrimidine and mixed oligonucleotides, and the affinity is sufficiently strong to disrupt duplex DNA. In addition, YdbC binds with lower affinity to ssRNA, making it a versatile nucleic acid-binding domain. The DUF2128 family is related to the eukaryotic nuclear protein positive cofactor 4 (PC4) family and to the PUR family both by fold similarity and molecular function.

Identification and characterization of TYK2 pseudokinase domain stabilizers that allosterically inhibit TYK2 signaling.

Kinase inhibition continues to be a major focus of pharmaceutical research and discovery due to the central role of these proteins in the regulation of cellular processes. One family of kinases of pharmacological interest, due to its role in activation of immunostimulatory pathways, is the Janus kinase family. Small molecule inhibitors targeting the individual kinase proteins within this family have long been sought-after therapies. High sequence and structural similarity of the family members makes selective inhibitors difficult to identify but critical because of their inter-related multiple cellular regulatory pathways. Herein, we describe the identification of inhibitors of the important Janus kinase, TYK2, a regulator of type I interferon response. In addition, the biochemical and structural confirmation of the direct interaction of these small molecules with the TYK2 pseudokinase domain is described and a potential mechanism of allosteric regulation of TYK2 activity through stabilization of the pseudokinase domain is proposed.

Regulation of response regulator autophosphorylation through interdomain contacts.

DNA-binding response regulators (RRs) of the OmpR/PhoB subfamily alternate between inactive and active conformational states, with the latter having enhanced DNA-binding affinity. Phosphorylation of an aspartate residue in the receiver domain, usually via phosphotransfer from a cognate histidine kinase, stabilizes the active conformation. Many of the available structures of inactive OmpR/PhoB family proteins exhibit extensive interfaces between the N-terminal receiver and C-terminal DNA-binding domains. These interfaces invariably involve the α4-ß5-α5 face of the receiver domain, the locus of the largest differences between inactive and active conformations and the surface that mediates dimerization of receiver domains in the active state. Structures of receiver domain dimers of DrrB, DrrD, and MtrA have been determined, and phosphorylation kinetics were analyzed. Analysis of phosphotransfer from small molecule phosphodonors has revealed large differences in autophosphorylation rates among OmpR/PhoB RRs. RRs with substantial domain interfaces exhibit slow rates of phosphorylation. Rates are greatly increased in isolated receiver domain constructs. Such differences are not observed between autophosphorylation rates of full-length and isolated receiver domains of a RR that lacks interdomain interfaces, and they are not observed in histidine kinase-mediated phosphotransfer. These findings suggest that domain interfaces restrict receiver domain conformational dynamics, stabilizing an inactive conformation that is catalytically incompetent for phosphotransfer from small molecule phosphodonors. Inhibition of phosphotransfer by domain interfaces provides an explanation for the observation that some RRs cannot be phosphorylated by small molecule phosphodonors in vitro and provides a potential mechanism for insulating some RRs from small molecule-mediated phosphorylation in vivo.

Structure of the Staphylococcus aureus AgrA LytTR domain bound to DNA reveals a beta fold with an unusual mode of binding.

The LytTR domain is a DNA-binding motif found within the AlgR/AgrA/LytR family of transcription factors that regulate virulence factor and toxin gene expression in pathogenic bacteria. This previously uncharacterized domain lacks sequence similarity with proteins of known structure. The crystal structure of the DNA-binding domain of Staphylococcus aureus AgrA complexed with a DNA pentadecamer duplex has been determined at 1.6 A resolution. The structure establishes a 10-stranded beta fold for the LytTR domain and reveals its mode of interaction with DNA. Residues within loop regions of AgrA contact two successive major grooves and the intervening minor groove on one face of the oligonucleotide duplex, inducing a substantial bend in the DNA. Loss of DNA binding upon substitution of key interacting residues in AgrA supports the observed binding mode. This mode of protein-DNA interaction provides a potential target for future antimicrobial drug design.

Defining the mode, energetics and specificity with which a macrocyclic hexaoxazole binds to human telomeric G-quadruplex DNA.

Oxazole-containing macrocycles represent a promising class of anticancer agents that target G-quadruplex DNA. We report the results of spectroscopic studies aimed at defining the mode, energetics and specificity with which a hexaoxazole-containing macrocycle (HXDV) binds to the intramolecular quadruplex formed by the human telomeric DNA model oligonucleotide d(T2AG3)4 in the presence of potassium ions. HXDV binds solely to the quadruplex nucleic acid form, but not to the duplex or triplex form. HXDV binds d(T2AG3)4 with a stoichiometry of two drug molecules per quadruplex, with these binding reactions being coupled to the destacking of adenine residues from the terminal G-tetrads. HXDV binding to d(T2AG3)4 does not alter the length of the quadruplex. These collective observations are indicative of a nonintercalative 'terminal capping' mode of interaction in which one HXDV molecule binds to each end of the quadruplex. The binding of HXDV to d(T2AG3)4 is entropy driven, with this entropic driving force reflecting contributions from favorable drug-induced alterations in the configurational entropy of the host quadruplex as well as in net hydration. The 'terminal capping' mode of binding revealed by our studies may prove to be a general feature of the interactions between oxazole-containing macrocyclic ligands (including telomestatin) and intramolecular DNA quadruplexes.

Targeting human telomeric G-quadruplex DNA with oxazole-containing macrocyclic compounds.

Oxazole-containing macrocycles, which include the natural product telomestatin, represent a promising class of anticancer agents that target G-quadruplex DNA. Two synthetic hexaoxazole-containing macrocyclic compounds (HXDV and HXLV-AC) have been characterized with regard to their cytotoxic activities versus human cancer cells, as well as the mode, thermodynamics, and specificity with which they bind to the intramolecular (3+1) G-quadruplex structural motif formed in the presence of K+ ions by human telomeric DNA. Both compounds exhibit cytotoxic activities versus human lymphoblast (RPMI 8402) and oral carcinoma (KB3-1) cells, with associated IC50 values ranging from 0.4 to 0.9microM. The compounds bind solely to the quadruplex nucleic acid form, but not to the duplex or triplex form. Binding to the quadruplex is associated with a stoichiometry of two ligand molecules per DNA molecule, with one ligand molecule binding to each end of the host quadruplex via a nonintercalative "terminal capping" mode of interaction. For both compounds, quadruplex binding is primarily entropy driven, while also being associated with a negative change in heat capacity. These thermodynamic properties reflect contributions from favorable ligand-induced alterations in the loop configurational entropies of the quadruplex, but not from changes in net hydration. The stoichiometry and mode of binding revealed by our studies have profound implications with regard to the number of ligand molecules that can potentially bind the 3-overhang region of human telomeric DNA.

Molecular determinants of antibiotic recognition and resistance by aminoglycoside phosphotransferase (3')-IIIa: a calorimetric and mutational analysis.

The growing threat from the emergence of multidrug resistant pathogens highlights a critical need to expand our currently available arsenal of broad-spectrum antibiotics. In this connection, new antibiotics must be developed that exhibit the abilities to circumvent known resistance pathways. An important step toward achieving this goal is to define the key molecular interactions that govern antibiotic resistance. Here, we use site-specific mutagenesis, coupled with calorimetric, NMR, and enzymological techniques, to define the key interactions that govern the binding of the aminoglycoside antibiotics neomycin and kanamycin B to APH(3')-IIIa (an antibiotic phosphorylating enzyme that confers resistance). Our mutational analyses identify the D261, E262, and C-terminal F264 residues of the enzyme as being critical for recognition of the two drugs as well as for the manifestation of the resistance phenotype. In addition, the E160 residue is more important for recognition of kanamycin B than neomycin, with mutation of this residue partially restoring sensitivity to kanamycin B but not to neomycin. By contrast, the D193 residue partially restores sensitivity to neomycin but not to kanamycin B, with the origins of this differential effect being due to the importance of D193 for catalyzing the phosphorylation of neomycin. These collective mutational results, coupled with (15)N NMR-derived pK(a) and calorimetrically derived binding-linked drug protonation data, identify the 1-, 3-, and 2'-amino groups of both neomycin and kanamycin B as being critical functionalities for binding to APH(3')-IIIa. These drug amino functionalities represent potential sites of modification in the design of next-generation compounds that can overcome APH(3')-IIIa-induced resistance.

Universally applicable methods for monitoring response regulator aspartate phosphorylation both in vitro and in vivo using Phos-tag-based reagents.

Recent development of the phosphate chelator, Phos-tag, together with Phos-tag pendant reagents, has provided new methods for detection of phosphorylated serine, threonine, tyrosine, and histidine residues in phosphoproteins. We have investigated the use of Phos-tag for detection and quantification of phospho-aspartate in response regulator proteins that function within two-component signaling systems. Alternative methods are especially important, because the labile nature of the acylphosphate bond in response regulator proteins has restricted the application of many traditional methods of phosphoprotein analysis. We demonstrate that Phos-tag gel stain can be used to detect phospho-Asp in response regulators and that Phos-tag acrylamide gel electrophoresis can be used to separate phosphorylated and unphosphorylated forms of response regulator proteins. The latter method, coupled to Western blot analysis, enables detection of specific phosphorylated proteins in complex mixtures such as cell lysates. Standards of phosphorylated proteins can be used to correct for hydrolysis of the labile phospho-Asp bond that invariably occurs during analysis. We have employed Phos-tag methods to characterize the phosphorylation state of the Escherichia coli response regulator PhoB both in vitro, using purified protein, and in vivo, by analyzing lysates of cells grown under different conditions of induction of the PhoR/PhoB phosphate assimilation pathway.

G-quadruplexes induce apoptosis in tumor cells.

Several G-rich oligodeoxynucleotides (ODNs), which are capable of forming G-quadruplexes, have been shown to exhibit antiproliferative activity against tumor cell lines and antitumor activity in nude mice carrying prostate and breast tumor xenografts. However, the molecular basis for their antitumor activity remains unclear. In the current study, we showed that a variety of telomeric G-tail oligodeoxynucleotides (TG-ODNs) exhibited antiproliferative activity against many tumor cells in culture. Systematic mutational analysis of the TG-ODNs suggests that the antiproliferative activity depends on the G-quadruplex conformation of these TG-ODNs. TG-ODNs were also shown to induce poly(ADP-ribose) polymerase-1 cleavage, phosphatidylserine flipping, and caspase activation, indicative of induction of apoptosis. TG-ODN-induced apoptosis was largely ataxia telangiectasia mutated (ATM) dependent. Furthermore, TG-ODN-induced apoptosis was inhibited by the c-Jun NH(2)-terminal kinase (JNK) inhibitor SP600125. Indeed, TG-ODNs were shown to activate the JNK pathway in an ATM-dependent manner as evidenced by elevated phosphorylation of JNK and c-Jun. Interestingly, a number of G-quadruplex ODNs (GQ-ODN) derived from nontelomeric sequences also induced ATM/JNK-dependent apoptosis, suggesting a possible common mechanism of tumor cell killing by GQ-ODNs.

Validation and Development of an Escherichia coli Riboflavin Pathway Phenotypic Screen Hit as a Small-Molecule Ligand of the Flavin Mononucleotide Riboswitch.

A riboflavin biosynthesis pathway-specific phenotypic screen using a library of compounds, all with unspecified antibiotic activity, identified one small molecule later named ribocil, for which intrinsic antibacterial activity against Escherichia coli was completely suppressed by addition of exogenous riboflavin to the bacterial growth medium. The ability of riboflavin to suppress the activity of ribocil, and further demonstration that ribocil inhibited riboflavin synthesis (IC50 = 0.3 µM), supported that a component of the riboflavin synthesis pathway was the molecular target. Remarkably, resistance mutation selection and whole-genome sequencing showed that the target of ribocil was not an enzyme in the riboflavin biosynthesis pathway, but instead the flavin mononucleotide riboswitch, a noncoding structural RNA element in the ribB gene that encodes a key riboflavin synthesis enzyme. Although ribocil is structurally distinct from the natural riboswitch regulatory ligand flavin mononucleotide, ribocil binding to the riboswitch results in efficient repression of ribB expression and inhibition of riboflavin biosynthesis and bacterial growth. A cell-based riboswitch regulated gene reporter assay as well as an in vitro riboswitch RNA aptamer-binding assay, both of which are described in detail here along with the riboflavin pathway-specific screen, were developed to further validate the mechanism of action of ribocil and to facilitate the discovery of more potent analogues.

Use of 2-aminopurine as a fluorescent tool for characterizing antibiotic recognition of the bacterial rRNA A-site.

Spectroscopic and calorimetric techniques have been employed to characterize the impact of incorporation of the fluorescent base analog 2-aminopurine into the 1492 or 1493 position of an E. coli rRNA A-site model oligonucleotide, as well as the energetics and dynamics associated with recognition of this A-site model oligomer by aminoglycoside antibiotics. The results of these studies indicate that incorporation of 2AP into either the 1492 or 1493 position does not perturb the structure or stability of the host RNA or its aminoglycoside binding affinity. In addition, the results also highlight drug-induced reduction in the mobilities of the bases at both positions 1492 and 1493 as a potentially key determinant of bactericidal potency.

Dual-Targeting Small-Molecule Inhibitors of the Staphylococcus aureus FMN Riboswitch Disrupt Riboflavin Homeostasis in an Infectious Setting.

Riboswitches are bacterial-specific, broadly conserved, non-coding RNA structural elements that control gene expression of numerous metabolic pathways and transport functions essential for cell growth. As such, riboswitch inhibitors represent a new class of potential antibacterial agents. Recently, we identified ribocil-C, a highly selective inhibitor of the flavin mononucleotide (FMN) riboswitch that controls expression of de novo riboflavin (RF, vitamin B2) biosynthesis in Escherichia coli. Here, we provide a mechanistic characterization of the antibacterial effects of ribocil-C as well as of roseoflavin (RoF), an antimetabolite analog of RF, among medically significant Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis. We provide genetic, biophysical, computational, biochemical, and pharmacological evidence that ribocil-C and RoF specifically inhibit dual FMN riboswitches, separately controlling RF biosynthesis and uptake processes essential for MRSA growth and pathogenesis. Such a dual-targeting mechanism is specifically required to develop broad-spectrum Gram-positive antibacterial agents targeting RF metabolism.

Defining the basis for the specificity of aminoglycoside-rRNA recognition: a comparative study of drug binding to the A sites of Escherichia coli and human rRNA.

2-Deoxystreptamine (2-DOS) aminoglycoside antibiotics exert their antimicrobial activities by targeting the decoding region A site of the rRNA and inhibiting protein synthesis. A prokaryotic specificity of action is critical to therapeutic utility of 2-DOS aminoglycosides as antibiotics. Here, isothermal titration calorimetry (ITC) and fluorescence studies are presented that provide insight into the molecular basis for this prokaryotic specificity of action. Specifically, the rRNA binding properties of the 2-DOS aminoglycosides paromomycin and G418 (geneticin) are compared, using both human and Escherichia coli rRNA A site model oligonucleotides as drug targets. Paromomycin and G418 differ with respect to their specificities of action, with only paromomycin exhibiting a specificity for prokaryotic versus human ribosomes. G418 binds to both the human and E. coli rRNA A sites with a markedly lower affinity than paromomycin, with the affinities of both drugs for the human rRNA A site being lower than those they exhibit for the E. coli rRNA A site. Paromomycin induces the destacking of the base at position 1492 (by E. coli numbering) upon binding to the E. coli rRNA A site, but not the human rRNA A site. By contrast, the binding of G418 induces the destacking of base 1492 when either rRNA A site serves as the drug target. In the aggregate, these results suggest that binding-induced base destacking at the rRNA A site is a critical factor in determining the prokaryotic specificity of aminoglycoside action, with binding affinity for the A site being of secondary importance.

Drug self-association modulates the cellular bioavailability of DNA minor groove-directed terbenzimidazoles.

The terbenzimidazoles are a class of anticancer agents that bind in the DNA minor groove. These compounds also exhibit a propensity for self-association, which can potentially impact their cellular bioavailabilities and activities. We have explored this possibility by using a broad range of biophysical and cytological techniques to characterize the self-association and cellular uptake properties of two terbenzimidazole analogues, 5-phenylterbenzimidazole (5PTB) and 5-phenyl-2'-(indolo-6-yl)bibenzimidazole (5P2'IBB). Concentration- and temperature-dependent fluorescence spectroscopy, dynamic light scattering, and transmission electron microscopy studies reveal that 5PTB and 5P2'IBB exhibit differing self-association properties. In this connection, 5PTB exhibits an enhanced propensity for self-association and forms larger and more stable aggregates than 5P2'IBB. In addition, the net uptake of 5PTB into human lymphoblast cells is diminished relative to that of 5P2'IBB. These observations suggest that the self-association properties of terbenzimidazoles modulate the cellular bioavailabilities of the compounds, with enhanced self-association propensity and aggregate size leading to reduced cellular bioavailability.

Moderate to high throughput in vitro binding kinetics for drug discovery.

This review provides a concise summary for state of the art, moderate to high throughput in vitro technologies being employed to study drug-target binding kinetics. These technologies cover a wide kinetic timescale spanning up to nine orders of magnitude from milliseconds to days. Automated stopped flow measures transient and (pre)steady state kinetics from milliseconds to seconds. For seconds to hours timescale kinetics we discuss surface plasmon resonance-based biosensor, global progress curve analysis for high throughput kinetic profiling of enzyme inhibitors and activators, and filtration plate-based radioligand or fluorescent binding assays for receptor binding kinetics. Jump dilution after pre-incubation is the preferred method for very slow kinetics lasting for days. The basic principles, best practices and simulated data for these technologies are described. Finally, the application of a universal label-free technology, liquid chromatography coupled tandem mass spectrometry (LC/MS/MS), is briefly reviewed. Select literature references are highlighted for in-depth understanding. A new reality is dawning wherein binding kinetics is an integral and routine part of mechanism of action elucidation and translational, quantitative pharmacology for drug discovery.

Coupling of drug protonation to the specific binding of aminoglycosides to the A site of 16 S rRNA: elucidation of the number of drug amino groups involved and their identities.

2-Deoxystreptamine (2-DOS) aminoglycoside antibiotics bind specifically to the central region of the 16S rRNA A site and interfere with protein synthesis. Recently, we have shown that the binding of 2-DOS aminoglycosides to an A site model RNA oligonucleotide is linked to the protonation of drug amino groups. Here, we extend these studies to define the number of amino groups involved as well as their identities. Specifically, we use pH-dependent 15N NMR spectroscopy to determine the pK(a) values of the amino groups in neomycin B, paromomycin I, and lividomycin A sulfate, with the resulting pK(a) values ranging from 6.92 to 9.51. For each drug, the 3-amino group was associated with the lowest pK(a), with this value being 6.92 in neomycin B, 7.07 in paromomycin I, and 7.24 in lividomycin A. In addition, we use buffer-dependent isothermal titration calorimetry (ITC) to determine the number of protons linked to the complexation of the three drugs with the A site model RNA oligomer at pH 5.5, 8.8, or 9.0. At pH 5.5, the binding of the three drugs to the host RNA is independent of drug protonation effects. By contrast, at pH 9.0, the RNA binding of paromomycin I and neomycin B is coupled to the uptake of 3.25 and 3.80 protons, respectively, with the RNA binding of lividomycin A at pH 8.8 being coupled to the uptake of 3.25 protons. A comparison of these values with the protonation states of the drugs predicted by our NMR-derived pK(a) values allows us to identify the specific drug amino groups whose protonation is linked to complexation with the host RNA. These determinations reveal that the binding of lividomycin A to the host RNA is coupled to the protonation of all five of its amino groups, with the RNA binding of paromomycin I and neomycin B being linked to the protonation of four and at least five amino groups, respectively. For paromomycin I, the protonation reactions involve the 1-, 3-, 2'-, and 2"'-amino groups, while, for neomycin B, the binding-linked protonation reactions involve at least the 1-, 3-, 2', 6'-, and 2"'-amino groups. Our results clearly identify drug protonation reactions as important thermodynamic participants in the specific binding of 2-DOS aminoglycosides to the A site of 16S rRNA.