Advancement of the 5-Amino-1-(Carbamoylmethyl)-1H-1,2,3-Triazole-4-Carboxamide Scaffold to Disarm the Bacterial SOS Response.

Many antibiotics, either directly or indirectly, cause DNA damage thereby activating the bacterial DNA damage (SOS) response. SOS activation results in expression of genes involved in DNA repair and mutagenesis, and the regulation of the SOS response relies on two key proteins, LexA and RecA. Genetic studies have indicated that inactivating the regulatory proteins of this response sensitizes bacteria to antibiotics and slows the appearance of resistance. However, advancement of small molecule inhibitors of the SOS response has lagged, despite their clear promise in addressing the threat of antibiotic resistance. Previously, we had addressed this deficit by performing a high throughput screen of ∼1.8 million compounds that monitored for inhibition of RecA-mediated auto-proteolysis of Escherichia coli LexA, the reaction that initiates the SOS response. In this report, the refinement of the 5-amino-1-(carbamoylmethyl)-1H-1,2,3-triazole-4-carboxamide scaffold identified in the screen is detailed. After development of a modular synthesis, a survey of key activity determinants led to the identification of an analog with improved potency and increased breadth, targeting auto-proteolysis of LexA from both E. coli and Pseudomonas aeruginosa. Comparison of the structure of this compound to those of others in the series suggests structural features that may be required for activity and cross-species breadth. In addition, the feasibility of small molecule modulation of the SOS response was demonstrated in vivo by the suppression of the appearance of resistance. These structure activity relationships thus represent an important step toward producing Drugs that Inhibit SOS Activation to Repress Mechanisms Enabling Resistance (DISARMERs).

Inhibitors of LexA Autoproteolysis and the Bacterial SOS Response Discovered by an Academic-Industry Partnership.

The RecA/LexA axis of the bacterial DNA damage (SOS) response is a promising, yet nontraditional, drug target. The SOS response is initiated upon genotoxic stress, when RecA, a DNA damage sensor, induces LexA, the SOS repressor, to undergo autoproteolysis, thereby derepressing downstream genes that can mediate DNA repair and accelerate mutagenesis. As genetic inhibition of the SOS response sensitizes bacteria to DNA damaging antibiotics and decreases acquired resistance, inhibitors of the RecA/LexA axis could potentiate our current antibiotic arsenal. Compounds targeting RecA, which has many mammalian homologues, have been reported; however, small-molecules targeting LexA autoproteolysis, a reaction unique to the prokaryotic SOS response, have remained elusive. Here, we describe the logistics and accomplishments of an academic-industry partnership formed to pursue inhibitors against the RecA/LexA axis. A novel fluorescence polarization assay reporting on RecA-induced self-cleavage of LexA enabled the screening of 1.8 million compounds. Follow-up studies on select leads show distinct activity patterns in orthogonal assays, including several with activity in cell-based assays reporting on SOS activation. Mechanistic assays demonstrate that we have identified first-in-class small molecules that specifically target the LexA autoproteolysis step in SOS activation. Our efforts establish a realistic example for navigating academic-industry partnerships in pursuit of anti-infective drugs and offer starting points for dedicated lead optimization of SOS inhibitors that could act as adjuvants for current antibiotics.

Acinetobacter baumannii OxPhos inhibitors as selective anti-infective agents.

The Gram-negative bacterium Acinetobacter baumannii is an opportunistic pathogen in humans and infections are poorly treated by current therapy. Recent emergence of multi-drug resistant strains and the lack of new antibiotics demand an immediate action for development of new anti-Acinetobacter agents. To this end, oxidative phosphorylation (OxPhos) was identified as a novel target for drug discovery research. Consequently, a library of ∼10,000 compounds was screened using a membrane-based ATP synthesis assay. One hit identified was the 2-iminobenzimidazole 1 that inhibited the OxPhos of A. baumannii with a modestly high selectivity against mitochondrial OxPhos, and displayed an MIC of 25µM (17µg/mL) against the pathogen. The 2-iminobenzimidazole 1 was found to inhibit the type 1 NADH-quinone oxidoreductase (NDH-1) of A. baumannii OxPhos by a biochemical approach. Among various derivatives that were synthesized to date, des-hydroxy analog 5 is among the most active with a relatively tight SAR requirement for the N'-aminoalkyl side chain. Analog 5 also showed less cytotoxicity against NIH3T3 and HepG2 mammalian cell lines, demonstrating the potential for this series of compounds as anti-Acinetobacter agents. Additional SAR development and target validation is underway.

Environmental contaminants perturb fragile protein assemblies and inhibit normal protein function.

The molecular mechanisms whereby small molecules that contaminate our environment cause physiological effects are largely unknown, in terms of both targets and mechanisms. The essential human enzyme porphobilinogen synthase (HsPBGS, a.k.a. 5-aminolevulinate dehydratase, ALAD) functions in heme biosynthesis. HsPBGS catalytic activity is regulated allosterically via an equilibrium of inactive hexamers and active octamers, and we have shown that certain drugs and drug-like small molecules can inhibit HsPBGS in vitro by stabilizing the hexamer. Here we address whether components of the National Toxicology Program library of environmental contaminants can stabilize the HsPBGS hexamer and inhibit activity in vitro. Native polyacrylamide gel electrophoresis was used to screen the library (1,408 compounds) for components that alter the oligomeric distribution of HsPBGS. Freshly purchased samples of 37 preliminary hits were used to confirm the electrophoretic results and to determine the dose-dependence of the perturbation of oligomeric distribution. Seventeen compounds were identified which alter the oligomeric distribution toward the hexamer and also inhibit HsPBGS catalytic activity, including the most potent HsPBGS inhibitor yet characterized (Mutagen X, IC50 = 1.4 µM). PBGS dysfunction is associated with the inborn error of metabolism know as ALAD porphyria and with lead poisoning. The identified hexamer-stabilizing inhibitors could potentiate these diseases. Allosteric regulation of activity via an equilibrium of alternate oligomers has been proposed for many proteins. Based on the precedent set herein, perturbation of these oligomeric equilibria by small molecules (such as environmental contaminants) can be considered as a mechanism of toxicity.

Dynamic dissociating homo-oligomers and the control of protein function.

Homo-oligomeric protein assemblies are known to participate in dynamic association/disassociation equilibria under native conditions, thus creating an equilibrium of assembly states. Such quaternary structure equilibria may be influenced in a physiologically significant manner either by covalent modification or by the non-covalent binding of ligands. This review follows the evolution of ideas about homo-oligomeric equilibria through the 20th and into the 21st centuries and the relationship of these equilibria to allosteric regulation by the non-covalent binding of ligands. A dynamic quaternary structure equilibria is described where the dissociated state can have alternate conformations that cannot reassociate to the original multimer; the alternate conformations dictate assembly to functionally distinct alternate multimers of finite stoichiometry. The functional distinction between different assemblies provides a mechanism for allostery. The requirement for dissociation distinguishes this morpheein model of allosteric regulation from the classical MWC concerted and KNF sequential models. These models are described alongside earlier dissociating allosteric models. The identification of proteins that exist as an equilibrium of diverse native quaternary structure assemblies has the potential to define new targets for allosteric modulation with significant consequences for further understanding and/or controlling protein structure and function. Thus, a rationale for identifying proteins that may use the morpheein model of allostery is presented and a selection of proteins for which published data suggests this mechanism may be operative are listed.

Diverse clinical compounds alter the quaternary structure and inhibit the activity of an essential enzyme.

An in vitro evaluation of the Johns Hopkins Clinical Compound Library demonstrates that certain drugs can alter the quaternary structure of an essential human protein. Human porphobilinogen synthase (HsPBGS) is an essential enzyme involved in heme biosynthesis; it exists as an equilibrium of high-activity octamers, low-activity hexamers, and alternate dimer configurations that dictate the stoichiometry and architecture of further assembly. Decreased HsPBGS activity is implicated in toxicities associated with lead poisoning and 5-aminolevulinate dehydratase (ALAD) porphyria, the latter of which involves hexamer-favoring HsPBGS variants. A medium-throughput native PAGE mobility-shift screen coupled with evaluation of hits as HsPBGS inhibitors revealed 12 drugs that stabilize the HsPBGS hexamer and inhibit HsPBGS activity in vitro. A detailed characterization of these effects is presented. Drug inhibition of HsPBGS in vivo by inducing hexamer formation would constitute an unprecedented mechanism for side effects. We suggest that small-molecule perturbation of quaternary structure equilibria be considered as a general mechanism for drug action and side effects.

Allosteric inhibition of human porphobilinogen synthase.

Porphobilinogen synthase (PBGS) catalyzes the first common step in tetrapyrrole (e.g. heme, chlorophyll) biosynthesis. Human PBGS exists as an equilibrium of high activity octamers, low activity hexamers, and alternate dimer configurations that dictate the stoichiometry and architecture of further assembly. It is posited that small molecules can be found that inhibit human PBGS activity by stabilizing the hexamer. Such molecules, if present in the environment, could potentiate disease states associated with reduced PBGS activity, such as lead poisoning and ALAD porphyria, the latter of which is associated with human PBGS variants whose quaternary structure equilibrium is shifted toward the hexamer (Jaffe, E. K., and Stith, L. (2007) Am. J. Hum. Genet. 80, 329-337). Hexamer-stabilizing inhibitors of human PBGS were identified using in silico prescreening (docking) of approximately 111,000 structures to a hexamer-specific surface cavity of a human PBGS crystal structure. Seventy-seven compounds were evaluated in vitro; three provided 90-100% conversion of octamer to hexamer in a native PAGE mobility shift assay. Based on chemical purity, two (ML-3A9 and ML-3H2) were subjected to further evaluation of their effect on the quaternary structure equilibrium and enzymatic activity. Naturally occurring ALAD porphyria-associated human PBGS variants are shown to have an increased susceptibility to inhibition by both ML-3A9 and ML-3H2. ML-3H2 is a structural analog of amebicidal drugs, which have porphyria-like side effects. Data support the hypothesis that human PBGS hexamer stabilization may explain these side effects. The current work identifies allosteric ligands of human PBGS and, thus, identifies human PBGS as a medically relevant allosteric enzyme.

Kinetics and thermodynamics of the interchange of the morpheein forms of human porphobilinogen synthase.

A morpheein is a homo-oligomeric protein that can adopt different nonadditive quaternary assemblies (morpheein forms) with different functionalities. The human porphobilinogen synthase (PBGS) morpheein forms are a high activity octamer, a low activity hexamer, and two structurally distinct dimer conformations. Conversion between hexamer and octamer involves dissociation to dimers, conformational change at the dimer level, followed by association to the alternate assembly. The current work promotes an alternative and novel view of the physiologically relevant dimeric structures, which are derived from the crystal structures, but are distinct from the asymmetric units of their crystal forms. Using a well characterized heteromeric system (WT+F12L; Tang, L. et al. (2005) J. Biol. Chem. 280, 15786-15793), extensive study of the human PBGS morpheein reequilibration process now reveals that the intervening dimers do not dissociate to monomers. The morpheein equilibria of wild type (WT) human PBGS are found to respond to changes in pH, PBGS concentration, and substrate turnover. Notably, the WT enzyme is predominantly an octamer at neutral pH, but increasing pH results in substantial conversion to lower order oligomers. Most significantly, the free energy of activation for the conversion of WT+F12L human PBGS heterohexamers to hetero-octamers is determined to be the same as that for the catalytic conversion of substrate to product by the octamer, remarkably suggesting a common rate-limiting step for both processes, which is postulated to be the opening/closing of the active site lid.

Characterization of three distinct catalytic forms of human tryptase-beta: their interrelationships and relevance.

Human tryptase-beta (HTbeta) is a serine protease that is isolated as a tetramer of four identical, catalytically active subunits (HTbeta-AT). Tetramer activity is not affected by protein-based physiological inhibitors but instead may be regulated by an autoinactivation process we have called spontaneous inactivation. Unless stabilized by heparin or high salt, the active tetramer converts to an inactive state consisting of an inactive-destabilized tetramer that reversibly dissociates to inactive monomers upon dilution. We refer to this mixture of inactive species as siHTbeta and show in this study that previous reports of monomeric catalytic forms are derived from this mixture. siHTbeta itself did not hydrolyze model substrates but unlike the tetramer did react slowly with the serpin alpha2-antiplasmin (alpha2-AP), suggesting a highly limited catalytic potential. In the presence of heparin (or other highly charged polysaccharides), we demonstrate that siHTbeta formed a well-defined complex with the heparin (siHTbeta-HC) that reacted 70-fold faster with alpha2-AP than siHTbeta and also hydrolyzed model substrates and fibrinogen. Formation of siHTbeta-HC was limited to dilute subunit solutions since high subunit concentrations resulted in the reformation of the active tetramer. By compensating for changes in the strength of heparin binding, siHTbeta-HC could be formed over the pH range of 6.0-8.5. The activity dependence on pH was bell-shaped with highest activity between pH 6.8 and pH 7.5. In contrast, HTbeta-AT activity showed no dependence upon heparin, increased over the pH range of 6.0-8.5, and was much higher than that of siHTbeta-HC especially above pH 6.8. HTbeta-AT incubated with excess heparin of different size (3-15 kDa) was functionally stable at 25 degrees C but lost activity regardless of heparin size at 37 degrees C above pH 6.8. The change in stability, which is likely due to weakened heparin binding, did not result in the formation of a stable catalytic monomer. These results confirm that siHTbeta is for the most part an inactive species and that any active monomer is a consequence of heparin binding to siHTbeta under dilute conditions where unfavorable thermodynamics and/or kinetics restrict formation of active tetramer. Heparin binding under these conditions drives a limited reorganization of the active site to a conformation that is catalytic but not the equivalent of a subunit within the active tetramer.

X-ray structures of free and leupeptin-complexed human alphaI-tryptase mutants: indication for an alpha-->beta-tryptase transition.

Tryptases alpha and beta are trypsin-like serine proteinases expressed in large amounts by mast cells. Beta-tryptase is a tetramer that has enzymatic activity, but requires heparin binding to maintain functional and structural stability, whereas alpha-tryptase has little, if any, enzymatic activity but is a stable tetramer in the absence of heparin. As shown previously, these differences can be mainly attributed to the different conformations of the 214-220 segment. Interestingly, the replacement of Asp216 by Gly, which is present in beta-tryptase, results in enzymatically active but less stable alpha-tryptase mutants. We have solved the crystal structures of both the single (D216G) and the double (K192Q/D216G) mutant forms of recombinant human alphaI-tryptase in complex with the peptide inhibitor leupeptin, as well as the structure of the non-inhibited single mutant. The inhibited mutants exhibited an open functional substrate binding site, while in the absence of an inhibitor, the open (beta-tryptase-like) and the closed (alpha-tryptase-like) conformations were present simultaneously. This shows that both forms are in a two-state equilibrium, which is influenced by the residues in the vicinity of the active site and by inhibitor/substrate binding. Novel insights regarding the observed stability differences as well as a potential proteolytic activity of wild-type alpha-tryptase, which may possess a cryptic active site, are discussed.

The interaction of human tryptase-beta with small molecule inhibitors provides new insights into the unusual functional instability and quaternary structure of the protease.

Human tryptase-beta (HTbeta) is a serine protease with an atypical tetrameric structure and an unusual dependence on heparin binding or high salt for functional and structural stability. In the absence of heparin and at physiological salt, pH, and temperature, HTbeta rapidly loses activity by a reversible process that we have called spontaneous inactivation. The role of tetramer dissociation in this process is controversial. Using small irreversible or competitive inhibitors of HTbeta as stabilizing ligands, we were able to examine tetramer stability under inactivating (decay) conditions in the absence of heparin and to define further the process of spontaneous inactivation. Size exclusion chromatography showed that interaction with inhibitors stabilized the tetramer. Using sedimentation equilibrium, spontaneously inactivated HTbeta (si-HTbeta) was shown to be a destabilized tetramer that dissociates upon dilution and which in the presence of a competitive inhibitor re-formed a stable tetramer. Addition of inhibitors to si-HTbeta rescued catalytic activity as was shown after inhibitor displacement. At high concentrations of si-HTbeta (4-5 microM), the binding of inhibitor alone provided sufficient free energy for complete reactivation and tetramer stabilization, whereas at low si-HTbeta concentration (0.1 microM) where the destabilized tetramer would be mostly dissociated, reactivation required more free energy which was provided by the binding of both an inhibitor and heparin. The results demonstrate that HTbeta is a tetramer in the absence of heparin and that tetramer dissociation is a consequence of and not a prerequisite for inactivation. Heparin binding likely stabilizes the tetramer by favoring a functionally active conformation with stable intersubunit contacts, rather than by simply cross-linking active monomers.

Calcium-dependent conformation of desmoglein 1 is required for its cleavage by exfoliative toxin.

In bullous impetigo, Staphylococcus aureus spreads under the stratum corneum of skin by elaboration of exfoliative toxin, which hydrolyzes only one peptide bond in a highly structured calcium-binding domain of desmoglein 1, resulting in loss of its function. We investigated the basis of this exquisite specificity. Exfoliative toxin cannot cleave desmoglein 1 pretreated at 56 degrees C or higher or at low or high pH, suggesting that the proper conformation of desmoglein 1 is critical for its cleavage. Because cleavage occurs in an area of desmoglein 1 stabilized by calcium, we determined if the conformation necessary for cleavage is calcium-dependent. Depletion of calcium from desmoglein 1 completely inhibited its cleavage by exfoliative toxin, even after calcium was added back. A change in conformation of desmoglein 1 by calcium depletion was shown, with immunofluorescence and enzyme-linked immunoassay, by loss of binding of PF sera, which recognize conformational epitopes. This change in conformation was confirmed by tryptophan fluorometry and circular dichroism, and was irreversible with repletion of calcium. These data suggest that the specificity of exfoliative toxin cleavage of desmoglein 1 resides not only in simple amino acid sequences but also in its calcium-dependent conformation.

Potent bivalent inhibition of human tryptase-beta by a synthetic inhibitor.

Human tryptase-beta (HTbeta) is a unique serine protease exhibiting a frame-like tetramer structure with four active sites directed toward a central pore. Potent inhibition of HTbeta has been attained using CRA-2059. This compound has two phenylguanidinium head groups connected via a linker capable of spanning between two active sites. The properties of the CRA-2059:HTbeta interaction were defined in this study. Tight-binding reversible inhibition was observed with an inhibition constant (Ki) of 620 pM, an association rate constant of 7x10(7) M(-1) s(-1) and a relatively slow dissociation rate constant of 0.04 s(-1). Bivalent inhibition was demonstrated by displacement of p-aminobenzamidine from the primary specificity pocket with a stoichiometry, [CRA-2059]0/[HTbeta]0, of 0.5. The potency of the bivalent interaction was illustrated by CRA-2059 inhibition of HTbeta, 24% or 53% inhibited by pre-incubation with an irreversible inhibitor. Two interactions were observed consistent with mono- and bi-valent binding; the Ki value for bivalent inhibition was at least 10(4)-fold lower than that for monovalent inhibition. Comparison of the affinities of CRA-2059 and phenylguanidine for HTbeta finds an approximate doubling of the free energy change upon bivalent binding. This doubling suggests that the linker portion minimally hinders the binding of CRA-2059 to HTbeta. The potency of CRA-2059 is thus attributable to effective bivalent binding.

Diverse stability and catalytic properties of human tryptase alpha and beta isoforms are mediated by residue differences at the S1 pocket.

Recombinant human tryptases (rHTs) corresponding to alpha and beta isoforms were characterized. rHTbeta was similar to tryptase isolated from skin (HST); it was a tetramer, hydrolyzed model substrates efficiently, and was functionally unstable when incubated under physiological conditions. Activity was lost rapidly (t(1/2) approximately 1 min) by a reversible process similar to that observed for the spontaneous inactivation of HST. Circular dichroism (CD) and intrinsic fluorescence emission (IFE) spectra of active rHTbeta corresponded to those of active HST and upon spontaneous inactivation IFE decreased in parallel to activity loss. rHTalpha differed from HST in catalytic ability and stability. rHTalpha did not react with model substrates, an active site titrant, or a competitive inhibitor of HST/rHTbeta. IFE and CD spectra were similar to those of the active and not the spontaneously inactivated form of HST. Under physiological conditions, rHTalpha IFE decreased at a rate 900-fold slower than that observed for HST, and rHTalpha remained tetrameric when examined by size exclusion chromatography at physiological salt concentration. Thus, rHTalpha is a stable "inactive" form of HT. Three active site variants of rHTalpha, K192Q, D216G, and K192Q-D216G were characterized. Residues 192 and 216 (chymotrypsinogen numbers for residues 191 and 215 of rHTalpha) lie at the entrance to the primary specificity (S1) pocket, and the mutations converted them to the residues of HTbeta. While K192Q displayed the same properties as rHTalpha, the catalytic and stability characteristics of D216G and K192Q-D216G progressively approached those of HST. Thus, the contrasting stability/activity properties of rHTalpha and rHTbeta are largely related to differences at the S1 pocket. On the basis of the properties of the variants, we suggest that the side chain of Asp216 is blocking and stabilizing the S1 pocket and that this stabilization is sufficient to prevent spontaneous inactivation.

Evidence for diversity of substrate specificity among members of the chymase family of serine proteases.

The term chymase is used to signify a chymotrypsin-like protease stored within the secretory granules of mast cells. Primarily based on amino acid sequence homology, 18 chymases have been identified among different animals. This study, which compares the structure of the primary specificity pocket (S1 subsite), defines a subgroup of four chymases likely to have a substrate specificity with more elastase- than chymotrypsin-like qualities. This difference is due, primarily, to finding a Val instead of a Gly at residue 199, a position corresponding to Gly216 in bovine chymotrypsin and Val216 in neutrophil and porcine elastases. Chymases with Val at 199 are found only in animals expressing multiple chymases, consistent with the premise that their substrate specificity differs from that of chymases with Gly at 199.

Heterogeneity in serpin-protease complexes as demonstrated by differences in the mechanism of complex breakdown.

Serpins trap their target proteases in the form of an acyl-enzyme complex. The trap is kinetic, however, and thus serpin-protease complexes ultimately break down, releasing a cleaved inactive serpin and an active protease. The rates of this deacylation process vary greatly depending on the serpin-protease pair with half-lives ranging from minutes to months. The reasons for the diversity in breakdown rates are not clearly understood. In the current study, pH and solvent isotope effects were utilized to probe the mechanism of breakdown for an extremely stable complex and several unstable complexes. Two different patterns for the pH dependence of k(bkdn), the first-order rate constant of breakdown, were found. The stable complex, which breaks down at neutral pH with a half-life of approximately 2 weeks, exhibited a pH-k(bkdn) profile consistent with solvent-hydroxide ion mediated ester hydrolysis. There was no evidence for the participation of the catalytic machinery in the breakdown of this complex, suggesting extensive distortion of the active site. The unstable complexes, which break down with half-lives ranging from minutes to hours, exhibited a bell-shaped pH profile for k(bkdn), typical of the pH-rate profiles of free serine proteases. In the low to neutral pH range k(bkdn) increased with increasing pH in a manner characteristic of His57-mediated catalysis. In the alkaline pH range a decrease in k(bkdn) was observed, consistent with the titration of the Ile16-Asp194 salt bridge (chymotrypsinogen numbering). The alkaline pH dependence was not exhibited in pH-rate profiles of free or substrate-bound HNE, indicating that the salt bridge was significantly destabilized in the complexed protease. These results indicate that breakdown is catalytically mediated in the unstable complexes although, most likely, the protease is not in its native conformation and the catalytic machinery functions inefficiently. However, a mechanism in which breakdown is determined by the equilibrium between distorted and undistorted forms of the complexed protease cannot be completely dismissed. Overall, the results of this study suggest that the protease structure in unstable complexes is distorted to a lesser extent than in stable complexes.