Search

The relationship between drug-target residence time and the post-antibiotic effect (PAE) provides insights into target vulnerability. To probe the vulnerability of bacterial acetyl-CoA carboxylase (ACC), a series of heterobivalent inhibitors were synthesized based on pyridopyrimidine

The hydrogen bonding network that surrounds the flavin in Blue Light Utilizing FAD (BLUF) photoreceptors plays a crucial role in sensing and communicating the changes in the electronic structure of the flavin to the protein matrix upon light absorption. The network contains a highly conserved tyrosine that is essential for photoactivation. Using time-resolved infrared spectroscopy (TRIR) and unnatural amino acid (UAA) incorporation, we investigated the photoactivation mechanism and the role of the conserved tyrosine (Y6) in the forward reaction of the photoactivated adenylyl cyclase (AC) from Oscillatoria Acuminata (OaPAC). Our work elucidates the direct connection between the photoactivation process in the BLUF domain and the structural and functional implications on the partner protein for the first time. The TRIR results demonstrate formation of FADH● as an intermediate species on the photoactivation pathway which decays to form the signaling state. Using fluorotyrosine analogs to modulate the physical properties of Y6, the TRIR data reveal that a change in the pKa and/or reduction potential of Y6 has a profound effect on the forward reaction, consistent with a mechanism involving proton transfer or proton-coupled electron transfer from Y6 to the electronically excited FAD. Decreasing the pKa from 9.9 to

The translation of time-dependent drug-target occupancy to extended pharmacological activity at low drug concentration depends on factors such as target vulnerability and the rate of target turnover. Previously, we demonstrated that the postantibiotic effect (PAE) caused by inhibitors of bacterial drug targets could be used to assess target vulnerability, and that high levels of target vulnerability coupled with relatively low rates of target resynthesis resulted in a strong correlation between drug-target residence time and the PAE following compound washout. Although the residence time of inhibitors on UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) in Pseudomonas aeruginosa (paLpxC) results in significant PAE, inhibitors of the equivalent enzyme in Escherichia coli (ecLpxC) do not cause a PAE. Hyperactivity of the fatty acid biosynthesis enzyme FabZ or the inclusion of sub-MIC levels of azithromycin lead to the observation of a PAE for three inhibitors of ecLpxC. FabZ hyperactivity has been shown to stabilize ecLpxC, and using mass spectrometry, we demonstrate that the appearance of a PAE can be directly linked to a 3-fold increase in the stability of ecLpxC. These studies substantiate the importance of target turnover in time-dependent drug activity.

UDP-3

The opportunistic human pathogen, A. baumannii, senses and responds to light using the blue light sensing A (BlsA) photoreceptor protein. BlsA is a blue-light-using flavin adenine dinucleotide (BLUF) protein that is known to regulate a wide variety of cellular functions through interactions with different binding partners. Using immunoprecipitation of tagged BlsA in A. baumannii lysates, we observed a number of proteins that interact with BlsA, including several transcription factors. In addition to a known binding partner, the iron uptake regulator Fur, we identified the biofilm response regulator BfmR as a putative BlsA-binding partner. Using microscale thermophoresis, we determined that both BfmR and Fur bind to BlsA with nanomolar binding constants. To better understand how BlsA interacts with and regulates these transcription factors, we solved the X-ray crystal structures of BlsA in both a ground (dark) state and a photoactivated light state. Comparison of the light- and dark-state structures revealed that, upon photoactivation, the two α-helices comprising the variable domain of BlsA undergo a distinct conformational change. The flavin-binding site, however, remains largely unchanged from dark to light. These structures, along with docking studies of BlsA and Fur, reveal key mechanistic details about how BlsA propagates the photoactivation signal between protein domains and on to its binding partner. Taken together, our structural and biophysical data provide important insights into how BlsA controls signal transduction in A. baumannii and provides a likely mechanism for blue-light-dependent modulation of biofilm formation and iron uptake.

Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light, oxygen, voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output partner. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 μs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in loss of the interaction between the side chain of N414 and the backbone C=O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.

Deep-seated bacterial infections caused by pathogens such as Staphylococcus aureus are difficult to diagnose and treat and are thus a major threat to human health. In previous work we demonstrated that positron emission tomography (PET) imaging with 2-[18F]F-p-aminobenzoic acid (2-[18F]F-PABA) could noninvasively identify, localize, and monitor S. aureus infection with excellent sensitivity and specificity in a rodent soft tissue infection model. However, 2-[18F]F-PABA is rapidly N-acetylated and eliminated, and in an attempt to improve radiotracer accumulation in bacteria we adopted a prodrug strategy in which the acid was protected by an ester and the amine was replaced with a nitro group. Metabolite analysis indicated that the nitro group of ethyl 2-[18F]fluoro-4-nitrobenzoate (2-[18F]F-ENB) is converted to the corresponding amine by bacteria-specific nitroreductases while the ester is hydrolyzed in vivo into the acid. PET/CT imaging of 2-[18F]F-ENB and the corresponding acid 2-[18F]F-NB in a rat soft tissue infection model demonstrated colocalization of the radiotracer with the bioluminescent signal arising from S. aureus Xen29, and demonstrated that the tracer could differentiate S. aureus infection from sterile inflammation. Significantly, the accumulation of both 2-[18F]F-ENB and 2-[18F]F-NB at the site of infection was 17-fold higher than at the site of sterile inflammation compared to 8-fold difference observed for 2-[18F]F-PABA, supporting the proposal that the active radiotracer in vivo is 2-[18F]F-NB. Collectively, these data suggest that 2-[18F]F-ENB and 2-[18F]F-NB have the potential for translation to humans as a rapid, noninvasive diagnostic tool to identify and localize S. aureus infections.

Target vulnerability correlates the level of drug-target engagement required to generate a pharmacological response. High vulnerability targets are those that require only a relatively small fraction of occupancy to achieve the desired pharmacological outcome, whereas low vulnerability targets require high levels of engagement. Here we demonstrate that the slope of the correlation between drug-target residence time and the post-antibiotic effect (PAE) can be used to define the vulnerability of bacterial targets. For macrolides, a steep slope is observed between residence time on the E. coli ribosome and the PAE, indicating that the ribosome is a highly vulnerable drug target. Analysis of the residence time-PAE data for erythromycin, azithromycin, spiramycin, and telithromycin using a mechanistic pharmacokinetic-pharmacodynamic model that integrates drug-target kinetics into predictions of drug activity, lead to the successful prediction of the cellular PAE for tylosin which has the longest residence time (7.1 h) and PAE (5.8 h). Although the macrolide data support a connection between residence time, PAE and bactericidality, many bactericidal β-lactam antibiotics do not give a PAE illustrating the role of factors such as protein resynthesis in the expression of target vulnerability.

Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule.

Blue light absorbing flavoproteins play important roles in a variety of photobiological processes. Consequently, there have been numerous investigations of their excited state structure and dynamics, in particular by time resolved vibrational spectroscopy. The isoalloxazine chromophore of the flavoprotein co-factors has been studied in detail by time resolved Raman, lending it a benchmark status for mode assignments in excited electronic states of large molecules. However, detailed comparisons of calculated and measured spectra have proven challenging, as there are many more modes calculated than are observed, and the role of resonance enhancement is difficult to characterise in excited electronic states. Here we employ a recently developed approach due to Elles and co-workers. (J. Phys. Chem. A 2018, 122, 8308–8319) for the calculation of resonance enhanced Raman spectra of excited states, and apply it to the lowest singlet and triplet excited states of the isoalloxazine chromophore. There is generally good agreement between calculated and observed enhancements, which allows assignment of vibrational bands of the flavoprotein co-factors to be refined. However, some prominently enhanced bands are found to be absent from the calculations, suggesting the need for further development of the theory.

Real-time observation of structure changes associated with protein function remains a major challenge. Ultrafast pump-probe methods record dynamics in light activated proteins, but the assignment of spectroscopic observables to specific structure changes can be difficult. The BLUF (blue light using flavin) domain proteins are an important class of light sensing flavoprotein. Here, we incorporate the unnatural amino acid (UAA) azidophenylalanine (AzPhe) at key positions in the H-bonding environment of the isoalloxazine chromophore of two BLUF domains, namely, PixD and AppABLUF; both proteins retain the red-shift on irradiation characteristic of photoactivity. Steady state and ultrafast time resolved infrared difference measurements of the azido mode reveal site-specific information on the nature and dynamics of light driven structure change. AzPhe dynamics are thus shown to be an effective probe of BLUF domain photoactivation, revealing significant differences between the two proteins and a differential response of the two sites to chromophore excitation.

Pharmacokinetic/pharmacodynamic (PK/PD) models predict the effect time course resulting from a drug dose. In this review, we summarize the development of mechanistic PK/PD models that explicitly integrate the kinetics of drug-target interactions into predictions of drug activity. Such mechanistic models are expected to have several advantages over approaches in which concentration and effect are linked using variations of the Hill equation, and where preclinical data are often used as a starting point for modeling drug activity. Instead, explicit use of the full kinetic scheme for drug binding enables time-dependent changes in target occupancy to be calculated using the kinetics of drug-target interactions and drug PK, providing a more precise picture of target engagement and drug action in the non-equilibrium environment of the human body. The mechanistic PK/PD models also generate target vulnerability functions that link target occupancy and effect, and inform on the sensitivity of a target to engagement by a drug. Key factors such as the rate of target turnover can also be integrated into the modeling which, together with target vulnerability, provide additional information on the PK profile required to achieve the desired pharmacological effect and on the utility of kinetic selectivity in developing drugs for specific targets.

The enoyl-acyl carrier protein (ACP) reductase (ENR) is a key enzyme within the bacterial fatty-acid synthesis pathway. It has been demonstrated that small-molecule inhibitors carrying the diphenylether (DPE) scaffold bear a great potential for the development of highly specific and effective drugs against this enzyme class. Interestingly, different substitution patterns of the DPE scaffold have been shown to lead to varying effects on the kinetic and thermodynamic behavior toward ENRs from different organisms. Here, we investigated the effect of a 4'-pyridone substituent in the context of the slow tight-binding inhibitor SKTS1 on the inhibition of the Staphylococcus aureus enoyl-ACP-reductase saFabI and the closely related isoenzyme from Mycobacterium tuberculosis, InhA, and explored a new interaction site of DPE inhibitors within the substrate-binding pocket. Using high-resolution crystal structures of both complexes in combination with molecular dynamics (MD) simulations, kinetic measurements, and quantum mechanical (QM) calculations, we provide evidence that the 4'-pyridone substituent adopts different tautomeric forms when bound to the two ENRs. We furthermore elucidate the structural determinants leading to significant differences in the residence time of SKTS1 on both enzymes.

Tryptophan and tyrosine radical intermediates play crucial roles in many biological charge transfer processes. Particularly in flavoprotein photochemistry, short-lived reaction intermediates can be studied by the complementary techniques of ultrafast visible and infrared spectroscopy. The spectral properties of tryptophan radical are well established, and the formation of neutral tyrosine radicals has been observed in many biological processes. However, only recently, the formation of a cation tyrosine radical was observed by transient visible spectroscopy in a few systems. Here, we assigned the infrared vibrational markers of the cationic and neutral tyrosine radical at 1483 and 1502 cm−1 (in deuterated buffer), respectively, in a variant of the bacterial methyl transferase TrmFO, and in the native glucose oxidase. In addition, we studied a mutant of AppABLUF blue-light sensor domain from Rhodobacter sphaeroides in which only a direct formation of the neutral radical was observed. Our studies highlight the exquisite sensitivity of transient infrared spectroscopy to low concentrations of specific radicals.

International audience; Triclosan and isoniazid are known antitubercular compounds that have proven to be also active against Leishmania parasites. On these grounds, a collection of 37 diverse 1,2,3-triazoles based on the antitubercular molecules triclosan and 5-octyl-2-phenoxyphenol (8PP) were designed in search of novel structures with leishmanicidal activity and prepared using different alkynes and azides. The 37 compounds were assayed against Leishmania donovani, the etiological agent of leishmaniasis, yielding some analogs with activity at micromolar concentrations and against M. tuberculosis H37Rv resulting in scarce active compounds with an MIC of 20 μM. To study the mechanism of action of these catechols, we analyzed the inhibition activity of the library on the M. tuberculosis enoyl-ACP reductase (ENR) InhA, obtaining poor inhibition of the enzyme. The cytotoxicity against Vero cells was also tested, resulting in none of the compounds being cytotoxic at concentrations of up to 20 μM. Derivative 5f could be considered a valuable starting point for future antileishmanial drug development. The validation of a putative leishmanial InhA orthologue as a therapeutic target needs to be further investigated

The opportunistic human pathogen, A. baumannii, senses and responds to light using the blue light sensing A (BlsA) photoreceptor protein. BlsA is a Blue Light Using FAD (BLUF) that is known to regulate a wide variety of cellular functions through interactions with different binding partners. Using immunoprecipitation of tagged BlsA in A. baumannii lysates, we observed a number of proteins that interact with BlsA, including several transcription factors. In addition to a known binding partner, the iron uptake regulator Fur, we identified the biofilm response regulator BfmR as a putative BlsA binding partner. Using microscale thermophoresis, we determined that both BfmR and Fur bind to BlsA with nanomolar binding constants. To better understand how BlsA interacts with and regulates these transcription factors, we solved the X-ray crystal structures of BlsA in both a ground (dark) state and a photo-activated light state. Comparison of the light- and dark-state structures revealed that, upon photoactivation, the two α-helices comprising the variable domain of BlsA undergo a distinct conformational change. The flavin binding site, however, remains largely unchanged from dark to light. These structures, along with docking studies of BlsA and Fur, reveal key mechanistic details about how BlsA propagates the photoactivation signal between protein domains and on to its binding partner. Taken together, our structural and biophysical data provide important insights into how BlsA controls signal transduction in A. baumannii and provides a likely mechanism for blue-light dependent modulation of biofilm formation and iron uptake.

Flavoproteins are important blue light sensors in photobiology and play a key role in optogenetics. The characterization of their excited state structure and dynamics is thus an important objective. Here, we present a detailed study of excited state vibrational spectra of flavin mononucleotide (FMN), in solution and bound to the LOV-2 (Light-Oxygen-Voltage) domain of Avena sativa phototropin. Vibrational frequencies are determined for the optically excited singlet state and the reactive triplet state, through resonant ultrafast femtosecond stimulated Raman spectroscopy (FSRS). To assign the observed spectra, vibrational frequencies of the excited states are calculated using density functional theory, and both measurement and theory are applied to four different isotopologues of FMN. Excited state mode assignments are refined in both states, and their sensitivity to deuteration and protein environment are investigated. We show that resonant FSRS provides a useful tool for characterizing photoactive flavoproteins and is able to highlight chromophore localized modes and to record hydrogen/deuterium exchange.

Deep seated bacterial infections caused by pathogens such as Staphylococcus aureus are difficult to diagnose and treat and are thus a major threat to human health. In previous work we demonstrated that positron emission tomography (PET) imaging with 2-[(18)F]F-p-aminobenzoic acid (2-[(18)F]F-PABA) could non-invasively identify, localize and monitor S. aureus infection with excellent sensitivity and specificity in a rodent soft tissue infection model. However, 2-[(18)F]F-PABA is rapidly N-acetylated and eliminated, and in an attempt to improve radiotracer accumulation in bacteria we adopted a pro-drug strategy in which the acid was protected by an ester and the amine was replaced with a nitro group. Metabolite analysis indicated that the nitro group of ethyl 2-[(18)F]fluoro-4-nitrobenzoate (2-[(18)F]F-ENB) is converted to the corresponding amine by bacteria-specific nitroreductases while the ester is hydrolyzed in vivo into the acid. PET/CT imaging of 2-[(18)F]F-ENB and the corresponding acid 2-[(18)F]F-NB in a rat soft tissue infection model demonstrated colocalization of the radiotracer with the bioluminescent signal arising from S. aureus Xen29, and demonstrated that the tracer could differentiate S. aureus infection from sterile inflammation. Significantly, the accumulation of both 2-[(18)F]F-ENB and 2-[(18)F]F-NB at the site of infection was 17-fold higher than at the site of sterile inflammation compared to 8-fold difference observed for 2-[(18)F]F-PABA, supporting the proposal that the active radiotracer in vivo is 2-[(18)F]F-NB. Collectively, these data suggest that 2-[(18)F]F-ENB and 2-[(18)F]F-NB have the potential for translation to humans as a rapid, noninvasive diagnostic tool to identify and localize S. aureus infections.

Ultrafast spectroscopy can measure real-time protein dynamics, but yields limited structural information. Here we use unnatural amino acid substitution to place an IR absorber at specific locations in light activated proteins. Ultrafast pump - transient IR probe spectroscopy then yields unique site-specific data on protein structural dynamics.

Staphylococcus aureus is the leading cause of life-threatening infections, frequently originating from unknown or deep-seated foci. Source control and institution of appropriate antibiotics remain challenges, especially with infections due to methicillin-resistant S. aureus (MRSA). In this study, we developed a radiofluorinated analog of para-aminobenzoic acid (2-[18F]F-PABA) and demonstrate that it is an efficient alternative substrate for the S. aureus dihydropteroate synthase (DHPS). 2-[18F]F-PABA rapidly accumulated in vitro within laboratory and clinical (including MRSA) strains of S. aureus but not in mammalian cells. Biodistribution in murine and rat models demonstrated localization at infection sites and rapid renal elimination. In a rat model, 2-[18F]F-PABA positron emission tomography (PET) rapidly differentiated S. aureus infection from sterile inflammation and could also detect therapeutic failures associated with MRSA. These data suggest that 2-[18F]F-PABA has the potential for translation to...

Time-dependent target occupancy is a function of both the thermodynamics and kinetics of drug-target interactions. However, while the optimization of thermodynamic affinity through approaches such as structure-based drug design is now relatively straight forward, less is understood about the molecular interactions that control the kinetics of drug complex formation and breakdown since this depends on both the ground and transition state energies on the binding reaction coordinate. In this opinion we highlight several recent examples that shed light on current approaches that are elucidating the factors that control the life-time of the drug-target complex.

The development of therapies for the treatment of neurological cancer faces a number of major challenges including the synthesis of small molecule agents that can penetrate the blood-brain barrier (BBB). Given the likelihood that in many cases drug exposure will be lower in the CNS than in systemic circulation, it follows that strategies should be employed that can sustain target engagement at low drug concentration. Time dependent target occupancy is a function of both the drug and target concentration as well as the thermodynamic and kinetic parameters that describe the binding reaction coordinate, and sustained target occupancy can be achieved through structural modifications that increase target (re)binding and/or that decrease the rate of drug dissociation. The discovery and deployment of compounds with optimized kinetic effects requires information on the structure-kinetic relationships that modulate the kinetics of binding, and the molecular factors that control the translation of drug-target kinetics to time-dependent drug activity in the disease state. This Review first introduces the potential benefits of drug-target kinetics, such as the ability to delineate both thermodynamic and kinetic selectivity, and then describes factors, such as target vulnerability, that impact the utility of kinetic selectivity. The Review concludes with a description of a mechanistic PK/PD model that integrates drug-target kinetics into predictions of drug activity.

A critical goal of lead compound selection and optimization is to maximize target engagement whilst minimizing off-target binding. Since target engagement is a function of both the thermodynamics and kinetics of drug-target interactions, it follows that the structures of both the ground states and transition states on the binding reaction coordinate are needed to rationally modulate the lifetime of the drug-target complex. Previously, we predicted the structure of the rate-limiting transition state that controlled the time-dependent inhibition of the enoyl-ACP reductase InhA. This led to the discovery of a triazole-containing diphenyl ether with an increased residence time on InhA due to transition state destabilization rather than ground state stabilization. In the present work, we have evaluated the inhibition of InhA by 14 triazole-based diphenyl ethers and used a combination of enzyme kinetics and X-ray crystallography to generate a structure-kinetic relationship (SKR) for time-dependent binding. We show that the triazole motif slows the rate of formation for the final drug-target complex by up to three orders of magnitude. In addition, we identify a novel inhibitor with a residence time on InhA of 220 min which is 3.5-fold longer than that of the INH-NAD adduct formed by the tuberculosis drug, isoniazid. This study provides a clear example in which the lifetime of the drug-target complex is controlled by interactions in the transition state for inhibitor binding rather than the ground state of the enzyme-inhibitor complex, and demonstrates the important role that on-rates can play in drug-target residence time.

Correlating target engagement with in vivo drug activity remains a central challenge in efforts to improve the efficiency of drug discovery. Previously we described a mechanistic pharmacokinetic–pharmacodynamic (PK/PD) model that used drug–target binding kinetics to successfully predict the in vivo efficacy of antibacterial compounds in models of Pseudomonas aeruginosa and Staphylococcus aureus infection. In the present work we extend this model to quantitatively correlate the engagement of Bruton's tyrosine kinase (Btk) by the covalent inhibitor CC-292 with the ability of this compound to reduce ankle swelling in an animal model of arthritis. The modeling studies include the rate of Btk turnover and reveal the vulnerability of Btk to engagement by CC-292.

A stereoselective synthesis has been developed to provide all four side-chain stereoisomers of difluoroindanediol 2, the mixture of which was previously identified as an inhibitor of the o-succinylbenzoate-CoA synthetase MenE in bacterial menaquinone biosynthesis, having promising in vitro activity against methicillin-resistant Staphylococcus aureus and Mycobacterium tuberculosis. Only the (1R,3S)-diastereomer inhibited the biochemical activity of MenE, consistent with computational docking studies, and this diastereomer also exhibited in vitro antibacterial activity comparable to that of the mixture. However, mechanism-of-action studies suggest that this inhibitor and its diastereomers may act via other mechanisms beyond inhibition of menaquinone biosynthesis.

The flavin cofactor performs many functions in the cell based on the ability of the isoalloxazine ring to undergo one- or two-electron reduction and form covalent adducts with reactants such as amino acids. In addition, the strong visible absorption of the cofactor is also the basis for flavin-dependent photoreceptors. Vibrational spectroscopy is uniquely suited to studying the mechanism of flavoproteins since the frequency of the vibrational modes is very sensitive to the electronic structure and environment of the isoalloxazine ring. This chapter describes the mechanistic information that can be gained using vibrational spectroscopy as well experimental challenges and approaches that are used to obtain and interpret the complex data contained in a vibrational spectrum.

Benzoxaboroles are a class of boron-containing compounds with a broad range of biological activities. A subset of benzoxaboroles have antimicrobial activity due primarily to their ability to inhibit leucyl-tRNA synthetase (LeuRS) via the oxaborole tRNA trapping mechanism, which involves formation of a stable tRNA(Leu)–benzoxaborole adduct in which the boron atom interacts with the 2′- and 3′-oxygen atoms of the 3′-terminal tRNA adenosine. We sought to identify other antibacterial targets for this promising class of compounds by means of mode of action studies, and we selected a nitrophenyl sulfonamide-based oxaborole (PT638) as a probe molecule because it had potent antibacterial activity (MIC of 0.4 μg/mL against methicillin-resistant Staphylococcus aureus) but did not inhibit LeuRS (IC(50) > 100 μM). Analogues of PT638 were synthesized to explore the importance of the sulfonamide linker and the impact of altering the functionalization of the phenyl ring. These structure–activity relationship studies revealed that the nitro substituent was essential for activity. To identify the target for PT638, we raised resistant strains of S. aureus and whole genome sequencing revealed mutations in leuRS, suggesting that the target for this compound was indeed LeuRS, despite the lack of enzyme inhibition. Subsequent analysis of PT638 metabolism demonstrated that bacterial nitroreductases readily converted this compound into the amino analogue, which inhibited LeuRS with an IC(50) of 3.0 ± 1.2 μM demonstrating that PT638 is thus a prodrug.

N-Acyl sulfamoyladenosines (acyl-AMS) have been used extensively to inhibit adenylate-forming enzymes that are involved in a wide range of biological processes. These acyl-AMS inhibitors are nonhydrolyzable mimics of the cognate acyl adenylate intermediates that are bound tightly by adenylate-forming enzymes. However, the anionic acyl sulfamate moiety presents a pharmacological liability that may be detrimental to cell permeability and pharmacokinetic profiles. We have previously developed the acyl sulfamate OSB-AMS (1) as a potent inhibitor of the adenylate-forming enzyme MenE, an o-succinylbenzoate-CoA (OSB-CoA) synthetase that is required for bacterial menaquinone biosynthesis. Herein, we report the use of computational docking to develop novel, non-acyl sulfamate inhibitors of MenE. A m-phenyl ether-linked analogue (5) was found to be the most potent inhibitor (IC50 = 8 μM; Kd = 244 nM), and its X-ray co-crystal structure was determined to characterize its binding mode in comparison to the computational prediction. This work provides a framework for the development of potent non-acyl sulfamate inhibitors of other adenylate-forming enzymes in the future., Graphical Abstract

[Image: see text] The accurate and precise determination of binding interactions plays a central role in fields such as drug discovery where structure–activity relationships guide the selection and optimization of drug leads. Binding is often assessed by monitoring the response caused by varying one of the binding partners in a functional assay or by using methods where the concentrations of free and/or bound ligand can be directly determined. In addition, there are also many approaches where binding leads to a change in the properties of the binding partner(s) that can be directly quantified such as an alteration in mass or in a spectroscopic signal. The analysis of data resulting from these techniques invariably relies on computer software that enable rapid fitting of the data to nonlinear multiparameter equations. The objective of this Perspective is to serve as a reminder of the basic assumptions that are used in deriving these equations and thus that should be considered during assay design and subsequent data analysis. The result is a set of guidelines for authors considering submitting their work to journals such as ACS Infectious Diseases.

The flavin cofactor performs many functions in the cell based on the ability of the isoalloxazine ring to undergo one- or two-electron reduction and form covalent adducts with reactants such as amino acids. In addition, the strong visible absorption of the cofactor is also the basis for flavin-dependent photoreceptors. Vibrational spectroscopy is uniquely suited to studying the mechanism of flavoproteins since the frequency of the vibrational modes is very sensitive to the electronic structure and environment of the isoalloxazine ring. This chapter describes the mechanistic information that can be gained using vibrational spectroscopy as well experimental challenges and approaches that are used to obtain and interpret the complex data contained in a vibrational spectrum.

The enoyl-ACP reductase (ENR) catalyzes the last reaction in the elongation cycle of the bacterial type II fatty acid biosynthesis (FAS-II) pathway. While the FabI ENR is a well-validated drug target in organisms such as Mycobacterium tuberculosis and Staphylococcus aureus, alternate ENR isoforms have been discovered in other pathogens, including the FabV enzyme that is the sole ENR in Yersinia pestis (ypFabV). Previously, we showed that the prototypical ENR inhibitor triclosan was a poor inhibitor of ypFabV and that inhibitors based on the 2-pyridone scaffold were more potent [Hirschbeck, M. (2012) Structure 20 (1), 89-100]. These studies were performed with the T276S FabV variant. In the work presented here, we describe a detailed examination of the mechanism and inhibition of wild-type ypFabV and the T276S variant. The T276S mutation significantly reduces the affinity of diphenyl ether inhibitors for ypFabV (20-fold → 100-fold). In addition, while T276S ypFabV generally displays an affinity for 2-pyridone inhibitors higher than that of the wild-type enzyme, the 4-pyridone scaffold yields compounds with similar affinity for both wild-type and T276S ypFabV. T276 is located at the N-terminus of the helical substrate-binding loop, and structural studies coupled with site-directed mutagenesis reveal that alterations in this residue modulate the size of the active site portal. Subsequently, we were able to probe the mechanism of time-dependent inhibition in this enzyme family by extending the inhibition studies to include P142W ypFabV, a mutation that results in a gain of slow-onset inhibition for the 4-pyridone PT156.

4-Oxo-4-phenyl-but-2-enoates inhibit MenB, the 1,4-dihydroxyl-2-naphthoyl-CoA synthase in the bacterial menaquinone (MK) biosynthesis pathway, through the formation of an adduct with coenzyme A (CoA). Here, we show that the corresponding methyl butenoates have MIC values as low as 0.35–0.75 µg/mL against drug sensitive and resistant strains of Staphylococcus aureus. Mode of action studies on the most potent compound, methyl 4-(4-chlorophenyl)-4-oxobut-2-enoate (1), reveal that 1 is converted into the corresponding CoA adduct in S. aureus cells, and that this adduct binds to the S. aureus MenB (saMenB) with a Kd value of 2 µM. The antibacterial spectrum of 1 is limited to bacteria that utilize MK for respiration, and the activity of 1 can be complemented with exogenous MK or menadione. Finally, treatment of methicillin-resistant S. aureus (MRSA) with 1 results in the small colony variant phenotype and thus 1 phenocopies knockout of the menB gene. Taken together the data indicate that the antibacterial activity of 1 results from a specific effect on MK biosynthesis. We also evaluated the in vivo efficacy of 1 using two mouse models of MRSA infection. Notably, compound 1 increased survival in a systemic infection model and resulted in a dose-dependent decrease in bacterial load in a thigh infection model, validating MenB as a target for the development of new anti-MRSA candidates.

PT70 is a diaryl ether inhibitor of InhA, the enoyl-ACP reductase in the Mycobacterium tuberculosis fatty acid biosynthesis pathway. It has a residence time of 24 min on the target, and also shows antibacterial activity in a mouse model of tuberculosis infection. Due to the interest in studying target tissue pharmacokinetics of PT70, we developed a method to radiolabel PT70 with carbon-11 and have studied its pharmacokinetics in mice and baboons using positron emission tomography.

Staphylococcus aureus is the leading cause of life-threatening infections, frequently originating from unknown or deep-seated foci. Source control and institution of appropriate antibiotics remain challenges, especially with infections due to methicillin-resistant S. aureus (MRSA). In this study, we developed a radiofluorinated analog of para-aminobenzoic acid (2-[18F]F-PABA) and demonstrate that it is an efficient alternative substrate for the S. aureus dihydropteroate synthase (DHPS). 2-[18F]F-PABA rapidly accumulated in vitro within laboratory and clinical (including MRSA) strains of S. aureus but not in mammalian cells. Biodistribution in murine and rat models demonstrated localization at infection sites and rapid renal elimination. In a rat model, 2-[18F]F-PABA positron emission tomography (PET) rapidly differentiated S. aureus infection from sterile inflammation and could also detect therapeutic failures associated with MRSA. These data suggest that 2-[18F]F-PABA has the potential for translation to humans as a rapid, noninvasive diagnostic tool to identify, localize, and monitor S. aureus infections.

Photochromic fluorescent proteins play key roles in super-resolution microscopy and optogenetics. The light-driven structural changes that modulate the fluorescence involve both trans-to-cis isomerization and proton transfer. The mechanism, timescale and relative contribution of chromophore and protein dynamics are currently not well understood. Here, the mechanism of off-to-on-state switching in dronpa is studied using femtosecond-to-millisecond time-resolved infrared spectroscopy and isotope labelling. Chromophore and protein dynamics are shown to occur on multiple timescales, from picoseconds to hundreds of microseconds. Following excitation of the trans chromophore, a ground-state primary product is formed within picoseconds. Surprisingly, the characteristic vibrational spectrum of the neutral cis isomer appears only after several tens of nanoseconds. Further fluctuations in protein structure around the neutral cis chromophore are required to form a new intermediate, which promotes the final proton-transfer reaction. These data illustrate the interplay between chromophore dynamics and the protein environment underlying fluorescent protein photochromism.

AN12855 is a novel cofactor-independent inhibitor of Mycobacterium tuberculosis InhA. AN12855 has potent activity against M. tuberculosis, good oral bioavailability, and comparable efficacy to isoniazid in infection models., New antitubercular agents are needed to combat the spread of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis. The frontline antitubercular drug isoniazid (INH) targets the mycobacterial enoyl-ACP reductase, InhA. Resistance to INH is predominantly through mutations affecting the prodrug-activating enzyme KatG. Here, we report the identification of the diazaborines as a new class of direct InhA inhibitors. The lead compound, AN12855, exhibited in vitro bactericidal activity against replicating bacteria and was active against several drug-resistant clinical isolates. Biophysical and structural investigations revealed that AN12855 binds to and inhibits the substrate-binding site of InhA in a cofactor-independent manner. AN12855 showed good drug exposure after i.v. and oral delivery, with 53% oral bioavailability. Delivered orally, AN12855 exhibited dose-dependent efficacy in both an acute and chronic murine model of tuberculosis infection that was comparable with INH. Combined, AN12855 is a promising candidate for the development of new antitubercular agents.