Enhancing Proton-Coupled Electron Transfer in Blue Light Using FAD Photoreceptor AppA.

The Blue Light Using FAD (BLUF) photoreceptor utilizes a noncovalently bound FAD to absorb light and trigger the initial ultrafast events in receptor activation. FAD undergoes 1 and 2 electron reduction as an enzyme redox cofactor, and studies on the BLUF photoreceptor PixD revealed the formation of flavin radicals (FAD and FADH) during the photocycle, supporting a general mechanism for BLUF operation that involves PCET from a conserved Tyr to the oxidized FAD. However, no radical intermediates are observed in the closely related BLUF proteins AppA and BlsA, and replacing the conserved Tyr with fluoro-Tyr analogs that increase the acidity of the phenol OH has a minor effect on AppA photoactivation in contrast to PixD where the photocycle is halted at FAD.

Preclinical Positron Emission Tomography (PET) of Prosthetic Joint Infection Using a Nitro-Prodrug of 2-[F]F--Aminobenzoic Acid ([F]F-PABA).

Deep-seated bacterial infections are difficult to detect and diagnose due to the lack of specific clinical imaging modalities. Therefore, the bacteria-specific positron emission tomography radiotracer 2-[F]fluoro-4-nitrobenzoic acid ([F]FNB) was developed, which is reduced to 2-[F]fluoro-4-aminobenzoic acid ([F]F-PABA) by bacterial nitroreductases and has improved pharmacokinetics compared to the parent compound. PET imaging demonstrated that the uptake of 2-[F]fluoro-4-nitrobenzoic acid in a clinically relevant prosthetic joint infection model was up to ∼4-fold higher in the infected joint compared to the contralateral joint.

Ready-to-use iPSC-derived microglia progenitors for the treatment of CNS disease in mouse models of neuropathic mucopolysaccharidoses.

Mucopolysaccharidoses are inherited metabolic disorders caused by the deficiency in lysosomal enzymes required to break down glycosaminoglycans. Accumulation of glycosaminoglycans leads to progressive, systemic degenerative disease. The central nervous system is particularly affected, resulting in developmental delays, neurological regression, and early mortality.

Biological Evaluation of d-[F]Fluoroalanine and d-[F]Fluoroalanine- as Positron Emission Tomography Imaging Tracers for Bacterial Infection.

d-Amino acids such as d-alanine are substrates for bacterial peptidoglycan biosynthesis and are selectively taken up by bacteria and not mammalian cells. Consequently, d-amino acid metabolism is an attractive target for antibiotic discovery and the development of bacteria-specific imaging agents. d-Fluoroalanine and the deuterium-labeled analogue fludalanine (MK641) were originally explored as antibiotics by Merck but failed in clinical trials due to unaccepted toxicity.

Synthesis and Biological Evaluation of Fluorine-18 and Deuterium Labeled l-Fluoroalanines as Positron Emission Tomography Imaging Agents for Cancer Detection.

To fully explore the potential of F-labeled l-fluoroalanine for imaging cancer and other chronic diseases, a simple and mild radiosynthesis method has been established to produce optically pure l-3-[F]fluoroalanine (l-[F]FAla), using a serine-derivatized, five-membered-ring sulfamidate as the radiofluorination precursor. A deuterated analogue, l-3-[F]fluoroalanine-d (l-[F]FAla-d), was also prepared to improve metabolic stability. Both l-[F]FAla and l-[F]FAla-d were rapidly taken up by 9L/lacZ, MIA PaCa-2, and U87MG cells and were shown to be substrates for the alanine-serine-cysteine (ASC) amino acid transporter.

Elucidating the Signal Transduction Mechanism of the Blue-Light-Regulated Photoreceptor YtvA: From Photoactivation to Downstream Regulation.

The blue-light photoreceptor YtvA from has an N-terminal flavin mononucleotide (FMN)-binding light-oxygen-voltage (LOV) domain that is fused to a C-terminal sulfate transporter and anti-σ factor antagonist (STAS) output domain. To interrogate the signal transduction pathway that leads to photoactivation, the STAS domain was replaced with a histidine kinase, so that photoexcitation of the flavin could be directly correlated with biological activity. N94, a conserved Asn that is hydrogen bonded to the FMN C2═O group, was replaced with Ala, Asp, and Ser residues to explore the role of this residue in triggering the structural dynamics that activate the output domain.

Single Amino Acid Mutation Decouples Photochemistry of the BLUF Domain from the Enzymatic Function of OaPAC and Drives the Enzyme to a Switched-on State.

Photoactivated adenylate cyclases (PACs) are light-activated enzymes that combine a BLUF (blue-light using flavin) domain and an adenylate cyclase domain that are able to increase the levels of the important second messenger cAMP (cyclic adenosine monophosphate) upon blue-light excitation. The light-induced changes in the BLUF domain are transduced to the adenylate cyclase domain via a mechanism that has not yet been established. One critical residue in the photoactivation mechanism of BLUF domains, present in the vicinity of the flavin is the glutamine amino acid close to the N5 of the flavin.

Neutral ceramidase-active site inhibitor chemotypes and binding modes.

Ceramides impact a diverse array of biological functions and have been implicated in disease pathogenesis. The enzyme neutral ceramidase (nCDase) is a zinc-containing hydrolase and mediates the metabolism of ceramide to sphingosine (Sph), both in cells and in the intestinal lumen. nCDase inhibitors based on substrate mimetics, for example C6-urea ceramide, have limited potency, aqueous solubility, and micelle-free fraction.

Photocycle alteration and increased enzymatic activity in genetically modified photoactivated adenylate cyclase OaPAC.

Photoactivated adenylate cyclases (PACs) are light activated enzymes that combine blue light sensing capacity with the ability to convert ATP to cAMP and pyrophosphate (PPi) in a light-dependent manner. In most of the known PACs blue light regulation is provided by a blue light sensing domain using flavin which undergoes a structural reorganization after blue-light absorption. This minor structural change then is translated toward the C-terminal of the protein, inducing a larger conformational change that results in the ATP conversion to cAMP.

Evaluating the Impact of the Tyr158 p on the Mechanism and Inhibition of InhA, the Enoyl-ACP Reductase from .

InhA, the enoyl-ACP reductase, is a target for the tuberculosis (TB) drug isoniazid (INH). InhA inhibitors that do not require KatG activation avoid the most common mechanism of INH resistance, and there are continuing efforts to fully elucidate the enzyme mechanism to drive inhibitor discovery. InhA is a member of the short-chain dehydrogenase/reductase superfamily characterized by a conserved active site Tyr, Y158 in InhA.

Discovery of Novel Bruton's Tyrosine Kinase PROTACs with Enhanced Selectivity and Cellular Efficacy.

Bruton's tyrosine kinase (BTK) is a target for treating B-cell malignancies and autoimmune diseases, and several BTK inhibitors are already approved for use in humans. Heterobivalent BTK protein degraders are also in development, based on the premise that proteolysis targeting chimeras (PROTACs) may provide additional therapeutic benefits. However, most BTK PROTACs are based on the BTK inhibitor ibrutinib raising concerns about their selectivity profiles, given the known off-target effects of ibrutinib.

High Accuracy Prediction of PROTAC Complex Structures.

The design of PROteolysis-TArgeting Chimeras (PROTACs) requires bringing an E3 ligase into proximity with a target protein to modulate the concentration of the latter through its ubiquitination and degradation. Here, we present a method for generating high-accuracy structural models of E3 ligase-PROTAC-target protein ternary complexes. The method is dependent on two computational innovations: adding a "silent" convolution term to an efficient protein-protein docking program to eliminate protein poses that do not have acceptable linker conformations and clustering models of multiple PROTACs that use the same E3 ligase and target the same protein.

Synthesis and Preclinical Evaluation of a Novel Fluorine-18-Labeled Tracer for Positron Emission Tomography Imaging of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK) is a target for treating B-cell malignancies and autoimmune diseases. To aid in the discovery and development of BTK inhibitors and improve clinical diagnoses, we have developed a positron emission tomography (PET) radiotracer based on a selective BTK inhibitor, remibrutinib. [F]PTBTK3 is an aromatic, F-labeled tracer that was synthesized in 3 steps with a 14.

Heterobivalent Inhibitors of Acetyl-CoA Carboxylase: Drug Target Residence Time and Time-Dependent Antibacterial Activity.

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 and moiramide B () which bind to the biotin carboxylase and carboxyltransferase ACC active sites, respectively. The heterobivalent compound , which has a linker of 50 Å, was a tight binding inhibitor of ACC ( 0.

Unraveling the Photoactivation Mechanism of a Light-Activated Adenylyl Cyclase Using Ultrafast Spectroscopy Coupled with Unnatural Amino Acid Mutagenesis.

The hydrogen bonding network that surrounds the flavin in blue light using flavin adenine dinucleotide (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. Using time-resolved infrared spectroscopy (TRIR) and unnatural amino acid incorporation, we investigated the photoactivation mechanism and the role of the conserved tyrosine (Y6) in the forward reaction of the photoactivated adenylyl cyclase from (OaPAC). Our work elucidates the direct connection between BLUF photoactivation and the structural and functional implications on the partner protein for the first time.

Structure-Kinetic Relationship Studies for the Development of Long Residence Time LpxC Inhibitors.

UDP-3--(-3-hydroxymyristoyl)--acetylglucosamine deacetylase (LpxC) is a promising drug target in Gram-negative bacteria. Previously, we described a correlation between the residence time of inhibitors on LpxC (LpxC) and the post-antibiotic effect (PAE) caused by the inhibitors on the growth of . Given that drugs with prolonged activity following compound removal may have advantages in dosing regimens, we have explored the structure-kinetic relationship for LpxC inhibition by analogues of the pyridone methylsulfone () originally developed by Pfizer.

Photophysics of the Blue Light Using Flavin Domain.

Light activated proteins are at the heart of photobiology and optogenetics, so there is wide interest in understanding the mechanisms coupling optical excitation to protein function. In addition, such light activated proteins provide unique insights into the real-time dynamics of protein function. Using pump-probe spectroscopy, the function of a photoactive protein can be initiated by a sub-100 fs pulse of light, allowing subsequent protein dynamics to be probed from femtoseconds to milliseconds and beyond.

Impact of Target Turnover on the Translation of Drug-Target Residence Time to Time-Dependent Antibacterial Activity.

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--acyl--acetylglucosamine deacetylase (LpxC) in (paLpxC) results in significant PAE, inhibitors of the equivalent enzyme in (ecLpxC) do not cause a PAE.

Excited State Resonance Raman of Flavin Mononucleotide: Comparison of Theory and Experiment.

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 cofactors has been studied in detail by time-resolved Raman, lending it a benchmark status for mode assignments in excited electronic states of large molecules.

Exploring the chemical space of 1,2,3-triazolyl triclosan analogs for discovery of new antileishmanial chemotherapeutic agents.

Triclosan and isoniazid are known antitubercular compounds that have proven to be also active against 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 , the etiological agent of leishmaniasis, yielding some analogs with activity at micromolar concentrations and against H37Rv resulting in scarce active compounds with an MIC of 20 μM.

Identification of the vibrational marker of tyrosine cation radical using ultrafast transient infrared spectroscopy of flavoprotein systems.

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.

A Long Residence Time Enoyl-Reductase Inhibitor Explores an Extended Binding Region with Isoenzyme-Dependent Tautomer Adaptation and Differential Substrate-Binding Loop Closure.

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.

Structural Basis for the Regulation of Biofilm Formation and Iron Uptake in by the Blue-Light-Using Photoreceptor, BlsA.

The opportunistic human pathogen, , 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 lysates, we observed a number of proteins that interact with BlsA, including several transcription factors.

Unraveling the Mechanism of a LOV Domain Optogenetic Sensor: A Glutamine Lever Induces Unfolding of the Jα Helix.

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 domain.