PAICS ubiquitination recruits UBAP2 to trigger phase separation for purinosome assembly.

Purinosomes serve as metabolons to enhance de novo purine synthesis (DNPS) efficiency through compartmentalizing DNPS enzymes during stressed conditions. However, the mechanism underpinning purinosome assembly and its pathophysiological functions remains elusive. Here, we show that K6-polyubiquitination of the DNPS enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) by cullin-5/ankyrin repeat and SOCS box containing 11 (Cul5/ASB11)-based ubiquitin ligase plays a driving role in purinosome assembly. Upon several purinosome-inducing cues, ASB11 is upregulated by relieving the H3K9me3/HP1α-mediated transcriptional silencing, thus stimulating PAICS polyubiquitination. The polyubiquitinated PAICS recruits ubiquitin-associated protein 2 (UBAP2), a ubiquitin-binding protein with multiple stretches of intrinsically disordered regions, thereby inducing phase separation to trigger purinosome assembly for enhancing DNPS pathway flux. In human melanoma, ASB11 is highly expressed to facilitate a constitutive purinosome formation to which melanoma cells are addicted for supporting their proliferation, viability, and tumorigenesis in a xenograft model. Our study identifies a driving mechanism for purinosome assembly in response to cellular stresses and uncovers the impact of purinosome formation on human malignancies.

The design, synthesis, and inhibition of Clostridioides difficile spore germination by acyclic and bicyclic tertiary amide analogs of cholate.

Clostridioides difficile infection (CDI) is a major identifiable cause of antibiotic-associated diarrhea. In our previous study (J. Med. Chem., 2018, 61, 6759-6778), we have identified N-phenyl-cholan-24-amide as a potent inhibitor of spore germination. The most potent compounds in our previous work are N-arylamides. We were interested in the role that the conformation of the amide plays in activity. Previous research has shown that secondary N-arylamides exist exclusively in the coplanar trans conformation while tertiary N-methyl-N-arylamides exist in a non-planar, cis conformation. The N-methyl-N-phenyl-cholan-24-amide was 17-fold less active compared to the parent compounds suggesting the importance of the orientation of the phenyl ring. To lock the phenyl ring into a trans conformation, cyclic tertiary amides were prepared. Indoline and quinoline cholan-24-amides were both inhibitors of spore germination; however, the indoline analogs were most potent. Isoindoline and isoquinoline amides were inactive. We found that the simple indoline derivative gave an IC50 value of 1 µM, while the 5'-fluoro-substituted compound (5d) possessed an IC50 of 400 nM. To our knowledge, 5d is the most potent known spore germination inhibitor described to date. Taken together, our results indicate that the trans, coplanar conformation of the phenyl ring is required for potent inhibition.

A Fluorescence-Based Assay for N5 -Carboxyaminoimidazole Ribonucleotide Mutase.

The enzyme N5 -carboxylaminoinidazole ribonucleotide (N5 -CAIR) mutase is found in microbial de novo purine biosynthesis but is absent in humans making it an attractive antimicrobial target. N5 -CAIR mutase catalyzes the synthesis of carboxyaminoimidazole ribonucleotide (CAIR) from N5 -CAIR which is itself prepared from aminoimidazole ribonucleotide (AIR) by the enzyme N5 -CAIR synthetase. During our research on identifying inhibitors of N5 -CAIR mutase, we developed an innovative, fluorescence-based assay to measure the activity of this enzyme. This assay relies upon our recent serendipitous observation that AIR reversibly reacts with the compound isatin. Reaction of a fluorescently-tagged isatin with AIR resulted in a large increase in fluorescence intensity allowing a measurement of the concentration of AIR in solution. From this observation, we developed a reproducible, non-continuous assay that can replicate the known kinetic parameters of the enzyme and can readily detect a recognized inhibitor of the enzyme. This assay should find utility in screening for inhibitors targeting N5 -CAIR mutase.

An Aniline-Substituted Bile Salt Analog Protects both Mice and Hamsters from Multiple Clostridioides difficile Strains.

Clostridioides difficile infection (CDI) is the major identifiable cause of antibiotic-associated diarrhea. The emergence of hypervirulent C. difficile strains has led to increases in both hospital- and community-acquired CDI. Furthermore, the rate of CDI relapse from hypervirulent strains can reach up to 25%. Thus, standard treatments are rendered less effective, making new methods of prevention and treatment more critical. Previously, the bile salt analog CamSA (cholic acid substituted with m-aminosulfonic acid) was shown to inhibit spore germination in vitro and protect mice and hamsters from C. difficile strain 630. Here, we show that CamSA was less active in preventing spore germination by other C. difficile ribotypes, including the hypervirulent strain R20291. The strain-specific in vitro germination activity of CamSA correlated with its ability to prevent CDI in mice. Additional bile salt analogs were screened for in vitro germination inhibition activity against strain R20291, and the most active compounds were tested against other strains. An aniline-substituted bile salt analog, CaPA (cholic acid substituted with phenylamine), was found to be a better antigerminant than CamSA against eight different C. difficile strains. In addition, CaPA was capable of reducing, delaying, or preventing murine CDI signs with all strains tested. CaPA-treated mice showed no obvious toxicity and showed minor effects on their gut microbiome. CaPA's efficacy was further confirmed by its ability to prevent CDI in hamsters infected with strain 630. These data suggest that C. difficile spores respond to germination inhibitors in a strain-dependent manner. However, careful screening can identify antigerminants with broad CDI prophylaxis activity.

Studies on the Importance of the 7α-, and 12α- hydroxyl groups of N-Aryl-3α,7α,12α-trihydroxy-5ß-cholan-24-amides on their Antigermination Activity Against a Hypervirulent Strain of Clostridioides (Clostridium) difficile.

Chenodeoxycholic acid (CDCA) is a natural germination inhibitor for C. difficile spores. In our previous study (J. Med. Chem., 2018, 61, 6759-6778), we identified N-phenyl-3α,7α,12α-trihydroxy-5ß-cholan-24-amide as an inhibitor of C. difficile strain R20291 with an IC50 of 1.8 µM. Studies of bile salts on spore germination have shown that chenodeoxycholate, ursodeoxycholate and lithocholate are more potent inhibitors of germination compared to cholate. Given this, we created amide analogs of chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids using amines identified from our previous studies. We found that chenodeoxy- and deoxycholate derivatives were active with potencies equivalent to those for cholanamides. This indicates that only 2 out of the 3 hydroxyl groups are needed for activity and that the alpha stereochemistry at position 7 is required for inhibition of spore germination.

Alternative Splicing of RAD6B and Not RAD6A is Selectively Increased in Melanoma: Identification and Functional Characterization.

Rad6B, a principal component of the translesion synthesis pathway, and activator of canonical Wnt signaling, plays an essential role in cutaneous melanoma development and progression. As Rad6 is encoded by two genes, namely, UBE2A (RAD6A) and UBE2B (RAD6B), in humans, we compared their expressions in melanomas and normal melanocytes. While both genes are weakly expressed in normal melanocytes, Rad6B is more robustly expressed in melanoma lines and patient-derived metastatic melanomas than RAD6A. The characterization of RAD6B transcripts revealed coexpression of various splice variants representing truncated or modified functional versions of wild-type RAD6B in melanomas, but not in normal melanocytes. Notably, two RAD6B isoforms with intact catalytic domains, RAD6BΔexon4 and RAD6Bintron5ins, were identified. We confirmed that RAD6BΔexon4 and RAD6Bintron5ins variants are expressed as 14 and 15 kDa proteins, respectively, with functional in vivo ubiquitin conjugating activity. Whole exome sequence analysis of 30 patient-derived melanomas showed RAD6B variants coexpressed with wild-type RAD6B in all samples analyzed, and RAD6Bintron5ins variants were found in half the cases. These variants constitute the majority of the RAD6B transcriptome in contrast to RAD6A, which was predominantly wild-type. The expression of functional RAD6B variants only in melanomas reveals RAD6B's molecular heterogeneity and its association with melanoma pathogenesis.

Isatins Inhibit N5-CAIR Synthetase by a Substrate Depletion Mechanism.

The continued rise of antibiotic-resistant infections coupled with the limited pipeline of new antimicrobials highlights the pressing need for the development of new antibacterial agents. One potential pathway for new agents is de novo purine biosynthesis as studies have shown that bacteria and lower eukaryotes synthesize purines differently than humans. Microorganisms utilize two enzymes, N5-CAIR synthetase and N5-CAIR mutase, to convert 5-aminoimidazole ribonucleotide (AIR) into 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) through the intermediate N5-carboxy-5-aminoimidazole ribonucleotide (N5-CAIR). In contrast, vertebrates directly convert AIR to CAIR via the enzyme AIR carboxylase. A high-throughput screen against N5-CAIR synthetase identified a group of compounds with a 2,3-indolinedione (isatin) core that inhibited the enzyme. While initial studies suggested that isatins inhibited the enzyme by a noncompetitive mechanism, here we show that isatins inhibit N5-CAIR synthetase by a substrate depletion mechanism. Unexpectedly, we found that isatin reacts rapidly and reversibly with the substrate AIR. The rate of the reaction is dependent upon the substituents on the phenyl moiety of isatin, with 5- and 7-bromoisatin being faster than 4-bromoisatin. These studies suggest that care should be taken when exploring isatin compounds because the biological activity could be a result of their reactivity.

Physicochemical and In Vitro Evaluation of Drug Delivery of an Antibacterial Synthetic Benzophenone in Biodegradable PLGA Nanoparticles.

Due to the increasing incidents of antimicrobial-resistant pathogens, the development of new antibiotics and their efficient formulation for suitable administration is crucial. Currently, one group of promising antimicrobial compounds are the benzophenone tetra-amides which show good activity even against gram-positive, drug-resistant pathogens. These compounds suffer from poor water solubility and bioavailability. It is therefore important to develop dosage forms which can address this disadvantage while also maintaining efficacy and potentially generating long-term exposures to minimize frequent dosing. Biodegradable nanoparticles provide one solution, and we describe here the encapsulation of the experimental benzophenone-based antibiotic, SV7. Poly-lactic-co-glycolic-acid (PLGA) nanoparticles were optimized for their physicochemical properties, their encapsulation efficiency, sustained drug release as well as antimicrobial activity. The optimized formulation contained particles smaller than 200 nm with a slightly negative zeta potential which released 39% of their drug load over 30 days. This formulation maintains the antibacterial activity of SV7 while minimizing the impact on mammalian cells.

The Design, Synthesis, and Characterizations of Spore Germination Inhibitors Effective against an Epidemic Strain of Clostridium difficile.

Clostridium difficile infections (CDI), particularly those caused by the BI/NAP1/027 epidemic strains, are challenging to treat. One method to address this disease is to prevent the development of CDI by inhibiting the germination of C. difficile spores. Previous studies have identified cholic amide m-sulfonic acid, CamSA, as an inhibitor of spore germination. However, CamSA is inactive against the hypervirulent strain R20291. To circumvent this problem, a series of cholic acid amides were synthesized and tested against R20291. The best compound in the series was the simple phenyl amide analogue which possessed an IC50 value of 1.8 µM, more than 225 times as potent as the natural germination inhibitor, chenodeoxycholate. This is the most potent inhibitor of C. difficile spore germination described to date. QSAR and molecular modeling analysis demonstrated that increases in hydrophobicity and decreases in partial charge or polar surface area were correlated with increases in potency.

Intramolecular charge-assisted hydrogen bond strength in pseudochair carboxyphosphate.

Carboxyphosphate, a suspected intermediate in ATP-dependent carboxylases, has not been isolated nor observed directly by experiment. Consequently, little is known concerning its structure, stability, and ionization state. Recently, carboxyphosphate as either a monoanion or dianion has been shown computationally to adopt a novel pseudochair conformation featuring an intramolecular charge-assisted hydrogen bond (CAHB). In this work, additive and subtractive correction schemes to the commonly employed open-closed method are used to estimate the strength of the CAHB. Truhlar's Minnesota M06-2X functional with Dunning's aug-cc-pVTZ basis set has been used for geometry optimization, energy evaluation, and frequency analysis. The CHARMM force field has been used to approximate the Pauli repulsive terms in the closed and open forms of carboxyphosphate. From our additive correction scheme, differential Pauli repulsion contributions between the pseudochair (closed) and open conformations of carboxyphosphate are found to be significant in determining the CAHB strength. The additive correction modifies the CAHB prediction (ΔEclosed-open) of -14 kcal/mol for the monoanion and -12 kcal/mol for the dianion to -22.9 and -18.4 kcal/mol, respectively. Results from the subtractive technique reinforce those from our additive procedure, where the predicted CAHB strength ranges from -17.8 to -25.4 kcal/mol for the monoanion and from -15.7 to -20.9 kcal/mol for the dianion. Ultimately, we find that the CAHB in carboxyphosphate meets the criteria for short-strong hydrogen bonds. However, carboxyphosphate has a unique energy profile that does not result in the symmetric double-well behavior of low-barrier hydrogen bonds. These findings provide deeper insight into the pseudochair conformation of carboxyphosphate, and lead to an improved mechanistic understanding of this intermediate in ATP-dependent carboxylases.

Site-directed mutagenesis of catalytic residues in N(5)-carboxyaminoimidazole ribonucleotide synthetase.

N(5)-CAIR synthetase, an essential enzyme in microorganisms, converts 5-aminoimidazole ribonucleotide (AIR) and bicarbonate to N(5)-CAIR with the aid of ATP. Previous X-ray crystallographic analyses of Aspergillus clavatus N(5)-CAIR synthetase postulated that R271, H273, and K353 were important for bicarbonate binding and for catalysis. As reported here, site-directed mutagenesis of these residues revealed that R271 and H273 are, indeed, critical for bicarbonate binding and catalysis whereas all K353 mutations, even ones conservative in nature, are inactive. Studies on the R271K mutant protein revealed cooperative substrate inhibition for ATP with a Ki of 1.2 mM. Kinetic investigation of the H273A mutant protein indicated that it was cooperative with respect to AIR; however, this effect was not seen in either the wild-type or any of the other mutant proteins. Cooperative ATP-dependent inhibition of wild-type N(5)-CAIR synthetase was also detected with ATP displaying a Ki of 3.3 mM. Taken together, these results indicate that N(5)-CAIR synthetase operates maximally within a narrow concentration of ATP.

Cyclam-based polymeric copper chelators for gene delivery and potential PET imaging.

A series of reducible polycationic copper chelators (RPCs) based on 1,4,8,11-tetraazacyclotetradecane (cyclam) were synthesized by Michael addition. Molecular weight of the polycations was controlled by reaction stoichiometry and reaction conditions, resulting in polymers with molecular weights ranging from 4400 to 13 800. The cyclam moieties in the polycations retained their ability to form complexes with Cu(II). The presence of disulfide bonds in the polycations resulted in substantially lower cytotoxicity than control 25 kDa poly(ethyleneimine). RPC as well as their complexes with Cu(II) exhibited high transfection activity in vitro. The reported polycationic Cu(II) chelates represent promising nucleic acid delivery vectors with potential for future theranostic applications.

Thieno[2,3-d]pyrimidinedione derivatives as antibacterial agents.

Several thieno[2,3-d]pyrimidinediones have been synthesized and examined for antibacterial activity against a range of gram-positive and gram-negative pathogens. Two compounds displayed potent activity (2-16 mg/L) against multi-drug resistant gram-positive organisms, including methicillin resistant, vancomycin-intermediate, vancomycin-resistant Staphylococcus aureus (MRSA, VISA, VRSA) and vancomycin-resistant enterococci (VRE). Only one of these agents possessed moderate activity (16-32 mg/L) against gram-negative strains. An examination of the cytotoxicity of these agents revealed that they displayed low toxicity (40-50 mg/L) against mammalian cells and very low hemolytic activity (2-7%). Taken together, these studies suggest that thieno[2,3-d]pyrimidinediones are interesting scaffolds for the development of novel gram-positive antibacterial agents.

The ATP-grasp enzymes.

The ATP-grasp enzymes consist of a superfamily of 21 proteins that contain an atypical ATP-binding site, called the ATP-grasp fold. The ATP-grasp fold is comprised of two α+ß domains that "grasp" a molecule of ATP between them and members of the family typically have an overall structural design containing three common conserved focal domains. The founding members of the family consist of biotin carboxylase, d-ala-d-ala ligase and glutathione synthetase, all of which catalyze the ATP-assisted reaction of a carboxylic acid with a nucleophile via the formation of an acylphosphate intermediate. While most members of the superfamily follow this mechanistic pathway, studies have demonstrated that two enzymes catalyze only the phosphoryl transfer step and thus are kinases instead of ligases. Members of the ATP-grasp superfamily are found in several metabolic pathways including de novo purine biosynthesis, gluconeogenesis, and fatty acid synthesis. Given the critical nature of these enzymes, researchers have actively sought the development of potent inhibitors of several members of the superfamily as antibacterial and anti-obseity agents. In this review, we will discuss the structure, function, mechanism, and inhibition of the ATP-grasp enzymes.

Examination of a synthetic benzophenone membrane-targeted antibiotic.

The enormous success of antibiotics is seriously threatened by the development of resistance to most of the drugs available on the market. Thus, novel antibiotics are needed that are less prone to bacterial resistance and are directed toward novel biological targets. Antimicrobial peptides (AMPs) have attracted considerable attention due to their unique mode of action and broad spectrum activity. However, these agents suffer from liability to proteases and the high cost of manufacturing has impeded their development. Previously, we have reported on a novel class of benzophenone-based antibiotics and early studies suggested that these agents might target the bacterial membrane. In this study, we present our work on the mechanism of action of these novel membrane targeted antibiotics. These compounds have good affinities to polyanionic components of the cell wall such as lipoteichoic acid (LTA) and lipopolysaccharide (LPS). We found that these agents release potassium ions from treated bacteria; thus, resulting in disruption of the bacterial membrane potential. Benzophenone-based membrane targeted antibiotics (BPMTAs) cause membrane disruption in synthetic lipid vesicles that mimic Gram-positive or Gram-negative bacteria. The compounds display no hemolytic activity up to a concentration that is 100 times the MIC values and they are capable of curing mice of a lethal MRSA infection. Repeated attempts to develop a mutant resistant to these agents has failed. Taken together, BPMTAs represent a promising new class of membrane-targeted antibacterial agents.

Characterization of insulin ILPR sequences for their ability to adopt a G-quadruplex structure.

A major genetic factor linked to the progression of type 1 diabetes occurs in the insulin-linked polymorphic repeat region (ILPR) located 363 bp upstream of the human insulin gene. Genetic studies have shown that individuals with class I repeats (30-60) are predisposed to the development of type 1 diabetes while individuals with longer repeats are protected. Previous research has suggested that some sequences found within the ILPR can adopt a G-quadruplex structure, and this finding has lead to speculation that G-quadruplexes may control insulin expression in certain circumstances. Unfortunately, relatively little study has been done on whether sequences found in the ILPR can adopt a quadruplex fold. In this study, we have utilized circular dichroism, thermal difference spectroscopy and ultraviolet (UV) melting studies to examine the first seven common repeat sequences (A-G) found in the ILPR. We find that sequences A-E adopt a quadruplex fold while sequences F and G likely do not. Examination of sequence B and a single nucleotide variant, B2, revealed that both folded into a G-quadruplex. This result casts doubt on previous studies suggesting that the formation of a quadruplex was related to the ability of ILPR sequences to regulate transcription.

Structural and functional studies of Aspergillus clavatus N(5)-carboxyaminoimidazole ribonucleotide synthetase .

N(5)-Carboxyaminoimidazole ribonucleotide synthetase (N(5)-CAIR synthetase), a key enzyme in microbial de novo purine biosynthesis, catalyzes the conversion of aminoimidazole ribonucleotide (AIR) to N(5)-CAIR. To date, this enzyme has been observed only in microorganisms, and thus, it represents an ideal target for antimicrobial drug development. Here we report the cloning, crystallization, and three-dimensional structural analysis of Aspergillus clavatus N(5)-CAIR synthetase solved in the presence of either Mg(2)ATP or MgADP and AIR. These structures, determined to 2.1 and 2.0 A, respectively, revealed that AIR binds in a pocket analogous to that observed for other ATP-grasp enzymes involved in purine metabolism. On the basis of these models, a site-directed mutagenesis study was subsequently conducted that focused on five amino acid residues located in the active site region of the enzyme. These investigations demonstrated that Asp 153 and Lys 353 play critical roles in catalysis without affecting substrate binding. All other mutations affected substrate binding and, in some instances, catalysis as well. Taken together, the structural and kinetic data presented here suggest a catalytic mechanism whereby Mg(2)ATP and bicarbonate first react to form the unstable intermediate carboxyphosphate. This intermediate subsequently decarboxylates to CO(2) and inorganic phosphate, and the amino group of AIR, through general base assistance by Asp 153, attacks CO(2) to form N(5)-CAIR.

Solution-phase parallel synthesis of novel membrane-targeted antibiotics.

The increase in the incidence of antibiotic-resistant infections is a major concern to healthcare workers and requires the development of novel antibacterial agents. Recently, we described a series of benzophenone-containing antibiotics which displayed activity against antibiotic-resistant bacteria. We have shown that these agents function by disrupting the bacterial membrane. To further explore these compounds, a practical and efficient solution-phase parallel synthesis method was developed which allowed us to prepare combinatorial libraries of these agents. Using this method, we prepared 218 compounds in 58 reactions. All of the compounds were characterized by HPLC and MALDI-TOF mass spectrometry. Analysis of this library for antibacterial activity identified six compounds which displayed MIC values of 2.0 mg/L against Staphylococcus aureus. Examination of the structure-function relationships of these agents revealed that cationic groups were required and that cyclic, aliphatic amines were crucial for activity. Using the information generated here, we speculate on how the various structural features of the molecule are necessary for the interaction with the bacterial membrane.

Design, synthesis, and structure-activity relationships of benzophenone-based tetraamides as novel antibacterial agents.

The increase in the incidence of both hospital- and community-acquired antibiotic-resistant infections is a major concern to the healthcare community. There have been only two new classes of antibiotics approved by the FDA over the past 40 years, and clearly there is a growing need for additional antimicrobial agents. In this paper, we present our work on the discovery of a class of benzophenone containing compounds that possess good activity against MRSA, VISA, VRSA, and VRE and moderate activity against E. coli. These compounds display MIC values in the 0.5-2.0 mg/L range and are not cytotoxic against mammalian cells. Extensive structure-activity relationship studies revealed that the benzophenone was absolutely essential for antibacterial activity as was the presence of a cationic group. Although these agents display DNA binding activity, we observed that these compounds do not inhibit any macromolecular synthesis reliant upon DNA nor do they inhibit lipid or cell wall biosynthesis. Instead, we found that these agents cause membrane depolarization, indicating that the bacterial membrane was the primary site of action for these agents. Our studies suggest that caution should be taken in assigning the mechanism of action for DNA binding antibiotics.

Identification of inhibitors of N5-carboxyaminoimidazole ribonucleotide synthetase by high-throughput screening.

The increasing risk of drug-resistant bacterial infections indicates that there is a growing need for new and effective antimicrobial agents. One promising, but unexplored area in antimicrobial drug design is de novo purine biosynthesis. Recent research has shown that de novo purine biosynthesis in microbes is different from that in humans. The differences in the pathways are centered around the synthesis of 4-carboxyaminoimidazole ribonucleotide (CAIR) which requires the enzyme N(5)-carboxyaminoimidazole ribonucleotide (N(5)-CAIR) synthetase. Humans do not require and have no homologs of this enzyme. Unfortunately, no studies aimed at identifying small-molecule inhibitors of N(5)-CAIR synthetase have been published. To remedy this problem, we have conducted high-throughput screening (HTS) against Escherichia coliN(5)-CAIR synthetase using a highly reproducible phosphate assay. HTS of 48,000 compounds identified 14 compounds that inhibited the enzyme. The hits identified could be classified into three classes based on chemical structure. Class I contains compounds with an indenedione core. Class II contains an indolinedione group, and Class III contains compounds that are structurally unrelated to other inhibitors in the group. We determined the Michaelis-Menten kinetics for five compounds representing each of the classes. Examination of compounds belonging to Class I indicates that these compounds do not follow normal Michaelis-Menten kinetics. Instead, these compounds inhibit N(5)-CAIR synthetase by reacting with the substrate AIR. Kinetic analysis indicates that the Class II family of compounds are non-competitive with both AIR and ATP. One compound in Class III is competitive with AIR but uncompetitive with ATP, whereas the other is non-competitive with both substrates. Finally, these compounds display no inhibition of human AIR carboxylase:SAICAR synthetase indicating that these agents are selective inhibitors of N(5)-CAIR synthetase.