Binding Studies of AICAR and Human Serum Albumin by Spectroscopic, Theoretical, and Computational Methodologies.

The interactions of small molecule drugs with plasma serum albumin are important because of the influence of such interactions on the pharmacokinetics of these therapeutic agents. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) is one such drug candidate that has recently gained attention for its promising clinical applications as an anti-cancer agent. This study sheds light upon key aspects of AICAR's pharmacokinetics, which are not well understood. We performed in-depth experimental and computational binding analyses of AICAR with human serum albumin (HSA) under simulated biochemical conditions, using ligand-dependent fluorescence sensitivity of HSA. This allowed us to characterize the strength and modes of binding, mechanism of fluorescence quenching, validation of FRET, and intermolecular interactions for the AICAR-HSA complexes. We determined that AICAR and HSA form two stable low-energy complexes, leading to conformational changes and quenching of protein fluorescence. Stern-Volmer analysis of the fluorescence data also revealed a collision-independent static mechanism for fluorescence quenching upon formation of the AICAR-HSA complex. Ligand-competitive displacement experiments, using known site-specific ligands for HSA's binding sites (I, II, and III) suggest that AICAR is capable of binding to both HSA site I (warfarin binding site, subdomain IIA) and site II (flufenamic acid binding site, subdomain IIIA). Computational molecular docking experiments corroborated these site-competitive experiments, revealing key hydrogen bonding interactions involved in stabilization of both AICAR-HSA complexes, reaffirming that AICAR binds to both site I and site II.

Cheminformatic comparison of approved drugs from natural product versus synthetic origins.

Despite the recent decline of natural product discovery programs in the pharmaceutical industry, approximately half of all new drug approvals still trace their structural origins to a natural product. Herein, we use principal component analysis to compare the structural and physicochemical features of drugs from natural product-based versus completely synthetic origins that were approved between 1981 and 2010. Drugs based on natural product structures display greater chemical diversity and occupy larger regions of chemical space than drugs from completely synthetic origins. Notably, synthetic drugs based on natural product pharmacophores also exhibit lower hydrophobicity and greater stereochemical content than drugs from completely synthetic origins. These results illustrate that structural features found in natural products can be successfully incorporated into synthetic drugs, thereby increasing the chemical diversity available for small-molecule drug discovery.

A diversity-oriented synthesis approach to macrocycles via oxidative ring expansion.

Macrocycles are key structural elements in numerous bioactive small molecules and are attractive targets in the diversity-oriented synthesis of natural product-based libraries. However, efficient and systematic access to diverse collections of macrocycles has proven difficult using classical macrocyclization reactions. To address this problem, we have developed a concise, modular approach to the diversity-oriented synthesis of macrolactones and macrolactams involving oxidative cleavage of a bridging double bond in polycyclic enol ethers and enamines. These substrates are assembled in only four or five synthetic steps and undergo ring expansion to afford highly functionalized macrocycles bearing handles for further diversification. In contrast to macrocyclization reactions of corresponding seco acids, the ring expansion reactions are efficient and insensitive to ring size and stereochemistry, overcoming key limitations of conventional approaches to systematic macrocycle synthesis. Cheminformatic analysis indicates that these macrocycles access regions of chemical space that overlap with natural products, distinct from currently targeted synthetic drugs.

An interdisciplinary course on computer-aided drug discovery to broaden student participation in original scientific research.

We present a new highly interdisciplinary project-based course in computer aided drug discovery (CADD). This course was developed in response to a call for alternative pedagogical approaches during the COVID-19 pandemic, which caused the cancellation of a face-to-face summer research program sponsored by the Louisiana Biomedical Research Network (LBRN). The course integrates guided research and educational experiences for chemistry, biology, and computer science students. We implement research-based methods with publicly available tools in bioinformatics and molecular modeling to identify and prioritize promising antiviral drug candidates for COVID-19. The purpose of this course is three-fold: I. Implement an active learning and inclusive pedagogy that fosters student engagement and research mindset; II. Develop student interdisciplinary research skills that are highly beneficial in a broader scientific context; III. Demonstrate that pedagogical shifts (initially incurred during the COVID-19 pandemic) can furnish longer-term instructional benefits. The course, which has now been successfully taught a total of five times, incorporates four modules, including lectures/discussions, live demos, inquiry-based assignments, and science communication.

Niraparib Shows Superior Tissue Distribution and Efficacy in a Prostate Cancer Bone Metastasis Model Compared with Other PARP Inhibitors.

Patients with prostate cancer whose tumors bear deleterious mutations in DNA-repair pathways often respond to PARP inhibitors. Studies were conducted to compare the activity of several PARP inhibitors in vitro and their tissue exposure and in vivo efficacy in mice bearing PC-3M-luc-C6 prostate tumors grown subcutaneously or in bone. Niraparib, olaparib, rucaparib, and talazoparib were compared in proliferation assays, using several prostate tumor cell lines and in a cell-free PARP-trapping assay. PC-3M-luc-C6 cells were approximately 12- to 20-fold more sensitive to PARP inhibition than other prostate tumor lines, suggesting that these cells bear a DNA damage repair defect. The tissue exposure and efficacy of these PARP inhibitors were evaluated in vivo in PC-3M-luc-C6 subcutaneous and bone metastasis tumor models. A steady-state pharmacokinetic study in PC-3M-luc-C6 tumor-bearing mice showed that all of the PARP inhibitors had favorable subcutaneous tumor exposure, but niraparib was differentiated by superior bone marrow exposure compared with the other drugs. In a PC-3M-luc-C6 subcutaneous tumor efficacy study, niraparib, olaparib, and talazoparib inhibited tumor growth and increased survival to a similar degree. In contrast, in the PC-3M-luc-C6 bone metastasis model, niraparib showed the most potent inhibition of bone tumor growth compared with the other therapies (67% vs. 40%-45% on day 17), and the best survival improvement over vehicle control [hazard ratio (HR), 0.28 vs. HR, 0.46-0.59] and over other therapies (HR, 1.68-2.16). These results show that niraparib has superior bone marrow exposure and greater inhibition of tumor growth in bone, compared with olaparib, rucaparib, and talazoparib.

Discovery of Potent and Orally Bioavailable Pyridine N-Oxide-Based Factor XIa Inhibitors through Exploiting Nonclassical Interactions.

Activated factor XI (FXIa) inhibitors are promising novel anticoagulants with low bleeding risk compared with current anticoagulants. The discovery of potent FXIa inhibitors with good oral bioavailability has been challenging. Herein, we describe our discovery effort, utilizing nonclassical interactions to improve potency, cellular permeability, and oral bioavailability by enhancing the binding while reducing polar atoms. Beginning with literature-inspired pyridine N-oxide-based FXIa inhibitor 1, the imidazole linker was first replaced with a pyrazole moiety to establish a polar C-H···water hydrogen-bonding interaction. Then, structure-based drug design was employed to modify lead molecule 2d in the P1' and P2' regions, with substituents interacting with key residues through various nonclassical interactions. As a result, a potent FXIa inhibitor 3f (Ki = 0.17 nM) was discovered. This compound demonstrated oral bioavailability in preclinical species (rat 36.4%, dog 80.5%, and monkey 43.0%) and displayed a dose-dependent antithrombotic effect in a rabbit arteriovenous shunt model of thrombosis.

Binding Isotope Effects for Interrogating Enzyme-Substrate Interactions.

Equilibrium binding isotope effects (BIEs) report on the bond vibrational status of enzyme substrates in the Michaelis complex prior to the transition state and how they differ from the solution state. Accordingly, BIEs provide an experimental means of interrogating enzyme-substrate interactions and inform on the influence of enzyme-mediated atomic distortions in modulating substrate reactivity. In this chapter, we outline a rapid equilibrium dialysis method that our lab has used to measure BIEs for several enzyme systems. Implementation of the rapid equilibrium dialysis approach is described in the context of our recent studies on the substrate bonding environment for the human protein lysine N-methyltransferase NSD2. A summary of BIE effects provides context for the range of experimental values.

Kinetic Isotope Effects and Transition State Structure for Human Phenylethanolamine N-Methyltransferase.

Phenylethanolamine N-methyltransferase (PNMT) catalyzes the S-adenosyl-l-methionine (SAM)-dependent conversion of norepinephrine to epinephrine. Epinephrine has been associated with critical processes in humans including the control of respiration and blood pressure. Additionally, PNMT activity has been suggested to play a role in hypertension and Alzheimer's disease. In the current study, labeled SAM substrates were used to measure primary methyl-14C and 36S and secondary methyl-3H, 5'-3H, and 5'-14C intrinsic kinetic isotope effects for human PNMT. The transition state of human PNMT was modeled by matching kinetic isotope effects predicted via quantum chemical calculations to intrinsic values. The model provides information on the geometry and electrostatics of the human PNMT transition state structure and indicates that human PNMT catalyzes the formation of epinephrine through an early SN2 transition state in which methyl transfer is rate-limiting.

Transition State Structure of RNA Depurination by Saporin L3.

Saporin L3 from the leaves of the common soapwort is a catalyst for hydrolytic depurination of adenine from RNA. Saporin L3 is a type 1 ribosome inactivating protein (RIP) composed only of a catalytic domain. Other RIPs have been used in immunotoxin cancer therapy, but off-target effects have limited their development. In the current study, we use transition state theory to understand the chemical mechanism and transition state structure of saporin L3. In favorable cases, transition state structures guide the design of transition state analogues as inhibitors. Kinetic isotope effects (KIEs) were determined for an A14C mutant of saporin L3. To permit KIE measurements, small stem-loop RNAs that contain an AGGG tetraloop structure were enzymatically synthesized with the single adenylate bearing specific isotopic substitutions. KIEs were measured and corrected for forward commitment to obtain intrinsic values. A model of the transition state structure for depurination of stem-loop RNA (5'-GGGAGGGCCC-3') by saporin L3 was determined by matching KIE values predicted via quantum chemical calculations to a family of intrinsic KIEs. This model indicates saporin L3 displays a late transition state with the N-ribosidic bond to the adenine nearly cleaved, and the attacking water nucleophile weakly bonded to the ribosyl anomeric carbon. The transition state retains partial ribocation character, a feature common to most N-ribosyl transferases. However, the transition state geometry for saporin L3 is distinct from ricin A-chain, the only other RIP whose transition state is known.

Transition State Structure and Inhibition of Rv0091, a 5'-Deoxyadenosine/5'-methylthioadenosine Nucleosidase from Mycobacterium tuberculosis.

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a bacterial enzyme that catalyzes the hydrolysis of the N-ribosidic bond in 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH). MTAN activity has been linked to quorum sensing pathways, polyamine biosynthesis, and adenine salvage. Previously, the coding sequence of Rv0091 was annotated as a putative MTAN in Mycobacterium tuberculosis. Rv0091 was expressed in Escherichia coli, purified to homogeneity, and shown to be a homodimer, consistent with MTANs from other microorganisms. Substrate specificity for Rv0091 gave a preference for 5'-deoxyadenosine relative to MTA or SAH. Intrinsic kinetic isotope effects (KIEs) for the hydrolysis of [1'-(3)H], [1'-(14)C], [5'-(3)H2], [9-(15)N], and [7-(15)N]MTA were determined to be 1.207, 1.038, 0.998, 1.021, and 0.998, respectively. A model for the transition state structure of Rv0091 was determined by matching KIE values predicted via quantum chemical calculations to the intrinsic KIEs. The transition state shows a substantial loss of C1'-N9 bond order, well-developed oxocarbenium character of the ribosyl ring, and weak participation of the water nucleophile. Electrostatic potential surface maps for the Rv0091 transition state structure show similarity to DADMe-immucillin transition state analogues. DADMe-immucillin transition state analogues showed strong inhibition of Rv0091, with the most potent inhibitor (5'-hexylthio-DADMe-immucillinA) displaying a Ki value of 87 pM.

Binding Isotope Effects for para-Aminobenzoic Acid with Dihydropteroate Synthase from Staphylococcus aureus and Plasmodium falciparum.

Dihydropteroate synthase is a key enzyme in folate biosynthesis and is the target of the sulfonamide class of antimicrobials. Equilibrium binding isotope effects and density functional theory calculations indicate that the substrate binding sites for para-aminobenzoic acid on the dihydropteroate synthase enzymes from Staphylococcus aureus and Plasmodium falciparum present distinct chemical environments. Specifically, we show that para-aminobenzoic acid occupies a more sterically constrained vibrational environment when bound to dihydropteroate synthase from P. falciparum relative to that of S. aureus. Deletion of a nonhomologous, parasite-specific insert from the plasmodial dihydropteroate synthase abrogated the binding of para-aminobenzoic acid. The loop specific to P. falciparum is important for effective substrate binding and therefore plays a role in modulating the chemical environment at the substrate binding site.

Principal component analysis as a tool for library design: a case study investigating natural products, brand-name drugs, natural product-like libraries, and drug-like libraries.

Principal component analysis (PCA) is a useful tool in the design and planning of chemical libraries. PCA can be used to reveal differences in structural and physicochemical parameters between various classes of compounds by displaying them in a convenient graphical format. Herein, we demonstrate the use of PCA to gain insight into structural features that differentiate natural products, synthetic drugs, natural product-like libraries, and drug-like libraries, and show how the results can be used to guide library design.

Immucillin-H, a purine nucleoside phosphorylase transition state analog, causes non-lethal attenuation of growth in Staphylococcus aureus.

Purine nucleoside phosphorylase (PNP; EC: 2.4.2.1) is a key enzyme involved in the purine salvage pathway. A recent bioinformatic study by Yadav, P. K. et al. (Bioinformation 2012, 8(14), 664-672) reports PNP as an essential enzyme and potential drug target in community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). We conducted an analysis using the methodology outlined by the authors, but were unable to identify PNP as an essential gene product in CA-MRSA. In addition, the treatment of Staphylococcus aureus cultures with immucillin-H, a powerful inhibitor of PNP, resulted in the non-lethal attenuation of growth, suggesting that PNP activity is not essential for cell viability.

Mechanism and inhibition of saFabI, the enoyl reductase from Staphylococcus aureus.

Approximately one-third of the world's population carries Staphylococcus aureus. The recent emergence of extreme drug resistant strains that are resistant to the "antibiotic of last resort", vancomycin, has caused a further increase in the pressing need to discover new drugs against this organism. The S. aureus enoyl reductase, saFabI, is a validated target for drug discovery. To drive the development of potent and selective saFabI inhibitors, we have studied the mechanism of the enzyme and analyzed the interaction of saFabI with triclosan and two related diphenyl ether inhibitors. Results from kinetic assays reveal that saFabI is NADPH-dependent, and prefers acyl carrier protein substrates carrying fatty acids with long acyl chains. On the basis of product inhibition studies, we propose that the reaction proceeds via an ordered sequential ternary complex, with the ACP substrate binding first, followed by NADPH. The interaction of NADPH with the enzyme has been further explored by site-directed mutagenesis, and residues R40 and K41 have been shown to be involved in determining the specificity of the enzyme for NADPH compared to NADH. Finally, in preliminary inhibition studies, we have shown that triclosan, 5-ethyl-2-phenoxyphenol (EPP), and 5-chloro-2-phenoxyphenol (CPP) are all nanomolar slow-onset inhibitors of saFabI. These compounds inhibit the growth of S. aureus with MIC values of 0.03-0.06 microg/mL. Upon selection for resistance, three novel safabI mutations, A95V, I193S, and F204S, were identified. Strains containing these mutations had MIC values approximately 100-fold larger than that of the wild-type strain, whereas the purified mutant enzymes had K i values 5-3000-fold larger than that of wild-type saFabI. The increase in both MIC and K i values caused by the mutations supports the proposal that saFabI is the intracellular target for the diphenyl ether-based inhibitors.

Evidence from Raman spectroscopy that InhA, the mycobacterial enoyl reductase, modulates the conformation of the NADH cofactor to promote catalysis.

InhA, the enoyl reductase from Mycobacterium tuberculosis, catalyzes the NADH-dependent reduction of trans-2-enoyl-ACPs. In the present work, Raman spectroscopy has been used to identify catalytically relevant changes in the conformation of the nicotinamide ring that occur when NADH binds to InhA. For 4(S)-NADD, there is an 11 cm-1 decrease in the wavenumber of the C4-D stretching band (nuC-D) and a 50% decrease in the width of this band upon binding to InhA. While a similar reduction in line width is observed for the corresponding band arising from 4(R)-NADD, nuC-D for this isomer increases 34 cm-1 upon binding to InhA. These changes in nuC-D indicate that the nicotinamide ring adopts a bound conformation in which the 4(S)C-D bond is in a pseudoaxial orientation. Mutagenesis of F149, a conserved active site residue close to the cofactor, demonstrates that this enzyme-induced modulation in cofactor structure is directly linked to catalysis. In contrast to the wild-type enzyme, Raman spectra of NADD bound to F149A InhA resemble those of NADD in solution. Consequently, F149A is no longer able to optimally position the cofactor for hydride transfer, which correlates with the 30-fold decrease in kcat and 2-fold increase in D(V/KNADH) caused by this mutation. These studies thus substantiate the proposal that hydride transfer is promoted by pseudoaxial positioning of the NADH pro-4S bond, and indicate that catalysis of substrate reduction by InhA results, in part, from correct orientation of the cofactor in the ground state.

Structure of acyl carrier protein bound to FabI, the FASII enoyl reductase from Escherichia coli.

Acyl carrier proteins play a central role in metabolism by transporting substrates in a wide variety of pathways including the biosynthesis of fatty acids and polyketides. However, despite their importance, there is a paucity of direct structural information concerning the interaction of ACPs with enzymes in these pathways. Here we report the structure of an acyl-ACP substrate bound to the Escherichia coli fatty acid biosynthesis enoyl reductase enzyme (FabI), based on a combination of x-ray crystallography and molecular dynamics simulation. The structural data are in agreement with kinetic studies on wild-type and mutant FabIs, and reveal that the complex is primarily stabilized by interactions between acidic residues in the ACP helix alpha2 and a patch of basic residues adjacent to the FabI substrate-binding loop. Unexpectedly, the acyl-pantetheine thioester carbonyl is not hydrogen-bonded to Tyr(156), a conserved component of the short chain alcohol dehydrogenase/reductase superfamily active site triad. FabI is a proven target for drug discovery and the present structure provides insight into the molecular determinants that regulate the interaction of ACPs with target proteins.

High affinity InhA inhibitors with activity against drug-resistant strains of Mycobacterium tuberculosis.

Novel chemotherapeutics for treating multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (MTB) are required to combat the spread of tuberculosis, a disease that kills more than 2 million people annually. Using structure-based drug design, we have developed a series of alkyl diphenyl ethers that are uncompetitive inhibitors of InhA, the enoyl reductase enzyme in the MTB fatty acid biosynthesis pathway. The most potent compound has a Ki' value of 1 nM for InhA and MIC99 values of 2-3 microg mL(-1) (6-10 microM) for both drug-sensitive and drug-resistant strains of MTB. Overexpression of InhA in MTB results in a 9-12-fold increase in MIC99, consistent with the belief that these compounds target InhA within the cell. In addition, transcriptional response studies reveal that the alkyl diphenyl ethers fail to upregulate a putative efflux pump and aromatic dioxygenase, detoxification mechanisms that are triggered by the lead compound triclosan. These diphenyl ether-based InhA inhibitors do not require activation by the mycobacterial KatG enzyme, thereby circumventing the normal mechanism of resistance to the front line drug isoniazid (INH) and thus accounting for their activity against INH-resistant strains of MTB.