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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Identification of a key water molecule involved in the macrophage migration inhibitory factor‐catalyzed tautomerization of para ‐hydroxyphenylpyruvate using neutron crystallography

Abstract Neutron crystallography was used to determine a 2.5‐Å resolution all‐atom structure of macrophage migration inhibitory factor (MIF) interacting with 3‐(4‐hydroxyphenyl)‐pyruvate (HPP). MIF is a pro‐inflammatory, pro‐tumorigenic protein that may be an attractive therapeutic target. MIF catalyzes the interconversion of the keto and enol forms of HPP by a tautomerase reaction. Although HPP is evidently not a physiological substrate of MIF, many compounds that inhibit this activity in enzymatic assays have been found also to inhibit physiological activities of MIF. Therefore, the MIF‐catalyzed HPP tautomerization reaction is used in initial screening of compounds in the search for inhibitors of MIF physiological activity. The neutron diffraction‐derived crystal structure reveals the position of a water molecule involved in the tautomerization reaction, and also confirms the charged state of lysine‐32 in the active site. The structure confirms the previously proposed catalytic mechanism of MIF, with the N‐terminal Pro‐1 abstracting a proton to generate an HPP enolate intermediate which is subsequently protonated. The structure reported herein reveals that this proton is supplied by a neighboring water molecule. Along with the neutron structure, a room‐temperature synchrotron x‐ray crystal structure reveals a covalent adduct between HPP and MIF. While this adduct is a result of radiation‐induced chemistry, its formation confirms the catalytic role of the active site residue because a covalent complex could only form if the reactive carbon of the substrate is correctly positioned by the enzyme.

Schröder, Gabriela C. [Department of Molecular and↗
DOE OSTI2022

A systematic exploration of boceprevir-based main protease inhibitors as SARS-CoV-2 antivirals

Boceprevir is an HCV NSP3 inhibitor that was explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (M Pro ) and contains an α-ketoamide warhead, a P1 β-cyclobutylalanyl moiety, a P2 dimethylcyclopropylproline, a P3 tert-butylglycine, and a P4 N-terminal tert-butylcarbamide. By introducing modifications at all four positions, we synthesized 20 boceprevir-based M Pro inhibitors including PF-07321332 and characterized their M Pro inhibition potency in test tubes (in vitro) and 293T cells (in cellulo). Crystal structures of M Pro bound with 10 inhibitors and cytotoxicity and antiviral potency of 4 inhibitors were characterized as well. Replacing the P1 site with a β-(S-2-oxopyrrolidin-3-yl)-alanyl (Opal) residue and the warhead with an aldehyde leads to high in vitro potency. The original moieties at P2, P3 and the P4 N-terminal cap positions in boceprevir are better than other tested chemical moieties for high in vitro potency. In crystal structures, all inhibitors form a covalent adduct with the M Pro active site cysteine. Here, the P1 Opal residue, P2 dimethylcyclopropylproline and P4 N-terminal tert-butylcarbamide make strong hydrophobic interactions with M Pro , explaining high in vitro potency of inhibitors that contain these moieties. A unique observation was made with an inhibitor that contains a P4 N-terminal isovaleramide. In its M Pro complex structure, the P4 N-terminal isovaleramide is tucked deep in a small pocket of M Pro that originally recognizes a P4 alanine side chain in a substrate. Although all inhibitors show high in vitro potency, they have drastically different in cellulo potency to inhibit ectopically expressed M Pro in human 293T cells. In general, inhibitors with a P4 N-terminal carbamide or amide have low in cellulo potency. This trend is reversed when the P4 N-terminal cap is changed to a carbamate. The installation of a P3 O-tert-butyl-threonine improves in cellulo potency. Three molecules that contain a P4 N-terminal carbamate were advanced to cytotoxicity tests on 293T cells and antiviral potency tests on three SARS-CoV-2 variants. They all have relatively low cytotoxicity and high antiviral potency with EC 50 values around 1 μM. A control compound with a nitrile warhead and a P4 N-terminal amide has undetectable antiviral potency. Based on all observations, we conclude that a P4 N-terminal carbamate in a boceprevir derivative is key for high antiviral potency against SARS-CoV-2.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Acetylcholinesterase: Structure, dynamics, and interactions with organophosphorus compounds

Acetylcholinesterase (AChE) is an enzyme that hydrolyzes the neurotransmitter acetylcholine (ACh), removing it from the synaptic cleft after the transmission of an electrical signal, making it an essential component of chemical neurotransmission. AChE is a serine hydrolase, containing a catalytic triad of Ser/His/Glu. AChE is a prime target for pharmaceuticals treating a variety of neurological disorders. It is also the target of synthetic organophosphorus (OP) compounds that have been used as pesticides and chemical warfare agents. OP compounds contain a potent leaving group, such as fluorine, and act by forming a covalent adduct with the catalytic serine of the AChE active site. A wealth of structural information is available for AChE, including over 300 structures, including a subset of structures in complex with drugs as well as OP compounds. This review will highlight the interactions between OP compounds and AChE from a structural and computational perspective, with a discussion of access to the active site, as well as side reactions that lead to dealkylation of the OP-catalytic serine adduct, a process known as aging. We conclude that while the majority of the conformational changes needed to accommodate the OP compounds are localized to the acyl loop in the crystal structures, molecular dynamics simulations highlight the potential for a far more dynamic enzyme.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Design, Synthesis, and Biological Activity of Novel Ornithine Decarboxylase (ODC) Inhibitors

We here describe the design, synthesis, and biological activity of novel ornithine decarboxylase (ODC) inhibitors that show significantly higher potency in vitro than α-difluoromethylornithine (DFMO), a U.S. Food and Drug Administration (FDA) approved drug. We report two X-ray structures of ODC complexed with new ODC inhibitors, computational docking, molecular dynamics, and binding free energy calculations to validate the experimental models. The X-ray structures reveal that covalent adducts with pyridoxal phosphate (PLP) are formed in the active site of the human ODC enzyme, as verified by their preparation and enzymatic testing. Finally, we verified that the cellular activity of endogenous ODC was inhibited, and polyamine levels were reduced. Given that ODC is a clinically validated target, combined with the fact that DFMO is currently the only ODC inhibitor in clinical use for several indications, the further development of more potent ODC inhibitors with superior activity and physical properties is warranted.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Cryo-EM analysis of S. aureus TarL, a polymerase in wall teichoic acid biogenesis central to virulence and antibiotic resistance

Wall teichoic acid (WTA), a covalent adduct of Gram-positive bacterial cell wall peptidoglycan, contributes directly to virulence and antibiotic resistance in pathogenic species. Polymerization of the Staphylococcus aureus WTA ribitol-phosphate chain is catalyzed by TarL, a member of the largely uncharacterized TagF-like family of membrane-associated enzymes. We report the cryo–electron microscopy structure of TarL, showing a tetramer that forms an extensive membrane-binding platform of monotopic helices. TarL is composed of an amino-terminal immunoglobulin-like domain and a carboxyl-terminal glycosyltransferase-B domain for ribitol-phosphate polymerization. The active site of the latter is complexed to donor substrate cytidine diphosphate–ribitol, providing mechanistic insights into the catalyzed phosphotransfer reaction. Furthermore, the active site is surrounded by electropositive residues that serve to retain the lipid-linked acceptor for polymerization. Our data advance general insight into the architecture and membrane association of the still poorly characterized monotopic membrane protein class and present molecular details of ribitol-phosphate polymerization that may aid in the design of new antimicrobials.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

NNA-DNA adducts as biomarkers for detecting exposure to thirdhand smoke

NNA-derived specific adducts represent an integrated biomarker of exposure to thirdhand smoke (THS) as NNA is unique to THS. The NNA-dG covalent binding adduct could serve as such a biomarker, and play a role in identifying individuals exposed to THS, thus providing critical information for early detection and prevention.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Watson-Crick Base-Pairing Requirements for ssDNA Recognition and Processing in Replication-Initiating HUH Endonucleases

Replication-initiating HUH endonucleases (Reps) are sequence-specific nucleases that cleave and rejoin single-stranded DNA (ssDNA) during rolling-circle replication. These functions are mediated by covalent linkage of the Rep to its substrate post cleavage. Here, we describe the structures of the endonuclease domain from the Muscovy duck circovirus Rep in complex with its cognate ssDNA 10-mer with and without manganese in the active site. Structural and functional analyses demonstrate that divalent cations play both catalytic and structural roles in Reps by polarizing and positioning their substrate. Further structural comparisons highlight the importance of an intramolecular substrate Watson-Crick (WC) base pairing between the -4 and +1 positions. Subsequent kinetic and functional analyses demonstrate a functional dependency on WC base pairing between these positions regardless of the pair’s identity (i.e., A·T, T·A, G·C, or C·G), highlighting a structural specificity for substrate interaction. Finally, considering how well WC swaps were tolerated in vitro, we sought to determine to what extent the canonical -4T·+1A pairing is conserved in circular Rep-encoding single-stranded DNA viruses and found evidence of noncanonical pairings in a minority of these genomes. Altogether, our data suggest that substrate intramolecular WC base pairing is a universal requirement for separation and reunion of ssDNA in Reps.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Oxaliplatin–DNA Adducts as Predictive Biomarkers of FOLFOX Response in Colorectal Cancer: A Potential Treatment Optimization Strategy

FOLFOX is one of the most effective treatments for advanced colorectal cancer (CRC). However, cumulative oxaliplatin neurotoxicity often results in halting the therapy. Oxaliplatin functions predominantly via the formation of toxic covalent drug-DNA adducts. We hypothesize that oxaliplatin-DNA adduct levels formed in vivo in peripheral blood mononuclear cells (PBMC) are proportional to tumor shrinkage caused by FOLFOX therapy. We further hypothesize that adducts induced by sub-therapeutic "diagnostic microdoses" are proportional to those induced by therapeutic doses and are also predictive of response to FOLFOX therapy. These hypotheses were tested in CRC cell lines and a pilot clinical study. Four CRC cell lines were cultured with therapeutically relevant (100 µM) or diagnostic microdose (1 µM) concentrations of [ 14 C]oxaliplatin. The C-14 label enabled quantification of oxaliplatin-DNA adduct level with accelerator mass spectrometry (AMS). Oxaliplatin-DNA adduct formation was correlated with oxaliplatin cytotoxicity for each cell line as measured by the MTT viability assay. Six CRC patients received by IV a diagnostic microdose containing [ 14 C]oxaliplatin prior to treatment, as well as a second [ 14 C]oxaliplatin dose during FOLFOX chemotherapy, termed a "therapeutic dose." Oxaliplatin-DNA adduct levels from PBMC correlated significantly to mean tumor volume change of evaluable target lesions (5 of the 6 patients had measurable disease). Finally, oxaliplatin-DNA adduct levels were linearly proportional between microdose and therapeutically relevant concentrations in cell culture experiments and patient samples, as was plasma pharmacokinetics, indicating potential utility of diagnostic microdosing.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Interplay between Electrostatic Properties of Molecular Adducts and Their Positions at Carbon Nanotubes

Formation of the sp 3 -defect due to covalent functionalization of a single-walled carbon nanotube (SWCNT) results in a new red-shifted emissive excitons. Here, using density functional theory, we study the impact of the intrinsic molecular dipole moments and the local charges induced by various molecular adducts on the energy and localization properties of the optically active defect-based exciton in a (10,5) SWCNT. The interplay of two effects plays a role in the localization of the exciton and, therefore, its red shift, but at different scales: The source of the leading order contribution is the defect conformation, resulting in the red shift of the defect-associated exciton with respect to the E 11 band of the pristine SWCNT on the order of ~100 meV, while the individual dipoles and polarization properties of molecular adducts lead to significantly smaller red shifts on the order of ~10 meV. While the species-dependent trends in defect-induced charges do not directly correlate to the exciton red shift, the charge at the sp 3 -defect exhibits a distinct behavior between ortho- and para-defect configurations and is relevant to the chemical reactivity of the defect position depending on the adduct type. Overall, our computational results may be helpful for diverse synthetic strategies to fine-tune emission of SWCNTs toward desired applications.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Peptidomimetic nitrile warheads as SARS-CoV-2 3CL protease inhibitors

Tragically, the death toll from the COVID-19 pandemic continues to rise, and with variants being observed around the globe new therapeutics, particularly direct-acting antivirals that are easily administered, are desperately needed. Studies targeting the SARS-CoV-2 3CL protease, which is critical for viral replication, with different peptidomimetics and warheads is an active area of research for development of potential drugs. To date, however, only a few publications have evaluated the nitrile warhead as a viral 3CL protease inhibitor, with only modest activity reported. This article describes our investigation of P3 4-methoxyindole peptidomimetic analogs with select P1 and P2 groups with a nitrile warhead that are potent inhibitors of SARS-CoV-2 3CL protease and demonstrate in vitro SARS-CoV-2 antiviral activity. A selectivity for SARS-CoV-2 3CL protease over human cathepsins B, S and L was also observed with the nitrile warhead, which was superior to that with the aldehyde warhead. A co-crystal structure with SARS-CoV-2 3CL protease and a reversibility study indicate that a reversible, thioimidate adduct is formed when the catalytic sulfur forms a covalent bond with the carbon of the nitrile. Furthermore, this effort also identified efflux as a property limiting antiviral activity of these compounds, and together with the positive attributes described these results provide insight for further drug development of novel nitrile peptidomimetics targeting SARS-CoV-2 3CL protease.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Au 3 -to-Ag 3 coordinate-covalent bonding and other supramolecular interactions with covalent bonding strength

An efficient strategy for designing charge-transfer complexes using coinage metal cyclic trinuclear complexes (CTCs) is described herein. Due to opposite quadrupolar electrostatic contributions from metal ions and ligand substituents, [Au(m-Pz-(i-C 3 H7) 2 )] 3 ∙[Ag(m-Tz-(n-C 3 F 7 ) 2 )] 3 (Pz = pyrazolate, Tz = triazolate) has been obtained and its structure verified by single crystal X-ray diffraction – representing the 1st crystallographically-verified M 3 @M' 3 stacked adduct of monovalent coinage metal CTCs. Abundant supramolecular interactions with aggregate covalent bonding strength arise from a combination of M–M' (Au / Ag), metal–π, π–π interactions and hydrogen bonding in this charge-transfer complex, according to density functional theory analyses, yielding a computed binding energy of 66 kcal mol -1 between the two trimer moieties – a large value for intermolecular interactions between adjacent d 10 centres (nearly doubling the value for a recently claimed Au(I) / Cu(I) polar-covalent bond: Proc. Natl. Acad. Sci. U.S.A., 2017, 114, E5042) – which becomes 87 kcal mol -1 with benzene stacking. Surprisingly, DFT analysis suggests that: (a) some other literature precedents should have attained a stacked M 3 @M' 3 product akin to the one herein, with similar or even higher binding energy; and (b) a high overall intertrimer bonding energy by inferior electrostatic assistance, underscoring genuine orbital overlap between M and M' frontier molecular orbitals in such polar-covalent M–M' bonds in this family of molecules. The Au / Ag bonding is reminiscent of classical Werner-type coordinate-covalent bonds such as H 3 N: → Ag in [Ag(NH 3 ) 2 ] + , as demonstrated herein quantitatively. Solidstate and molecular modeling illustrate electron flow from the p-basic gold trimer to the p-acidic silver trimer with augmented contributions from ligand-to-ligand’ (LL'CT) and metal-to-ligand (MLCT) charge transfer.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Uncompetitive, adduct-forming SARM1 inhibitors are neuroprotective in preclinical models of nerve injury and disease

Axon degeneration is an early pathological event in many neurological diseases. The identification of the nicotinamide adenine dinucleotide (NAD) hydrolase SARM1 as a central metabolic sensor and axon executioner presents an exciting opportunity to develop novel neuroprotective therapies that can prevent or halt the degenerative process, yet limited progress has been made on advancing efficacious inhibitors. Here we describe a class of NAD-dependent active-site SARM1 inhibitors that function by intercepting NAD hydrolysis and undergoing covalent conjugation with the reaction product adenosine diphosphate ribose (ADPR). The resulting small-molecule ADPR adducts are highly potent and confer compelling neuroprotection in preclinical models of neurological injury and disease, validating this mode of inhibition as a viable therapeutic strategy. Additionally, we show that the most potent inhibitor of CD38, a related NAD hydrolase, also functions by the same mechanism, further underscoring the broader applicability of this mechanism in developing therapies against this class of enzymes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Mechanism of Vapor-Phase Infiltration of Organometallic Hf in Poly(Methyl Methacrylate) for Hybrid Resist Applications

Inorganic–organic hybrid thin films synthesized by vapor-phase infiltration (VPI) of metal oxides into organic photoresists, such as poly(methyl methacrylate) (PMMA), have recently demonstrated their utility in extreme ultraviolet lithography, critical for angstrom-era semiconductor device miniaturization. Hafnium oxide infiltration has been reported recently for this purpose, but its detailed VPI mechanism has remained largely unexplored. In this study, we investigated the VPI characteristics and mechanisms of tetrakis(dimethylamido)hafnium (TDMAHf)─the hafnium precursor predominantly used for VPI in the field─into PMMA and examined its impact on electron-beam lithography (EBL) exposure behavior. VPI was performed at temperatures ranging from 85 to 150 °C, with chemical interactions characterized using infrared reflection-absorption spectroscopy, and resist patterning performance was evaluated through EBL dose-sensitivity assessments. The results indicate that TDMAHf forms a reversible adduct with PMMA at temperatures up to 120 °C, whereas at 150 °C, covalent bond formation occurs, most likely via dealkylation that leads to acetate formation. EBL studies reveal that resist sensitivity is influenced by both infiltration temperature and developer selection, with aqueous isopropyl alcohol development demonstrating enhanced sensitivity compared to organic solvent-based development. The optimized infiltration protocol at 120 °C ensures a uniform inorganic distribution without compromising resist dissolution. These findings not only help refine hybrid resist patterning performance but also offer insights potentially applicable to the VPI of other homoleptic metal-amide organometallic VPI precursors that include TDMA ligands.

36 MATERIALS SCIENCE↗
DOE OSTI2022

Efficient Electrochemical Reduction of CO 2 to Formate in Methanol Solutions by Mn‐Functionalized Electrodes in the Presence of Amines**

Carbon cloth electrode modified by covalently attaching a manganese organometallic catalyst is used as cathode for the electrochemical reduction of CO 2 in methanol solutions. Six different industrial amines are employed as co-catalyst in millimolar concentrations to deliver a series of new reactive system. While such absorbents were so far believed to provide a CO 2 reservoir and act as sacrificial proton source, we herein demonstrate that this role can be played by methanol, and that the adduct formed between CO 2 and the amine can act as an effector or inhibitor toward the catalyst, thereby enhancing or reducing the production of formate. Pentamethyldiethylentriamine (PMDETA), identified as the best effector in our series, converts CO 2 in wet methanolic solution into bisammonium bicarbonate. Computational studies revealed that this adduct is responsible for a barrierless transformation of CO 2 to formate by the reduced form of the Mn catalyst covalently bonded to the electrode surface. As a consequence, selectivity can be switched on demand from CO to formate anion, and in the case of (PMDETA) an impressive TON HCOO- of 2.8×10 4 can be reached. This new valuable knowledge on an integrated capture and utilization system paves the way toward more efficient transformation of CO 2 into liquid fuel.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Autoprocessing and oxyanion loop reorganization upon GC373 and nirmatrelvir binding of monomeric SARS-CoV-2 main protease catalytic domain

The monomeric catalytic domain (residues 1–199) of SARS-CoV-2 main protease (MPro 1-199 ) fused to 25 amino acids of its flanking nsp4 region mediates its autoprocessing at the nsp4-MPro 1-199 junction. We report the catalytic activity and the dissociation constants of MPro 1-199 and its analogs with the covalent inhibitors GC373 and nirmatrelvir (NMV), and the estimated monomer-dimer equilibrium constants of these complexes. Mass spectrometry indicates the presence of the accumulated adduct of NMV bound to MPro WT and MPro 1-199 and not of GC373. A room temperature crystal structure reveals a native-like fold of the catalytic domain with an unwound oxyanion loop (E state). In contrast, the structure of a covalent complex of the catalytic domain-GC373 or NMV shows an oxyanion loop conformation (E* state) resembling the full-length mature dimer. These results suggest that the E-E* equilibrium modulates autoprocessing of the main protease when converting from a monomeric polyprotein precursor to the mature dimer.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Effect of Redox-Inactive Metal Ion–Nickel(III) Interactions on the Redox Properties and Proton-Coupled Electron Transfer Reactivity

We report Mononuclear nickel(II) and nickel(III) complexes of a bisamidate-bisalkoxide ligand, (NMe 4 ) 2 [Ni- II (HMPAB)] (1) and (NMe 4 )[Ni III (HMPAB)] (2), respectively, have been synthesized and characterized by various spectroscopic techniques including X-ray crystallography. The reaction of redox-inactive metal ions (M n+ = Ca 2+ , Mg 2+ , Zn 2+ , Y 3+ , and Sc 3+ ) with 2 resulted in 2-M n+ adducts, which was assessed by an array of spectroscopic techniques including X-ray absorption spectroscopy (XAS), electron paramagnetic resonance (EPR), and reactivity studies. The X-ray structure of Ca 2+ coordinated to Ni(III) complexes, 2Ca 2+ T, was determined and exhibited an average Ni-Ca distance of 3.1253 angstrom, close to the metal ions' covalent radius. XAS analysis of 2-Ca 2+ and 2-Y 3+ in solution further revealed an additional coordination to Ca and Y in the 2-M n+ adducts with shortened Ni-M distances of 2.15 and 2.11 Å, respectively, implying direct bonding interactions between Ni and Lewis acids (LAs). Such a short interatomic distance between Ni(III) and M is unprecedented and was not observed before. EPR analysis of 2 and 2-M n+ species, moreover, displayed rhombic signals with g av > 2.12 for all complexes, supporting the +III oxidation state of Ni. The Ni III /Ni II redox potential of 2 and 2-M n+ species was determined, and a plot of E 1/ 2 of 2-M n+ versus pK a of [M(H 2 O) n ] m+ exhibited a linear relationship, implying that the Ni III /Ni II potential of 2 can be tuned with different redox-inactive metal ions. Reactivity studies of 2 and 2-M n+ with different 4-X-2,6-ditert-butylphenol (4-XDTBP) and other phenol derivatives were performed, and based on kinetic studies, we propose the involvement of a proton-coupled electron transfer (PCET) pathway. Analysis of the reaction products after the reaction of 2 with 4-OMe-DTBP showed the formation of a Ni(II) complex (1a) where one of the alkoxide arms of the ligand is protonated. A pK a value of 24.2 was estimated for 1a. The reaction of 2-M n+ species was examined with 4-OMe-DTBP, and it was observed that the k 2 values of 2-M n+ species increase by increasing the Lewis acidity of redox-inactive metal ions. However, the obtained k 2 values for 2-M n+ species are much lower compared to the k 2 value for 2. Such a variation of PCET reactivity between 2 and 2-M n+ species may be attributed to the interactions between Ni(III) and LAs. Our findings show the significance of the secondary coordination sphere effect on the PCET reactivity of Ni(III) complexes and furnish important insights into the reaction mechanism involving high-valent nickel species, which are frequently invoked as key intermediates in Ni-mediated enzymatic reactions, solar-fuel catalysis, and biomimetic/synthetic transformation reactions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Small molecule microarray identifies inhibitors of tyrosyl-DNA phosphodiesterase 1 that simultaneously access the catalytic pocket and two substrate binding sites

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a member of the phospholipase D family of enzymes, which catalyzes the removal of both 3'- and 5'-DNA phosphodiester adducts. Importantly, it is capable of reducing the anticancer effects of type I topoisomerase (TOP1) inhibitors by repairing the stalled covalent complexes of TOP1 with DNA. It achieves this by promoting the hydrolysis of the phosphodiester bond between the Y723 residue of human TOP1 and the 3'-phosphate of its DNA substrate. Blocking TDP1 function is an attractive means of enhancing the efficacy of TOP1 inhibitors and overcoming drug resistance. Previously, we reported the use of an X-ray crystallographic screen of more than 600 fragments to identify small molecule variations on phthalic acid and hydroxyquinoline motifs that bind within the TDP1 catalytic pocket. Yet, the majority of these compounds showed limited (millimolar) TDP1 inhibitory potencies. We now report examining a 21 000-member library of drug-like Small Molecules in Microarray (SMM) format for their ability to bind Alexa Fluor 647 (AF647)-labeled TDP1. The screen identified structurally similar N,2-diphenylimidazo[1,2-a]pyrazin-3-amines as TDP1 binders and catalytic inhibitors. We then explored the core heterocycle skeleton using one-pot Groebke–Blackburn–Bienayme multicomponent reactions and arrived at analogs having higher inhibitory potencies. Solving TDP1 co-crystal structures of a subset of compounds showed their binding at the TDP1 catalytic site, while mimicking substrate interactions. Although our original fragment screen differed significantly from the current microarray protocol, both methods identified ligand–protein interactions containing highly similar elements. Importantly inhibitors identified through the SMM approach show competitive inhibition against TDP1 and access the catalytic phosphate-binding pocket, while simultaneously providing extensions into both the substrate DNA and peptide-binding channels. As such, they represent a platform for further elaboration of trivalent ligands, that could serve as a new genre of potent TDP1 inhibitors.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Fission yeast Duf89 and Duf8901 are cobalt/nickel-dependent phosphatase–pyrophosphatases that act via a covalent aspartyl–phosphate intermediate

Domain of Unknown Function 89 (DUF89) proteins are metal-dependent phosphohydrolases. Exemplary DUF89 enzymes differ in their metal and phosphosubstrate preferences. Here, we interrogated the activities and structures of two DUF89 paralogs from fission yeast—Duf89 and Duf8901. We find that Duf89 and Duf8901 are cobalt/nickel-dependent phosphohydrolases adept at hydrolyzing p-nitrophenylphosphate and PP i . Crystal structures of metal-free Duf89 and Co 2+ -bound Duf8901 disclosed two enzyme conformations that differed with respect to the position of a three-helix module, which is either oriented away from the active site in Duf89 or forms a lid over the active site in Duf8901. Lid closure results in a 16 Å movement of Duf8901 Asp195, vis-à-vis Asp199 in Duf89, that brings Asp195 into contact with an octahedrally coordinated cobalt. Reaction of Duf8901 with BeCl 2 and NaF in the presence of divalent cations Co 2+ , Ni 2+ , or Zn 2+ generated covalent Duf8901-(Asp248)–beryllium trifluoride (BeF 3 )•Co 2+ , Duf8901-(Asp248)–BeF 3 •Ni 2+ , or Duf8901-(Asp248)–BeF 3 •Zn 2+ adducts, the structures of which suggest a two-step catalytic mechanism via formation and hydrolysis of an enzyme-(aspartyl)–phosphate intermediate. Alanine mutations of Duf8901 Asp248, Asn249, Lys401, Asp286, and Asp195 that interact with BeF 3 •Co 2+ squelched p-nitrophenylphosphatase activity. A 1.8 Å structure of a Duf8901-(Asp248)–AlF 4 –OH 2 •Co 2+ transition-state mimetic suggests an associative mechanism in which Asp195 and Asp363 orient and activate the water nucleophile. Whereas deletion of the duf89 gene elicited a phenotype in which expression of phosphate homeostasis gene pho1 was derepressed, deleting duf8901 did not, thereby hinting that the DUF89 paralogs have distinct functional repertoires in vivo.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗