Crystal structures and fragment screening of SARS-CoV-2 NSP14 reveal details of exoribonuclease activation and mRNA capping and provide starting points for antiviral drug development.

NSP14 is a dual function enzyme containing an N-terminal exonuclease domain (ExoN) and C-terminal Guanine-N7-methyltransferase (N7-MTase) domain. Both activities are essential for the viral life cycle and may be targeted for anti-viral therapeutics. NSP14 forms a complex with NSP10, and this interaction enhances the nuclease but not the methyltransferase activity. We have determined the structure of SARS-CoV-2 NSP14 in the absence of NSP10 to 1.7 Å resolution. Comparisons with NSP14/NSP10 complexes reveal significant conformational changes that occur within the NSP14 ExoN domain upon binding of NSP10, including helix to coil transitions that facilitate the formation of the ExoN active site and provide an explanation of the stimulation of nuclease activity by NSP10. We have determined the structure of NSP14 in complex with cap analogue 7MeGpppG, and observe conformational changes within a SAM/SAH interacting loop that plays a key role in viral mRNA capping offering new insights into MTase activity. We perform an X-ray fragment screen on NSP14, revealing 72 hits bound to sites of inhibition in the ExoN and MTase domains. These fragments serve as excellent starting point tools for structure guided development of NSP14 inhibitors that may be used to treat COVID-19 and potentially other future viral threats.

Characterization of the SARS-CoV-2 ExoN (nsp14ExoN-nsp10) complex: implications for its role in viral genome stability and inhibitor identification.

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14-nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14-nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3'-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14-nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12-nsp7-nsp8 (nsp12-7-8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14-nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.

A phosphate binding pocket is a key determinant of exo- versus endo-nucleolytic activity in the SNM1 nuclease family.

The SNM1 nucleases which help maintain genome integrity are members of the metallo-ß-lactamase (MBL) structural superfamily. Their conserved MBL-ß-CASP-fold SNM1 core provides a molecular scaffold forming an active site which coordinates the metal ions required for catalysis. The features that determine SNM1 endo- versus exonuclease activity, and which control substrate selectivity and binding are poorly understood. We describe a structure of SNM1B/Apollo with two nucleotides bound to its active site, resembling the product state of its exonuclease reaction. The structure enables definition of key SNM1B residues that form contacts with DNA and identifies a 5' phosphate binding pocket, which we demonstrate is important in catalysis and which has a key role in determining endo- versus exonucleolytic activity across the SNM1 family. We probed the capacity of SNM1B to digest past sites of common endogenous DNA lesions and find that base modifications planar to the nucleobase can be accommodated due to the open architecture of the active site, but lesions axial to the plane of the nucleobase are not well tolerated due to constriction around the altered base. We propose that SNM1B/Apollo might employ its activity to help remove common oxidative lesions from telomeres.

Structural and mechanistic insights into the Artemis endonuclease and strategies for its inhibition.

Artemis (SNM1C/DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in dsDNA break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Artemis deficiency leads to congenital radiosensitive severe acquired immune deficiency (RS-SCID). Artemis belongs to a superfamily of nucleases containing metallo-ß-lactamase (MBL) and ß-CASP (CPSF-Artemis-SNM1-Pso2) domains. We present crystal structures of the catalytic domain of wildtype and variant forms of Artemis, including one causing RS-SCID Omenn syndrome. The catalytic domain of the Artemis has similar endonuclease activity to the phosphorylated full-length protein. Our structures help explain the predominantly endonucleolytic activity of Artemis, which contrasts with the predominantly exonuclease activity of the closely related SNM1A and SNM1B MBL fold nucleases. The structures reveal a second metal binding site in its ß-CASP domain unique to Artemis, which is amenable to inhibition by compounds including ebselen. By combining our structural data with that from a recently reported Artemis structure, we were able model the interaction of Artemis with DNA substrates. The structures, including one of Artemis with the cephalosporin ceftriaxone, will help enable the rational development of selective SNM1 nuclease inhibitors.

Mechanistic and Evolutionary Insights from the Reciprocal Promiscuity of Two Pyridoxal Phosphate-dependent Enzymes.

Enzymes that utilize the cofactor pyridoxal 5'-phosphate play essential roles in amino acid metabolism in all organisms. The cofactor is used by proteins that adopt at least five different folds, which raises questions about the evolutionary processes that might explain the observed distribution of functions among folds. In this study, we show that a representative of fold type III, the Escherichia coli alanine racemase (ALR), is a promiscuous cystathionine ß-lyase (CBL). Furthermore, E. coli CBL (fold type I) is a promiscuous alanine racemase. A single round of error-prone PCR and selection yielded variant ALR(Y274F), which catalyzes cystathionine ß-elimination with a near-native Michaelis constant (Km = 3.3 mm) but a poor turnover number (kcat ≈10 h(-1)). In contrast, directed evolution also yielded CBL(P113S), which catalyzes l-alanine racemization with a poor Km (58 mm) but a high kcat (22 s(-1)). The structures of both variants were solved in the presence and absence of the l-alanine analogue, (R)-1-aminoethylphosphonic acid. As expected, the ALR active site was enlarged by the Y274F substitution, allowing better access for cystathionine. More surprisingly, the favorable kinetic parameters of CBL(P113S) appear to result from optimizing the pKa of Tyr-111, which acts as the catalytic acid during l-alanine racemization. Our data emphasize the short mutational routes between the functions of pyridoxal 5'-phosphate-dependent enzymes, regardless of whether or not they share the same fold. Thus, they confound the prevailing model of enzyme evolution, which predicts that overlapping patterns of promiscuity result from sharing a common multifunctional ancestor.

Radiation Damage and Racemic Protein Crystallography Reveal the Unique Structure of the GASA/Snakin Protein Superfamily.

Proteins from the GASA/snakin superfamily are common in plant proteomes and have diverse functions, including hormonal crosstalk, development, and defense. One 63-residue member of this family, snakin-1, an antimicrobial protein from potatoes, has previously been chemically synthesized in a fully active form. Herein the 1.5 Šstructure of snakin-1, determined by a novel combination of racemic protein crystallization and radiation-damage-induced phasing (RIP), is reported. Racemic crystals of snakin-1 and quasi-racemic crystals incorporating an unnatural 4-iodophenylalanine residue were prepared from chemically synthesized d- and l-proteins. Breakage of the C-I bonds in the quasi-racemic crystals facilitated structure determination by RIP. The crystal structure reveals a unique protein fold with six disulfide crosslinks, presenting a distinct electrostatic surface that may target the protein to microbial cell surfaces.

Binding mode of the breakthrough inhibitor AZD9291 to epidermal growth factor receptor revealed.

The discovery of genetic drivers of lung cancer in patient sub-groups has led to their use as predictive biomarkers and as targets for selective drug therapy. Some of the most important lung cancer drivers are mutations in the EGFR gene, for example, the exon 19 deletions and the L858R variant that confer sensitivity to the front line drugs erlotinib and gefitinib; the acquired T790M variants confer drug resistance and a poor prognosis. A challenge then in targeting EGFR is to produce drugs that inhibit both sensitising variants and resistance variants, leaving wild type protein in healthy cells unaffected. One such agent is AstraZeneca's "breakthrough" AZD9291 molecule that shows a 200-fold selectivity for T790M/L858R over wild type EGFR. Our X-ray crystal structure reveals the binding mode of AZD9291 to the kinase domain of wild type EGFR.

The 1.65†Šresolution structure of the complex of AZD4547 with the kinase domain of FGFR1 displays exquisite molecular recognition.

The fibroblast growth factor receptor (FGFR) family are expressed widely in normal tissues and play a role in tissue repair, inflammation, angiogenesis and development. However, aberrant signalling through this family can lead to cellular proliferation, evasion of apoptosis and induction of angiogenesis, which is implicated in the development of many cancers and also in drug resistance. The high frequency of FGFR amplification or mutation in multiple cancer types is such that this family has been targeted for the discovery of novel, selective drug compounds, with one of the most recently discovered being AZD4547, a subnanomolar (IC50) FGFR1 inhibitor developed by AstraZeneca and currently in clinical trials. The 1.65 Šresolution crystal structure of AZD4547 bound to the kinase domain of FGFR1 has been determined and reveals extensive drug-protein interactions, an integral network of water molecules and the tight closure of the FGFR1 P-loop to form a long, narrow crevice in which the AZD4547 molecule binds.

Cell-active small molecule inhibitors validate the SNM1A DNA repair nuclease as a cancer target.

The three human SNM1 metallo-ß-lactamase fold nucleases (SNM1A-C) play key roles in DNA damage repair and in maintaining telomere integrity. Genetic studies indicate that they are attractive targets for cancer treatment and to potentiate chemo- and radiation-therapy. A high-throughput screen for SNM1A inhibitors identified diverse pharmacophores, some of which were shown by crystallography to coordinate to the di-metal ion centre at the SNM1A active site. Structure and turnover assay-guided optimization enabled the identification of potent quinazoline-hydroxamic acid containing inhibitors, which bind in a manner where the hydroxamic acid displaces the hydrolytic water and the quinazoline ring occupies a substrate nucleobase binding site. Cellular assays reveal that SNM1A inhibitors cause sensitisation to, and defects in the resolution of, cisplatin-induced DNA damage, validating the tractability of MBL fold nucleases as cancer drug targets.

Structure of AKR1C3 with 3-phenoxybenzoic acid bound.

Aldo-keto reductase 1C3 (AKR1C3) is a human enzyme that catalyzes the NADPH-dependent reduction of steroids and prostaglandins. AKR1C3 overexpression is associated with the proliferation of hormone-dependent cancers, most notably breast and prostate cancers. Nonsteroidal anti-inflammatory drugs (NSAIDs) and their analogues are well characterized inhibitors of AKR1C3. Here, the X-ray crystal structure of 3-phenoxybenzoic acid in complex with AKR1C3 is presented. This structure provides useful information for the future development of new anticancer agents by structure-guided drug design.

Structure, mechanism and crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase.

There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two "druggable" pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.

The SNM1A DNA repair nuclease.

Unrepaired, or misrepaired, DNA damage can contribute to the pathogenesis of a number of conditions, or disease states; thus, DNA damage repair pathways, and the proteins within them, are required for the safeguarding of the genome. Human SNM1A is a 5'-to-3' exonuclease that plays a role in multiple DNA damage repair processes. To date, most data suggest a role of SNM1A in primarily ICL repair: SNM1A deficient cells exhibit hypersensitivity to ICL-inducing agents (e.g. mitomycin C and cisplatin); and both in vivo and in vitro experiments demonstrate SNM1A and XPF-ERCC1 can function together in the 'unhooking' step of ICL repair. SNM1A further interacts with a number of other proteins that contribute to genome integrity outside canonical ICL repair (e.g. PCNA and CSB), and these may play a role in regulating SNM1As function, subcellular localisation, and post-translational modification state. These data also provide further insight into other DNA repair pathways to which SNM1A may contribute. This review aims to discuss all aspects of the exonuclease, SNM1A, and its contribution to DNA damage tolerance.

TAS-120 Cancer Target Binding: Defining Reactivity and Revealing the First Fibroblast Growth Factor Receptor†1 (FGFR1) Irreversible Structure.

1-[(3S)-3-[4-Amino-3-[2-(3,5-dimethoxyphenyl)ethynyl]-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-pyrrolidinyl]-2-propen-1-one (TAS-120) is an irreversible inhibitor of the fibroblast growth factor receptor (FGFR) family, and is currently under phase I/II clinical trials in patients with confirmed advanced metastatic solid tumours harbouring FGFR aberrations. This inhibitor specifically targets the P-loop of the FGFR tyrosine kinase domain, forming a covalent adduct with a cysteine side chain of the protein. Our mass spectrometry experiments characterise an exceptionally fast chemical reaction in forming the covalent complex. The structural basis of this reactivity is revealed by a sequence of three X-ray crystal structures: a free ligand structure, a reversible FGFR1 structure, and the first reported irreversible FGFR1 adduct structure. We hypothesise that the most significant reactivity feature of TAS-120 is its inherent ability to undertake conformational sampling of the FGFR P-loop. In designing novel covalent FGFR inhibitors, such a phenomenon presents an attractive strategy requiring appropriate positioning of an acrylamide group similarly to that of TAS-120.

Rotational Freedom, Steric Hindrance, and Protein Dynamics Explain BLU554 Selectivity for the Hinge Cysteine of FGFR4.

Aberration in FGFR4 signaling drives carcinogenesis and progression in a subset of hepatocellular carcinoma (HCC) patients, thereby making FGFR4 an attractive molecular target for this disease. Selective FGFR4 inhibition can be achieved through covalently targeting a poorly conserved cysteine residue in the FGFR4 kinase domain. We report mass spectrometry assays and cocrystal structures of FGFR4 in covalent complex with the clinical candidate BLU554 and with a series of four structurally related inhibitors that define the inherent reactivity and selectivity profile of these molecules. We further reveal the structure of FGFR1 with one of our inhibitors and show that off-target covalent binding can occur through an alternative conformation that supports targeting of a cysteine conserved in all members of the FGFR family. Collectively, we propose that rotational freedom, steric hindrance, and protein dynamics explain the exceptional selectivity profile of BLU554 for targeting FGFR4.

2-Oxo-3, 4-dihydropyrimido[4, 5-d]pyrimidinyl derivatives as new irreversible pan fibroblast growth factor receptor (FGFR) inhibitors.

A series of 2-oxo-3, 4-dihydropyrimido[4,5-d]-pyrimidinyl derivatives were designed and synthesized as new irreversible inhibitors of the FGFR family. One of the most promising compounds 2l potently inhibited FGFR1/2/3 with IC50 values of 1.06, 0.84 and 5.38 nM, respectively, whereas its potency against FGFR4 was diminished by an order of magnitude. Compound 2l strongly suppresses the proliferation of FGFR1-amplified H520 non-small cell lung cancer cells, FGFR2-amplified SUM52 breast cancer cells and FGFR3-amplified SW780 bladder cancer cells with low nanomolar IC50 values, but was significantly less potent against four FGFR-negative cancer cell lines, with low micromolar IC50 values. Biological investigation also confirmed the irreversible binding of the molecule with the FGFR1-3 target kinases. Compound 2l may serve as a promising new lead for further anticancer drug discovery.

Structures of Human Peroxiredoxin 3 Suggest Self-Chaperoning Assembly that Maintains Catalytic State.

Peroxiredoxins are antioxidant proteins primarily responsible for detoxification of hydroperoxides in cells. On exposure to various cellular stresses, peroxiredoxins can acquire chaperone activity, manifested as quaternary reorganization into a high molecular weight (HMW) form. Acidification, for example, causes dodecameric rings of human peroxiredoxin 3 (HsPrx3) to stack into long helical filaments. In this work, a 4.1-Å resolution structure of low-pH-instigated helical filaments was elucidated, showing a locally unfolded active site and partially folded C terminus. A 2.8-Å crystal structure of HsPrx3 was determined at pH 8.5 under reducing conditions, wherein dodecameric rings are arranged as a short stack, with symmetry similar to low-pH filaments. In contrast to previous observations, the crystal structure displays both a fully folded active site and ordered C terminus, suggesting that the HsPrx3 HMW form maintains catalytic activity. We propose a new role for the HMW form as a self-chaperoning assembly maintaining HsPrx3 function under stress.

Crystal structures of three classes of non-steroidal anti-inflammatory drugs in complex with aldo-keto reductase 1C3.

Aldo-keto reductase 1C3 (AKR1C3) catalyses the NADPH dependent reduction of carbonyl groups in a number of important steroid and prostanoid molecules. The enzyme is also over-expressed in prostate and breast cancer and its expression is correlated with the aggressiveness of the disease. The steroid products of AKR1C3 catalysis are important in proliferative signalling of hormone-responsive cells, while the prostanoid products promote prostaglandin-dependent proliferative pathways. In these ways, AKR1C3 contributes to tumour development and maintenance, and suggest that inhibition of AKR1C3 activity is an attractive target for the development of new anti-cancer therapies. Non-steroidal anti-inflammatory drugs (NSAIDs) are one well-known class of compounds that inhibits AKR1C3, yet crystal structures have only been determined for this enzyme with flufenamic acid, indomethacin, and closely related analogues bound. While the flufenamic acid and indomethacin structures have been used to design novel inhibitors, they provide only limited coverage of the NSAIDs that inhibit AKR1C3 and that may be used for the development of new AKR1C3 targeted drugs. To understand how other NSAIDs bind to AKR1C3, we have determined ten crystal structures of AKR1C3 complexes that cover three different classes of NSAID, N-phenylanthranilic acids (meclofenamic acid, mefenamic acid), arylpropionic acids (flurbiprofen, ibuprofen, naproxen), and indomethacin analogues (indomethacin, sulindac, zomepirac). The N-phenylanthranilic and arylpropionic acids bind to common sites including the enzyme catalytic centre and a constitutive active site pocket, with the arylpropionic acids probing the constitutive pocket more effectively. By contrast, indomethacin and the indomethacin analogues sulindac and zomepirac, display three distinctly different binding modes that explain their relative inhibition of the AKR1C family members. This new data from ten crystal structures greatly broadens the base of structures available for future structure-guided drug discovery efforts.

The interaction of human neuroglobin with hydrogen sulphide.

Neuroglobin has been identified to protect brain neurons from apoptotic stress. Hydrogen sulphide has a role in the brain as a neuromodulator, involving NMDA receptor activation. Here we report on studies of the in vitro interaction of ferric neuroglobin with hydrogen sulphide. Hydrogen sulphide binds very tightly to the heme group of neuroglobin in a biphasic reaction. The faster of the two reaction processes is concentration dependent whilst the slower process is not. The rate of hydrogen sulphide binding is pH sensitive and as the pH is reduced over the physiological range the rate of reaction increases by a factor of approximately 10. This change in reactivity appears to reflect the ionisation of the heme distal His ligand rather than a preference for the binding of H(2)S. We discuss the potential role of neuroglobin in the modulation of hydrogen sulphide sensitivity of neurons in the brain.