Molecular basis for the activation of the Fatty Acid Kinase complex of Staphylococcus aureus.

Gram-positive bacteria utilize a Fatty Acid Kinase (FAK) complex to harvest fatty acids from the environment. The complex, consisting of the fatty acid kinase, FakA, and an acyl carrier protein, FakB, is known to impact virulence and disease outcomes. However, FAK's structure and enzymatic mechanism remain poorly understood. Here, we used a combination of modeling, biochemical, and cell-based approaches to establish critical details of FAK activity. Solved structures of the apo and ligand-bound FakA kinase domain captured the protein state through ATP hydrolysis. Additionally, targeted mutagenesis of an understudied FakA Middle domain identified critical residues within a metal-binding pocket that contribute to FakA dimer stability and protein function. Regarding the complex, we demonstrated nanomolar affinity between FakA and FakB and generated computational models of the complex's quaternary structure. Together, these data provide critical insight into the structure and function of the FAK complex which is essential for understanding its mechanism.

Potent small molecule inhibitors against the 3C protease of foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is one of the most devastating diseases of livestock which can cause significant economic losses, especially when introduced to FMD-free countries. FMD virus (FMDV) belongs to the family Picornaviridae and is antigenically heterogeneous with seven established serotypes. The prevailing preventive and control strategies are limited to restriction of animal movement and elimination of infected or exposed animals, which can be potentially combined with vaccination. However, FMD vaccination has limitations including delayed protection and lack of cross-protection against different serotypes. Recently, antiviral drug use for FMD outbreaks has increasingly been recognized as a potential tool to augment the existing early response strategies, but limited research has been reported on potential antiviral compounds for FMDV. FMDV 3C protease (3Cpro) cleaves the viral-encoded polyprotein into mature and functional proteins during viral replication. The essential role of viral 3Cpro in viral replication and the high conservation of 3Cpro among different FMDV serotypes make it an excellent target for antiviral drug development. We have previously reported multiple series of inhibitors against picornavirus 3Cpro or 3C-like proteases (3CLpros) encoded by coronaviruses or caliciviruses. In this study, we conducted structure-activity relationship studies for our in-house focused compound library containing 3Cpro or 3CLpro inhibitors against FMDV 3Cpro using enzyme and cell-based assays. Herein, we report the discovery of aldehyde and α-ketoamide inhibitors of FMDV 3Cpro with high potency. These data inform future preclinical studies that are related to the advancement of these compounds further along the drug development pathway.IMPORTANCEFood-and-mouth disease (FMD) virus (FMDV) causes devastating disease in cloven-hoofed animals with a significant economic impact. Emergency response to FMD outbreaks to limit FMD spread is critical, and the use of antivirals may overcome the limitations of existing control measures by providing immediate protection for susceptible animals. FMDV encodes 3C protease (3Cpro), which is essential for virus replication and an attractive target for antiviral drug discovery. Here, we report a structure-activity relationship study on multiple series of protease inhibitors and identified potent inhibitors of FMDV 3Cpro. Our results suggest that these compounds have the potential for further development as FMD antivirals.

Mowat-Wilson Syndrome: Case Report and Review of ZEB2 Gene Variant Types, Protein Defects and Molecular Interactions.

Mowat-Wilson syndrome (MWS) is a rare genetic neurodevelopmental congenital disorder associated with various defects of the zinc finger E-box binding homeobox 2 (ZEB2) gene. The ZEB2 gene is autosomal dominant and encodes six protein domains including the SMAD-binding protein, which functions as a transcriptional corepressor involved in the conversion of neuroepithelial cells in early brain development and as a mediator of trophoblast differentiation. This review summarizes reported ZEB2 gene variants, their types, and frequencies among the 10 exons of ZEB2. Additionally, we summarized their corresponding encoded protein defects including the most common variant, c.2083 C>T in exon 8, which directly impacts the homeodomain (HD) protein domain. This single defect was found in 11% of the 298 reported patients with MWS. This review demonstrates that exon 8 encodes at least three of the six protein domains and accounts for 66% (198/298) of the variants identified. More than 90% of the defects were due to nonsense or frameshift changes. We show examples of protein modeling changes that occurred as a result of ZEB2 gene defects. We also report a novel pathogenic variant in exon 8 in a 5-year-old female proband with MWS. This review further explores other genes predicted to be interacting with the ZEB2 gene and their predicted gene-gene molecular interactions with protein binding effects on embryonic multi-system development such as craniofacial, spine, brain, kidney, cardiovascular, and hematopoiesis.

Mechanism-Based Macrocyclic Inhibitors of Serine Proteases.

Protease inhibitor drug discovery is challenged by the lack of cellular and oral permeability, selectivity, metabolic stability, and rapid clearance of peptides. Here, we describe the rational design, synthesis, and evaluation of peptidomimetic side-chain-cyclized macrocycles which we converted into covalent serine protease inhibitors with the addition of an electrophilic ketone warhead. We have identified potent and selective inhibitors of TMPRSS2, matriptase, hepsin, and HGFA and demonstrated their improved protease selectivity, metabolic stability, and pharmacokinetic (PK) properties. We obtained an X-ray crystal structure of phenyl ether-cyclized tripeptide VD4162 (8b) bound to matriptase, revealing an unexpected binding conformation. Cyclic biphenyl ether VD5123 (11) displayed the best PK properties in mice with a half-life of 4.5 h and compound exposure beyond 24 h. These new cyclic tripeptide scaffolds can be used as easily modifiable templates providing a new strategy to overcoming the obstacles presented by linear acyclic peptides in protease inhibitor drug discovery.

HSV-1 ICP0 dimer domain adopts a novel ß-barrel fold.

Infected cell protein 0 (ICP0) is an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) that possesses E3 ubiquitin ligase activity. ICP0 transactivates viral genes, in part, through its C-terminal dimer domain (residues 555-767). Deletion of this dimer domain results in reduced viral gene expression, lytic infection, and reactivation from latency. Since ICP0's dimer domain is associated with its transactivation activity and efficient viral replication, we wanted to determine the structure of this specific domain. The C-terminus of ICP0 was purified from bacteria and analyzed by X-ray crystallography to solve its structure. Each subunit or monomer in the ICP0 dimer is composed of nine ß-strands and two α-helices. Interestingly, two adjacent ß-strands from one monomer "reach" into the adjacent subunit during dimer formation, generating two ß-barrel-like structures. Additionally, crystallographic analyses indicate a tetramer structure is formed from two ß-strands of each dimer, creating a "stacking" of the ß-barrels. The structural protein database searches indicate the fold or structure adopted by the ICP0 dimer is novel. The dimer is held together by an extensive network of hydrogen bonds. Computational analyses reveal that ICP0 can either form a dimer or bind to SUMO1 via its C-terminal SUMO-interacting motifs but not both. Understanding the structure of the dimer domain will provide insights into the activities of ICP0 and, ultimately, the HSV-1 life cycle.

HSV-1 ICP0 Dimer Domain Adopts a Novel ß-barrel Fold.

Infected cell protein 0 (ICP0) is an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) that possesses E3 ubiquitin ligase activity. ICP0 transactivates viral genes, in part, through its C-terminal dimer domain (residues 555-767). Deletion of this dimer domain results in reduced viral gene expression, lytic infection, and reactivation from latency. Since ICP0's dimer domain is associated with its transactivation activity and efficient viral replication, we wanted to determine the structure of this specific domain. The C-terminus of ICP0 was purified from bacteria and analyzed by X-ray crystallography to solve its structure. Each subunit or monomer in the ICP0 dimer is composed of nine ß-strands and two α-helices. Interestingly, two adjacent ß-strands from one monomer "reach" into the adjacent subunit during dimer formation, generating two ß-barrel-like structures. Additionally, crystallographic analyses indicate a tetramer structure is formed from two ß-strands of each dimer, creating a "stacking" of the ß-barrels. The structural protein database searches indicate the fold or structure adopted by the ICP0 dimer is novel. The dimer is held together by an extensive network of hydrogen bonds. Computational analyses reveal that ICP0 can either form a dimer or bind to SUMO1 via its C-terminal SUMO-interacting motifs but not both. Understanding the structure of the dimer domain will provide insights into the activities of ICP0 and, ultimately, the HSV-1 life cycle.

Mixed Alkyl/Aryl Phosphonates Identify Metabolic Serine Hydrolases as Antimalarial Targets.

Malaria, caused by Plasmodium falciparum, remains a significant health burden. A barrier for developing anti-malarial drugs is the ability of the parasite to rapidly generate resistance. We demonstrated that Salinipostin A (SalA), a natural product, kills parasites by inhibiting multiple lipid metabolizing serine hydrolases, a mechanism with a low propensity for resistance. Given the difficulty of employing natural products as therapeutic agents, we synthesized a library of lipidic mixed alkyl/aryl phosphonates as bioisosteres of SalA. Two constitutional isomers exhibited divergent anti-parasitic potencies which enabled identification of therapeutically relevant targets. We also confirm that this compound kills parasites through a mechanism that is distinct from both SalA and the pan-lipase inhibitor, Orlistat. Like SalA, our compound induces only weak resistance, attributable to mutations in a single protein involved in multidrug resistance. These data suggest that mixed alkyl/aryl phosphonates are a promising, synthetically tractable anti-malarials with a low-propensity to induce resistance.

The N-terminal intrinsically disordered region of Ncb5or docks with the cytochrome b5 core to form a helical motif that is of ancient origin.

NADH cytochrome b5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b5 (b5 ) and cytochrome b5 reductase (b5 R) domains separated by a CHORD-Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full-length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high-resolution atomic structures of the b5 domain and a construct containing the CS and b5 R domains (CS/b5 R). Ncb5or also contains an N-terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L34 MDWIRL40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to a b5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N-terminal region naturally fused to the b5 domain, we were able to obtain a high-resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25-129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp120 (corresponding to invariant Trp37 in Ncb5or) to be part of an 11-residue α-helix (S116 QMDWLKLTRT126 ) packing against two of the four helices in the b5 domain that surround heme (α2 and α5). The Trp120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr85 and Tyr88 (α2), Cys124 (α5), and Leu47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp37 in nucleating the formation of the N-terminal helix, whose location in the N/b5 module suggests a role in regulating the function of this multi-domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b5 module as reflected by its existence in a class of cytochrome b5 proteins from three kingdoms among eukaryotes.

New cyclophilin D inhibitor rescues mitochondrial and cognitive function in Alzheimer's disease.

Mitochondrial dysfunction is an early pathological feature of Alzheimer disease and plays a crucial role in the development and progression of Alzheimer's disease. Strategies to rescue mitochondrial function and cognition remain to be explored. Cyclophilin D (CypD), the peptidylprolyl isomerase F (PPIase), is a key component in opening the mitochondrial membrane permeability transition pore, leading to mitochondrial dysfunction and cell death. Blocking membrane permeability transition pore opening by inhibiting CypD activity is a promising therapeutic approach for Alzheimer's disease. However, there is currently no effective CypD inhibitor for Alzheimer's disease, with previous candidates demonstrating high toxicity, poor ability to cross the blood-brain barrier, compromised biocompatibility and low selectivity. Here, we report a new class of non-toxic and biocompatible CypD inhibitor, ebselen, using a conventional PPIase assay to screen a library of ∼2000 FDA-approved drugs with crystallographic analysis of the CypD-ebselen crystal structure (PDB code: 8EJX). More importantly, we assessed the effects of genetic and pharmacological blockade of CypD on Alzheimer's disease mitochondrial and glycolytic bioenergetics in Alzheimer's disease-derived mitochondrial cybrid cells, an ex vivo human sporadic Alzheimer's disease mitochondrial model, and on synaptic function, inflammatory response and learning and memory in Alzheimer's disease mouse models. Inhibition of CypD by ebselen protects against sporadic Alzheimer's disease- and amyloid-ß-induced mitochondrial and glycolytic perturbation, synaptic and cognitive dysfunction, together with suppressing neuroinflammation in the brain of Alzheimer's disease mouse models, which is linked to CypD-related membrane permeability transition pore formation. Thus, CypD inhibitors have the potential to slow the progression of neurodegenerative diseases, including Alzheimer's disease, by boosting mitochondrial bioenergetics and improving synaptic and cognitive function.

Potent 3CLpro inhibitors effective against SARS-CoV-2 and MERS-CoV in animal models by therapeutic treatment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) are zoonotic betacoronaviruses that continue to have a significant impact on public health. Timely development and introduction of vaccines and antivirals against SARS-CoV-2 into the clinic have substantially mitigated the burden of COVID-19. However, a limited or lacking therapeutic arsenal for SARS-CoV-2 and MERS-CoV infections, respectively, calls for an expanded and diversified portfolio of antivirals against these coronavirus infections. In this report, we examined the efficacy of two potent 3CLpro inhibitors, 5d and 11d, in fatal animal models of SARS-CoV-2 and MERS-CoV to demonstrate their broad-spectrum activity against both viral infections. These compounds significantly increased the survival of mice in both models when treatment started 1 day post infection compared to no treatment which led to 100% fatality. Especially, the treatment with compound 11d resulted in 80% and 90% survival in SARS-CoV-2 and MERS-CoV-infected mice, respectively. Amelioration of lung viral load and histopathological changes in treated mice correlated well with improved survival in both infection models. Furthermore, compound 11d exhibited significant antiviral activities in K18-hACE2 mice infected with SARS-CoV-2 Omicron subvariant XBB.1.16. The results suggest that these are promising candidates for further development as broad-spectrum direct-acting antivirals against highly virulent human coronaviruses.IMPORTANCEHuman coronaviruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) continue to have a significant impact on public health. A limited or lacking therapeutic arsenal for SARS-CoV-2 and MERS-CoV infections calls for an expanded and diversified portfolio of antivirals against these coronavirus infections. We have previously reported a series of small-molecule 3C-like protease (3CLpro) inhibitors against human coronaviruses. In this report, we demonstrated the in vivo efficacy of 3CLpro inhibitors for their broad-spectrum activity against both SARS-CoV-2 and MERS-CoV infections using the fatal animal models. The results suggest that these are promising candidates for further development as broad-spectrum direct-acting antivirals against highly virulent human coronaviruses.

Histidine Protonation and Conformational Switching in Diphtheria Toxin Translocation Domain.

Protonation of key histidine residues has been long implicated in the acid-mediated cellular action of the diphtheria toxin translocation (T-) domain, responsible for the delivery of the catalytic domain into the cell. Here, we use a combination of computational (constant-pH Molecular Dynamics simulations) and experimental (NMR, circular dichroism, and fluorescence spectroscopy along with the X-ray crystallography) approaches to characterize the initial stages of conformational change happening in solution in the wild-type T-domain and in the H223Q/H257Q double mutant. This replacement suppresses the acid-induced transition, resulting in the retention of a more stable protein structure in solutions at pH 5.5 and, consequently, in reduced membrane-disrupting activity. Here, for the first time, we report the pKa values of the histidine residues of the T-domain, measured by NMR-monitored pH titrations. Most peaks in the histidine side chain spectral region are titrated with pKas ranging from 6.2 to 6.8. However, the two most up-field peaks display little change down to pH 6, which is a limiting pH for this protein in solution at concentrations required for NMR. These peaks are absent in the double mutant, suggesting they belong to H223 and H257. The constant-pH simulations indicate that for the T-domain in solution, the pKa values for histidine residues range from 3.0 to 6.5, with those most difficult to protonate being H251 and H257. Taken together, our experimental and computational data demonstrate that previously suggested cooperative protonation of all six histidines in the T-domain does not occur.

Structure of Rhizobium sp. 4-9 histamine dehydrogenase and analysis of the electron transfer pathway to an abiological electron acceptor.

Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp. 4-9 (HaDHR) is a member of a small family of dehydrogenases containing a covalently attached FMN, and the only member so far identified to date that does not exhibit substrate inhibition. In this study, we present the 2.1 Å resolution crystal structure of HaDHR. This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators. Alanine 437 was identified as the exit point of electrons from the Fe4S4 cluster. The enzyme was modified with a Ser436Cys mutation to facilitate covalent attachment of a ferrocene moiety. When modified with Fc-maleimide, this new construct demonstrated direct electron transfer from the enzyme to a gold electrode in a histamine concentration-dependent manner without the need for any additional electron mediators.

An African-Specific Variant of TP53 Reveals PADI4 as a Regulator of p53-Mediated Tumor Suppression.

TP53 is the most frequently mutated gene in cancer, yet key target genes for p53-mediated tumor suppression remain unidentified. Here, we characterize a rare, African-specific germline variant of TP53 in the DNA-binding domain Tyr107His (Y107H). Nuclear magnetic resonance and crystal structures reveal that Y107H is structurally similar to wild-type p53. Consistent with this, we find that Y107H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p53 target genes; this includes the epigenetic modifier PADI4, which deiminates arginine to the nonnatural amino acid citrulline. Surprisingly, we show that Y107H mice develop spontaneous cancers and metastases and that Y107H shows impaired tumor suppression in two other models. We show that PADI4 is itself tumor suppressive and that it requires an intact immune system for tumor suppression. We identify a p53-PADI4 gene signature that is predictive of survival and the efficacy of immune-checkpoint inhibitors. SIGNIFICANCE: We analyze the African-centric Y107H hypomorphic variant and show that it confers increased cancer risk; we use Y107H in order to identify PADI4 as a key tumor-suppressive p53 target gene that contributes to an immune modulation signature and that is predictive of cancer survival and the success of immunotherapy. See related commentary by Bhatta and Cooks, p. 1518. This article is highlighted in the In This Issue feature, p. 1501.

Novel antimicrobial strategies to treat multi-drug resistant Staphylococcus aureus infections.

Antimicrobial resistance is a major obstacle for the treatment of infectious diseases and currently represents one of the most significant threats to global health. Staphylococcus aureus remains a formidable human pathogen with high mortality rates associated with severe systemic infections. S. aureus has become notorious as a multidrug resistant bacterium, which when combined with its extensive arsenal of virulence factors that exacerbate disease, culminates in an incredibly challenging pathogen to treat clinically. Compounding this major health issue is the lack of antibiotic discovery and development, with only two new classes of antibiotics approved for clinical use in the last 20 years. Combined efforts from the scientific community have reacted to the threat of dwindling treatment options to combat S. aureus disease in several innovative and exciting developments. This review describes current and future antimicrobial strategies aimed at treating staphylococcal colonization and/or disease, examining therapies that show significant promise at the preclinical development stage to approaches that are currently being investigated in clinical trials.

Structure-guided design of direct-acting antivirals that exploit the gem-dimethyl effect and potently inhibit 3CL proteases of severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) and middle east respiratory syndrome coronavirus (MERS-CoV).

The high morbidity and mortality associated with SARS-CoV-2 infection, the etiological agent of COVID-19, has had a major impact on global public health. Significant progress has been made in the development of an array of vaccines and biologics, however, the emergence of SARS-CoV-2 variants and breakthrough infections are an ongoing major concern. Furthermore, there is an existing paucity of small-molecule host and virus-directed therapeutics and prophylactics that can be used to counter the spread of SARS-CoV-2, and any emerging and re-emerging coronaviruses. We describe herein our efforts to address this urgent need by focusing on the structure-guided design of potent broad-spectrum inhibitors of SARS-CoV-2 3C-like protease (3CLpro or Main protease), an enzyme essential for viral replication. The inhibitors exploit the directional effects associated with the presence of a gem-dimethyl group that allow the inhibitors to optimally interact with the S4 subsite of the enzyme. Several compounds were found to potently inhibit SARS-CoV-2 and MERS-CoV 3CL proteases in biochemical and cell-based assays. Specifically, the EC50 values of aldehyde 1c and its corresponding bisulfite adduct 1d against SARS-CoV-2 were found to be 12 and 10 nM, respectively, and their CC50 values were >50 µM. Furthermore, deuteration of these compounds yielded compounds 2c/2d with EC50 values 11 and 12 nM, respectively. Replacement of the aldehyde warhead with a nitrile (CN) or an α-ketoamide warhead or its corresponding bisulfite adduct yielded compounds 1g, 1eand1f with EC50 values 60, 50 and 70 nM, respectively. High-resolution cocrystal structures have identified the structural determinants associated with the binding of the inhibitors to the active site of the enzyme and, furthermore, have illuminated the mechanism of action of the inhibitors. Overall, the high Safety Index (SI) (SI=CC50/EC50) displayed by these compounds suggests that they are well-suited to conducting further preclinical studies.

Pseudomonas aeruginosa Dps (PA0962) Functions in H2O2 Mediated Oxidative Stress Defense and Exhibits In Vitro DNA Cleaving Activity.

We report the structural, biochemical, and functional characterization of the product of gene PA0962 from Pseudomonas aeruginosa PAO1. The protein, termed Pa Dps, adopts the Dps subunit fold and oligomerizes into a nearly spherical 12-mer quaternary structure at pH 6.0 or in the presence of divalent cations at neutral pH and above. The 12-Mer Pa Dps contains two di-iron centers at the interface of each subunit dimer, coordinated by conserved His, Glu, and Asp residues. In vitro, the di-iron centers catalyze the oxidation of Fe2+ utilizing H2O2 (not O2) as an oxidant, suggesting Pa Dps functions to aid P. aeruginosa to survive H2O2-mediated oxidative stress. In agreement, a P. aeruginosa Δdps mutant is significantly more susceptible to H2O2 than the parent strain. The Pa Dps structure harbors a novel network of Tyr residues at the interface of each subunit dimer between the two di-iron centers, which captures radicals generated during Fe2+ oxidation at the ferroxidase centers and forms di-tyrosine linkages, thus effectively trapping the radicals within the Dps shell. Surprisingly, incubating Pa Dps and DNA revealed unprecedented DNA cleaving activity that is independent of H2O2 or O2 but requires divalent cations and 12-mer Pa Dps.

Crystal structures of di-µ-chlorido-bis-({(E)-5-(ethyl-amino)-4-methyl-2-[(pyridin-2-yl)diazen-yl]phen-o-lato}copper(II)) and chlorido-bis-(1,10-phen-anthroline)copper(II) chloride tetra-hydrate.

The dark-red title complex crystallized from an equimolar methanol solution of (E)-5-(ethyl-amino)-4-methyl-2-[(pyridin-2-yl)diazen-yl]phenol and CuCl2(phen) (phen = 1,10-phenanthroline) as a centrosymmetric dimer, [CuCl(C14H15N4O)]2. The Cu atoms are bridged by two Cl ligands and have a slightly distorted square-pyramidal coordination, where two N atoms from the azo and the pyridine moieties, a phenolic O and a Cl atom comprise the base and the other Cl occupies the apex position. The apical Cu-Cl bond, 2.6192 (4) Å, is longer than the basal one, 2.2985 (3) Å, due to Jahn-Teller distortion. The dimers are associated via weak inter-molecular hydrogen bonds and π-π stacking inter-actions between phenyl and pyridine rings. A monomeric by-product of the same reaction, [CuCl(phen)2]Cl·4H2O, has a trigonal-bipyramidal coordination of Cu with equatorial Cl ligand, and extensive outer-sphere disorder. In the structure of 4, the packing of cations leaves continuous channels containing disordered Cl- anions and solvent mol-ecules. The identity of the solvent (water or a water/methanol mixture) was not certain. The disordered anion/solvent regions comprise 28% of the unit-cell volume. The disorder was approximated by five partly occupied positions of the Cl- anion and ten positions of O atoms with a total occupancy of 3, giving a total of 48 electrons per asymmetric unit, in agreement with the integral electron density of 47.8 electrons in the disordered region, as was estimated using the BYPASS-type solvent-masking program [van der Sluis & Spek (1990). Acta Cryst. A46, 194-201].

Solid Phase Synthesis of Fluorosulfate Containing Macrocycles for Chemoproteomic Workflows.

Macrocyclic peptides are attractive for chemoproteomic applications due to their modular synthesis and potential for high target selectivity. We describe a solid phase synthesis method for the efficient generation of libraries of small macrocycles that contain an electrophile and alkyne handle. The modular synthesis produces libraries that can be directly screened using simple SDS-PAGE readouts and then optimal lead molecules applied to proteomic analysis. We generated a library of 480 macrocyclic peptides containing the weakly reactive fluorosulfate (OSF) electrophile. Initial screening of a subset of the library containing each of the various diversity elements identified initial molecules of interest. The corresponding positional and confirmational isomers were then screened to select molecules that showed specific protein labeling patterns that were dependent on the probe structure. The most promising hits were applied to standard chemoproteomic workflows to identify protein targets. Our results demonstrate the feasibility of rapid, on-resin synthesis of diverse macrocyclic electrophiles to generate new classes of covalent ligands.

Broad-Spectrum Cyclopropane-Based Inhibitors of Coronavirus 3C-like Proteases: Biochemical, Structural, and Virological Studies.

The advent of SARS-CoV-2, the causative agent of COVID-19, and its worldwide impact on global health, have provided the impetus for the development of effective countermeasures that can be deployed against the virus, including vaccines, monoclonal antibodies, and direct-acting antivirals (DAAs). Despite these efforts, the current paucity of DAAs has created an urgent need for the creation of an enhanced and diversified portfolio of broadly acting agents with different mechanisms of action that can effectively abrogate viral infection. SARS-CoV-2 3C-like protease (3CLpro), an enzyme essential for viral replication, is a validated target for the discovery of SARS-CoV-2 therapeutics. In this report, we describe the structure-guided utilization of the cyclopropane moiety in the design of highly potent inhibitors of SARS-CoV-2 3CLpro, SARS-CoV-1 3CLpro, and MERS-CoV 3CLpro. High-resolution cocrystal structures were used to identify the structural determinants associated with the binding of the inhibitors to the active site of the enzyme and unravel the mechanism of action. Aldehydes 5c and 11c inhibited SARS-CoV-2 replication with EC50 values of 12 and 11 nM, respectively. Furthermore, the corresponding aldehyde bisulfite adducts 5d and 11d were equipotent with EC50 values of 13 and 12 nM, respectively. The safety index (SI) values for compounds 5c / 11c and 5d / 11d ranged between 7692 and 9090. Importantly, aldehydes 5c / 11c and bisulfite adducts 5d / 11d potently inhibited MERS-CoV 3CLpro with IC50 values of 80 and 120 nM, and 70 and 70 nM, respectively. Likewise, compounds 5c / 11c and 5d / 11d inhibited SARS-CoV-1 with IC50 values of 960 and 350 nM and 790 and 240 nM, respectively. Taken together, these studies suggest that the inhibitors described herein have low cytotoxicity and high potency and are promising candidates for further development as broad-spectrum direct-acting antivirals against highly pathogenic coronaviruses.

A KLK6 Activity-Based Probe Reveals a Role for KLK6 Activity in Pancreatic Cancer Cell Invasion.

Pancreatic cancer has the lowest survival rate of all common cancers due to late diagnosis and limited treatment options. Serine hydrolases are known to mediate cancer progression and metastasis through initiation of signaling cascades and cleavage of extracellular matrix proteins, and the kallikrein-related peptidase (KLK) family of secreted serine proteases have emerging roles in pancreatic ductal adenocarcinoma (PDAC). However, the lack of reliable activity-based probes (ABPs) to profile KLK activity has hindered progress in validation of these enzymes as potential targets or biomarkers. Here, we developed potent and selective ABPs for KLK6 by using a positional scanning combinatorial substrate library and characterized their binding mode and interactions by X-ray crystallography. The optimized KLK6 probe IMP-2352 (kobs/I = 11,000 M-1 s-1) enabled selective detection of KLK6 activity in a variety of PDAC cell lines, and we observed that KLK6 inhibition reduced the invasiveness of PDAC cells that secrete active KLK6. KLK6 inhibitors were combined with N-terminomics to identify potential secreted protein substrates of KLK6 in PDAC cells, providing insights into KLK6-mediated invasion pathways. These novel KLK6 ABPs offer a toolset to validate KLK6 and associated signaling partners as targets or biomarkers across a range of diseases.