Rational design of a new antibiotic class for drug-resistant infections.

The development of new antibiotics to treat infections caused by drug-resistant Gram-negative pathogens is of paramount importance as antibiotic resistance continues to increase worldwide1. Here we describe a strategy for the rational design of diazabicyclooctane inhibitors of penicillin-binding proteins from Gram-negative bacteria to overcome multiple mechanisms of resistance, including ß-lactamase enzymes, stringent response and outer membrane permeation. Diazabicyclooctane inhibitors retain activity in the presence of ß-lactamases, the primary resistance mechanism associated with ß-lactam therapy in Gram-negative bacteria2,3. Although the target spectrum of an initial lead was successfully re-engineered to gain in vivo efficacy, its ability to permeate across bacterial outer membranes was insufficient for further development. Notably, the features that enhanced target potency were found to preclude compound uptake. An improved optimization strategy leveraged porin permeation properties concomitant with biochemical potency in the lead-optimization stage. This resulted in ETX0462, which has potent in vitro and in vivo activity against Pseudomonas aeruginosa plus all other Gram-negative ESKAPE pathogens, Stenotrophomonas maltophilia and biothreat pathogens. These attributes, along with a favourable preclinical safety profile, hold promise for the successful clinical development of the first novel Gram-negative chemotype to treat life-threatening antibiotic-resistant infections in more than 25 years.

Analysis of the Substrate Specificity of the SMYD2 Protein Lysine Methyltransferase and Discovery of Novel Non-Histone Substrates.

The SMYD2 protein lysine methyltransferase methylates various histone and non-histone proteins and is overexpressed in several cancers. Using peptide arrays, we investigated the substrate specificity of the enzyme, revealing a recognition of leucine (or weaker phenylalanine) at the -1 peptide site and disfavor of acidic residues at the +1 to +3 sites. Using this motif, novel SMYD2 peptide substrates were identified, leading to the discovery of 32 novel peptide substrates with a validated target site. Among them, 19 were previously reported to be methylated at the target lysine in human cells, strongly suggesting that SMYD2 is the protein lysine methyltransferase responsible for this activity. Methylation of some of the novel peptide substrates was tested at the protein level, leading to the identification of 14 novel protein substrates of SMYD2, six of which were more strongly methylated than p53, the best SMYD2 substrate described so far. The novel SMYD2 substrate proteins are involved in diverse biological processes such as chromatin regulation, transcription, and intracellular signaling. The results of our study provide a fundament for future investigations into the role of this important enzyme in normal development and cancer.

Resistance mutations generate divergent antibiotic susceptibility profiles against translation inhibitors.

Mutations conferring resistance to translation inhibitors often alter the structure of rRNA. Reduced susceptibility to distinct structural antibiotic classes may, therefore, emerge when a common ribosomal binding site is perturbed, which significantly reduces the clinical utility of these agents. The translation inhibitors negamycin and tetracycline interfere with tRNA binding to the aminoacyl-tRNA site on the small 30S ribosomal subunit. However, two negamycin resistance mutations display unexpected differential antibiotic susceptibility profiles. Mutant U1060A in 16S Escherichia coli rRNA is resistant to both antibiotics, whereas mutant U1052G is simultaneously resistant to negamycin and hypersusceptible to tetracycline. Using a combination of microbiological, biochemical, single-molecule fluorescence transfer experiments, and X-ray crystallography, we define the specific structural defects in the U1052G mutant 70S E. coli ribosome that explain its divergent negamycin and tetracycline susceptibility profiles. Unexpectedly, the U1052G mutant ribosome possesses a second tetracycline binding site that correlates with its hypersusceptibility. The creation of a previously unidentified antibiotic binding site raises the prospect of identifying similar phenomena in antibiotic-resistant pathogens in the future.

Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology.

Millions of individuals are infected with and die from tuberculosis (TB) each year, and multidrug-resistant (MDR) strains of TB are increasingly prevalent. As such, there is an urgent need to identify novel drugs to treat TB infections. Current frontline therapies include the drug isoniazid, which inhibits the essential NADH-dependent enoyl-acyl-carrier protein (ACP) reductase, InhA. To inhibit InhA, isoniazid must be activated by the catalase-peroxidase KatG. Isoniazid resistance is linked primarily to mutations in the katG gene. Discovery of InhA inhibitors that do not require KatG activation is crucial to combat MDR TB. Multiple discovery efforts have been made against InhA in recent years. Until recently, despite achieving high potency against the enzyme, these efforts have been thwarted by lack of cellular activity. We describe here the use of DNA-encoded X-Chem (DEX) screening, combined with selection of appropriate physical properties, to identify multiple classes of InhA inhibitors with cell-based activity. The utilization of DEX screening allowed the interrogation of very large compound libraries (1011 unique small molecules) against multiple forms of the InhA enzyme in a multiplexed format. Comparison of the enriched library members across various screening conditions allowed the identification of cofactor-specific inhibitors of InhA that do not require activation by KatG, many of which had bactericidal activity in cell-based assays.

Discovery of 2,6-disubstituted pyrazine derivatives as inhibitors of CK2 and PIM kinases.

The design and synthesis of a novel series of 2,6-disubstituted pyrazine derivatives as CK2 kinase inhibitors is described. Structure-guided optimization of a 5-substituted-3-thiophene carboxylic acid screening hit (3a) led to the development of a lead compound (12b), which shows inhibition in both enzymatic and cellular assays. Subsequent design and hybridization efforts also led to the unexpected identification of analogs with potent PIM kinase activity (14f).

Negamycin induces translational stalling and miscoding by binding to the small subunit head domain of the Escherichia coli ribosome.

Negamycin is a natural product with broad-spectrum antibacterial activity and efficacy in animal models of infection. Although its precise mechanism of action has yet to be delineated, negamycin inhibits cellular protein synthesis and causes cell death. Here, we show that single point mutations within 16S rRNA that confer resistance to negamycin are in close proximity of the tetracycline binding site within helix 34 of the small subunit head domain. As expected from its direct interaction with this region of the ribosome, negamycin was shown to displace tetracycline. However, in contrast to tetracycline-class antibiotics, which serve to prevent cognate tRNA from entering the translating ribosome, single-molecule fluorescence resonance energy transfer investigations revealed that negamycin specifically stabilizes near-cognate ternary complexes within the A site during the normally transient initial selection process to promote miscoding. The crystal structure of the 70S ribosome in complex with negamycin, determined at 3.1 Å resolution, sheds light on this finding by showing that negamycin occupies a site that partially overlaps that of tetracycline-class antibiotics. Collectively, these data suggest that the small subunit head domain contributes to the decoding mechanism and that small-molecule binding to this domain may either prevent or promote tRNA entry by altering the initial selection mechanism after codon recognition and before GTPase activation.

Identification of azabenzimidazoles as potent JAK1 selective inhibitors.

We have identified a class of azabenzimidazoles as potent and selective JAK1 inhibitors. Investigations into the SAR are presented along with the structural features required to achieve selectivity for JAK1 versus other JAK family members. An example from the series demonstrated highly selective inhibition of JAK1 versus JAK2 and JAK3, along with inhibition of pSTAT3 in vivo, enabling it to serve as a JAK1 selective tool compound to further probe the biology of JAK1 selective inhibitors.

The role of a novel auxiliary pocket in bacterial phenylalanyl-tRNA synthetase druggability.

The antimicrobial activity of phenyl-thiazolylurea-sulfonamides against Staphylococcus aureus PheRS are dependent upon phenylalanine levels in the extracellular fluids. Inhibitor efficacy in animal models of infection is substantially diminished by dietary phenylalanine intake, thereby reducing the perceived clinical utility of this inhibitor class. The search for novel antibacterial compounds against Gram-negative pathogens led to a re-evaluation of this phenomenon, which is shown here to be unique to S. aureus. Inhibition of macromolecular syntheses and characterization of novel resistance mutations in Escherichia coli demonstrate that antimicrobial activity of phenyl-thiazolylurea-sulfonamides is mediated by PheRS inhibition, validating this enzyme as a viable drug discovery target for Gram-negative pathogens. A search for novel inhibitors of PheRS yielded three novel chemical starting points. NMR studies were used to confirm direct target engagement for phenylalanine-competitive hits. The crystallographic structure of Pseudomonas aeruginosa PheRS defined the binding modes of these hits and revealed an auxiliary hydrophobic pocket that is positioned adjacent to the phenylalanine binding site. Three viable inhibitor-resistant mutants were mapped to this pocket, suggesting that this region is a potential liability for drug discovery.

Discovery of AZ0108, an orally bioavailable phthalazinone PARP inhibitor that blocks centrosome clustering.

The propensity for cancer cells to accumulate additional centrosomes relative to normal cells could be exploited for therapeutic benefit in oncology. Following literature reports that suggested TNKS1 (tankyrase 1) and PARP16 may be involved with spindle structure and function and may play a role in suppressing multi-polar spindle formation in cells with supernumerary centrosomes, we initiated a phenotypic screen to look for small molecule poly (ADP-ribose) polymerase (PARP) enzyme family inhibitors that could produce a multi-polar spindle phenotype via declustering of centrosomes. Screening of AstraZeneca's collection of phthalazinone PARP inhibitors in HeLa cells using high-content screening techniques identified several compounds that produced a multi-polar spindle phenotype at low nanomolar concentrations. Characterization of these compounds across a broad panel of PARP family enzyme assays indicated that they had activity against several PARP family enzymes, including PARP1, 2, 3, 5a, 5b, and 6. Further optimization of these initial hits for improved declustering potency, solubility, permeability, and oral bioavailability resulted in AZ0108, a PARP1, 2, 6 inhibitor that potently inhibits centrosome clustering and is suitable for in vivo efficacy and tolerability studies.

Neoplastic and non-neoplastic lesions in biopsy samples from pet rabbits in Hong Kong: a retrospective analysis, 2019-2022.

Rabbits are popular pets in the urban environment of Hong Kong, ranking third behind cats and dogs. Here we describe the frequency of neoplastic and non-neoplastic lesions in biopsies from pet rabbits submitted to the CityU Veterinary Diagnostic Laboratory between 2019 and 2022, comprising 247 tissue samples from 243 rabbits collected by veterinarians in 19 veterinary clinics. Among the 243 rabbits, there were 128 females (65 spayed), 114 males (54 castrated); sex information was not provided for 1 rabbit. The rabbit breeds included 45 Lionhead, 35 Dwarf, 14 Lop, 11 Dwarf Lop, 5 French Lop, 3 Angora, 2 Dutch, 2 Holland Lop, and 1 each of Netherland Dwarf, Velveteen, Mini Lop, and New Zealand White. The mean ages of rabbits with neoplastic and non-neoplastic lesions were 7.1 and 5.7 y, respectively. The most common neoplastic lesions were adenocarcinoma (26.4%), trichoblastoma (21.4%), sarcoma (9.4%), and thymoma (8.2%). The most common non-neoplastic lesion was uterine cystic endometrial hyperplasia (14.8%), followed by dermal abscess formation in the ventral abdomen or skin of the head (12.5%). Although a broad spectrum of other lesions was described, our findings in biopsies from pet rabbits in Hong Kong are consistent with those in other jurisdictions.

Discovery of aminopiperidine-based Smac mimetics as IAP antagonists.

A series of structurally unique Smac mimetics that act as antagonists of inhibitor of apoptosis proteins (IAPs) has been discovered. While most previously described Smac mimetics contain the proline ring (or a similar cyclic motif) found in Smac, a key feature of the compounds described herein is that this ring has been removed. Despite this, compounds in this series potently bind to cIAP1 and elicit the expected phenotype of cIAP1 inhibition in cancer cells. Marked selectivity for cIAP1 over XIAP is observed for these compounds, which is attributed to a slight difference in the binding groove between the two proteins and the resulting steric interactions with the inhibitors. XIAP binding can be improved by constraining the inhibitor so that these unfavorable steric interactions are minimized.

Discovery of novel benzylidene-1,3-thiazolidine-2,4-diones as potent and selective inhibitors of the PIM-1, PIM-2, and PIM-3 protein kinases.

Novel substituted benzylidene-1,3-thiazolidine-2,4-diones (TZDs) have been identified as potent and highly selective inhibitors of the PIM kinases. The synthesis and SAR of these compounds are described, along with X-ray crystallographic, anti-proliferative, and selectivity data.

A novel strain of lumpy skin disease virus causes clinical disease in cattle in Hong Kong.

Lumpy skin disease virus (LSDV) is an emerging poxviral pathogen of cattle that is currently spreading throughout Asia. The disease situation is of high importance for farmers and policy makers in Asia. In October 2020, feral cattle in Hong Kong developed multi-focal cutaneous nodules consistent with lumpy skin disease (LSD). Gross and histological pathology further supported the diagnosis and samples were sent to the OIE Reference Laboratory at The Pirbright Institute for confirmatory testing. LSDV was detected using quantitative polymerase chain reaction (qPCR) and additional molecular analyses. This is the first report of LSD in Hong Kong. Whole genome sequencing (WGS) of the strain LSDV/Hong Kong/2020 and phylogenetic analysis were carried out in order to identify connections to previous outbreaks of LSD, and better understand the drivers of LSDV emergence. Analysis of the 90 core poxvirus genes revealed LSDV/Hong Kong/2020 was a novel strain most closely related to the live-attenuated Neethling vaccine strains of LSDV and more distantly related to wildtype LSDV isolates from Africa, the Middle East and Europe. Analysis of the more variable regions located towards the termini of the poxvirus genome revealed genes in LSDV/Hong Kong/2020 with different patterns of grouping when compared to previously published wildtype and vaccine strains of LSDV. This work reveals that the LSD outbreak in Hong Kong in 2020 was caused by a different strain of LSDV than the LSD epidemic in the Middle East and Europe in 2015-2018. The use of WGS is highly recommended when investigating LSDV disease outbreaks.

Biochemical characterization of human SET and MYND domain-containing protein 2 methyltransferase.

SET and MYND domain-containing protein 2 (SMYD2) is a protein lysine methyltransferase that catalyzes the transfer of methyl groups from S-adenosylmethionine (AdoMet) to acceptor lysine residues on histones and other proteins. To understand the kinetic mechanism and the function of individual domains, human SMYD2 was overexpressed, purified, and characterized. Substrate specificity and product analysis studies established SMYD2 as a monomethyltransferase that prefers nonmethylated p53 peptide substrate. Steady-state kinetic and product inhibition studies showed that SMYD2 operates via a rapid equilibrium random Bi Bi mechanism at a rate of 0.048 ± 0.001 s(-1), with K(M)s for AdoMet and the p53 peptide of 0.031 ± 0.01 µM and 0.68 ± 0.22 µM, respectively. Metal analyses revealed that SMYD2 contains three tightly bound zinc ions that are important for maintaining the structural integrity and catalytic activity of SMYD2. Catalytic activity was also shown to be dependent on the GxG motif in the S-sequence of the split SET domain, as a G18A/G20A double mutant and a sequence deletion within the conserved motif impaired AdoMet binding and significantly decreased enzymatic activity. The functional importance of other SMYD2 domains including the MYND domain, the cysteine-rich post-SET domain, and the C-terminal domain (CTD), were also investigated. Taken together, these results demonstrated the functional importance of distinct domains in the SMYD family of proteins and further advanced our understanding of the catalytic mechanism of this family.

Discovery of Novel, Potent Inhibitors of Hydroxy Acid Oxidase 1 (HAO1) Using DNA-Encoded Chemical Library Screening.

Inhibition of hydroxy acid oxidase 1 (HAO1) is a strategy to mitigate the accumulation of toxic oxalate that results from reduced activity of alanine-glyoxylate aminotransferase (AGXT) in primary hyperoxaluria 1 (PH1) patients. DNA-Encoded Chemical Library (DECL) screening provided two novel chemical series of potent HAO1 inhibitors, represented by compounds 3-6. Compound 5 was further optimized via various structure-activity relationship (SAR) exploration methods to 29, a compound with improved potency and absorption, distribution, metabolism, and excretion (ADME)/pharmacokinetic (PK) properties. Since carboxylic acid-containing compounds are often poorly permeable and have potential active glucuronide metabolites, we undertook a brief, initial exploration of acid replacements with the aim of identifying non-acid-containing HAO1 inhibitors. Structure-based drug design initiated with Compound 5 led to the identification of a nonacid inhibitor of HAO1, 31, which has weaker potency and increased permeability.

Discovery of a Series of 7-Azaindoles as Potent and Highly Selective CDK9 Inhibitors for Transient Target Engagement.

Optimization of a series of azabenzimidazoles identified from screening hit 2 and the information gained from a co-crystal structure of the azabenzimidazole-based lead 6 bound to CDK9 led to the discovery of azaindoles as highly potent and selective CDK9 inhibitors. With the goal of discovering a highly selective and potent CDK9 inhibitor administrated intravenously that would enable transient target engagement of CDK9 for the treatment of hematological malignancies, further optimization focusing on physicochemical and pharmacokinetic properties led to azaindoles 38 and 39. These compounds are highly potent and selective CDK9 inhibitors having short half-lives in rodents, suitable physical properties for intravenous administration, and the potential to achieve profound but transient inhibition of CDK9 in vivo.

Discovery of (2R)-N-[3-[2-[(3-Methoxy-1-methyl-pyrazol-4-yl)amino]pyrimidin-4-yl]-1H-indol-7-yl]-2-(4-methylpiperazin-1-yl)propenamide (AZD4205) as a Potent and Selective Janus Kinase 1 Inhibitor.

JAK1, JAK2, JAK3, and TYK2 belong to the JAK (Janus kinase) family. They play critical roles in cytokine signaling. Constitutive activation of JAK/STAT pathways is associated with a wide variety of diseases. Particularly, pSTAT3 is observed in response to the treatment with inhibitors of oncogenic signaling pathways such as EGFR, MAPK, and AKT and is associated with resistance or poorer response to agents targeting these pathways. Among the JAK family kinases, JAK1 has been shown to be the primary driver of STAT3 phosphorylation and signaling; therefore, selective JAK1 inhibition can be a viable means to overcome such treatment resistances. Herein, an account of the medicinal chemistry optimization from the promiscuous kinase screening hit 3 to the candidate drug 21 (AZD4205), a highly selective JAK1 kinase inhibitor, is reported. Compound 21 has good preclinical pharmacokinetics. Compound 21 displayed an enhanced antitumor activity in combination with an approved EGFR inhibitor, osimertinib, in a preclinical non-small-cell lung cancer (NSCLC) xenograft NCI-H1975 model.

Discovery of AZD4573, a Potent and Selective Inhibitor of CDK9 That Enables Short Duration of Target Engagement for the Treatment of Hematological Malignancies.

A CDK9 inhibitor having short target engagement would enable a reduction of Mcl-1 activity, resulting in apoptosis in cancer cells dependent on Mcl-1 for survival. We report the optimization of a series of amidopyridines (from compound 2), focusing on properties suitable for achieving short target engagement after intravenous administration. By increasing potency and human metabolic clearance, we identified compound 24, a potent and selective CDK9 inhibitor with suitable predicted human pharmacokinetic properties to deliver transient inhibition of CDK9. Furthermore, the solubility of 24 was considered adequate to allow i.v. formulation at the anticipated effective dose. Short-term treatment with compound 24 led to a rapid dose- and time-dependent decrease of pSer2-RNAP2 and Mcl-1, resulting in cell apoptosis in multiple hematological cancer cell lines. Intermittent dosing of compound 24 demonstrated efficacy in xenograft models derived from multiple hematological tumors. Compound 24 is currently in clinical trials for the treatment of hematological malignancies.

What's all the FLAP about?: 5-lipoxygenase-activating protein inhibitors for inflammatory diseases.

Leukotrienes have physiological roles in innate immune responses and pathological roles in inflammatory diseases, such as asthma, allergic rhinitis and atherosclerosis. Anti-leukotriene therapy has proven benefits in the treatment of respiratory disease, either through the inhibition of leukotriene synthesis or the selective antagonism of leukotriene receptors. The first committed step in the synthesis of leukotrienes is the oxidation of arachidonic acid (AA) by 5-lipoxygenase (5-LO), and the integral membrane protein 5-lipoxygenase-activating protein (FLAP) is an essential partner of 5-LO for this process. FLAP was molecularly identified via a photoaffinity probe and an affinity gel based on MK-886, a selective leukotriene inhibitor that has no activity against broken-cell preparations of 5-LO. Several FLAP inhibitors showed efficacy in early clinical trials in asthma but were not developed commercially for unpublished reasons. Recently, the FLAP (ALOX5AP) gene has been linked to risk for myocardial infarction, stroke and restenosis, reigniting pharmaceutical interest in this target. In addition, the recent determination of the crystal structure of inhibitor-bound FLAP offers exciting potential for novel FLAP inhibitor design.

The ER protein folding sensor UDP-glucose glycoprotein-glucosyltransferase modifies substrates distant to local changes in glycoprotein conformation.

We present in vitro data that explain the recognition mechanism of misfolded glycoproteins by UDP-glucose glycoprotein-glucosyltransferase (UGGT). The glycoprotein exo-(1,3)-beta-glucanase (beta-Glc) bearing two glycans unfolds in a pH-dependent manner to become a misfolded substrate for UGGT. In the crystal structure of this glycoprotein, the local hydrophobicity surrounding each glycosylation site coincides with the differential recognition of N-linked glycans by UGGT. We introduced a single F280S point mutation, producing a beta-Glc protein with full enzymatic activity that was both recognized as misfolded and monoglucosylated by UGGT. Contrary to current views, these data show that UGGT can modify N-linked glycans positioned at least 40 A from localized regions of disorder and sense subtle conformational changes within structurally compact, enzymatically active glycoprotein substrates.