A major resistance mechanism in Gram-negative bacteria is the production of β-lactamase enzymes. Originally recognized for their ability to hydrolyze penicillins, emergent β-lactamases can now confer resistance to other β-lactam drugs, including both cephalosporins and carbapenems. The emergence and global spread of β-lactamase-producing multi-drug-resistant “superbugs” has caused increased alarm within the medical community due to the high mortality rate associated with these difficult-to-treat bacterial infections. To address this unmet medical need, we initiated an iterative program combining medicinal chemistry, structural biology, biochemical testing, and microbiological profiling to identify broad-spectrum inhibitors of both serine- and metallo-β-lactamase enzymes. Lead optimization, beginning with narrower-spectrum, weakly active compounds, provided 20 (VNRX-5133, taniborbactam), a boronic-acid-containing pan-spectrum β-lactamase inhibitor. In vitro and in vivo studies demonstrated that 20 restored the activity of β-lactam antibiotics against carbapenem-resistant Pseudomonas aeruginosa and carbapenem-resistant Enterobacteriaceae. Taniborbactam is the first pan-spectrum β-lactamase inhibitor to enter clinical development.

Doublecortin-like kinase 1 (DCLK1) is a serine/threonine kinase that belongs to the family of microtubule-associated proteins. Originally identified for its role in neurogenesis, DCLK1 has recently been shown to regulate biological processes outside of the CNS. DCLK1 is among the 15 most common putative driver genes for gastric cancers and is highly mutated across various other human cancers. However, our present understanding of how DCLK1 dysfunction leads to tumorigenesis is limited. Here, we provide evidence that DCLK1 kinase activity negatively regulates microtubule polymerization. We present the crystal structure of the DCLK1 kinase domain at 1.7 Å resolution, providing detailed insight into the ATP-binding site that will serve as a framework for future drug design. This structure also allowed for the mapping of cancer-causing mutations within the kinase domain, suggesting that a loss of kinase function may contribute to tumorigenesis.

As shifts in the epidemiology of β-lactamase-mediated resistance continue, carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant Pseudomonas aeruginosa (CRPA) are the most urgent threats. Although approved β-lactam (BL)–β-lactamase inhibitor (BLI) combinations address widespread serine β-lactamases (SBLs), such as CTX-M-15, none provide broad coverage against either clinically important serine-β-lactamases (KPC, OXA-48) or clinically important metallo-β-lactamases (MBLs; e.g., NDM-1). VNRX-5133 (taniborbactam) is a new cyclic boronate BLI that is in clinical development combined with cefepime for the treatment of infections caused by β-lactamase-producing CRE and CRPA. Taniborbactam is the first BLI with direct inhibitory activity against Ambler class A, B, C, and D enzymes. From biochemical and structural analyses, taniborbactam exploits substrate mimicry while employing distinct mechanisms to inhibit both SBLs and MBLs. It is a reversible covalent inhibitor of SBLs with slow dissociation and a prolonged active-site residence time (half-life, 30 to 105 min), while in MBLs, it behaves as a competitive inhibitor, with inhibitor constant (Ki) values ranging from 0.019 to 0.081 μM. Inhibition is achieved by mimicking the transition state structure and exploiting interactions with highly conserved active-site residues. In microbiological testing, taniborbactam restored cefepime activity in 33/34 engineered Escherichia coli strains overproducing individual enzymes covering Ambler classes A, B, C, and D, providing up to a 1,024-fold shift in the MIC. Addition of taniborbactam restored the antibacterial activity of cefepime against all 102 Enterobacterales clinical isolates tested and 38/41 P. aeruginosa clinical isolates tested with MIC90s of 1 and 4 μg/ml, respectively, representing ≥256- and ≥32-fold improvements, respectively, in antibacterial activity over that of cefepime alone. The data demonstrate the potent, broad-spectrum rescue of cefepime activity by taniborbactam against clinical isolates of CRE and CRPA.

Conformational restrictions in the form of [i, i + 4] lactam bridges were sequentially incorporated into the shortest fragment of hPTH with recognized efficacy in the OVX rat model of osteoporosis, hPTH-(1-31)NH 2 (1). Cyclo(Lys 18 -Asp 22 )[Ala 1 ,Nle 8 ,Lys 18 ,Asp 22 ,Leu 27 ]hPTH(1-31)NH 2 (2) is a potent agonist of the PTH/PTHrP receptor located on the surface of ROS 17/2.8 cells as measured by its ability to stimulate adenylyl cyclase activity (EC 50 ) 0.29 nM). A second analogue, which constrains the entire C-terminal receptor binding domain, bicyclo(Lys 18 -Asp 22 ,Lys 26 -Asp 30 )[Ala 1 ,Nle 8 ,Lys 18 ,Asp 22 ,Leu 27 ] hPTH(1-31)NH 2 ( 6), is also shown to be a potent agonist (EC 50 ) 0.13 nM), thus providing further evidence for an extended helix as the relevant bioactive conformation in this region of the hormone. Adjacent lactam bridges were incorporated into the analogue bicyclo(Lys 13 -Asp 17 ,Lys 18 -Asp 22 )[Ala 1 ,Nle 8 ,Lys 18 ,Asp 17,22 ,Leu 27 ]hPTH(1-31)NH 2 (7) to evaluate the receptor's tolerance to conformational restriction in the midregion of the peptide. In fact, peptide 7 is also a highly potent agonist (EC 50 ) 0.43 nM) in the cAMP-based assay, which suggests that at least one bioactive form of the hormone requires a helical conformation extending from residue 13 to residue 22. Incorporation of all three lactam bridges afforded the most conformationally constrained PTH peptide agonist yet reported, tricyclo(Lys 13 -Asp 17 ,Lys 18 -Asp 22 ,Lys 26 -Asp 30 )[Ala 1 ,Nle 8 ,Lys 18 ,Asp 17,22 ,Leu 27 ]hPTH(1-31)NH 2 (9). Peptide 9 (EC 50 ) 0.14 nM) forces residues 13-30 into an extended helical conformation and is a more potent PTH receptor agonist than the parent linear hPTH(1-31)NH 2 (1, EC 50 ) 4.7 nM). Comparative circular dichroism studies indicate that peptide 9 is highly helical even in the absence of TFE, indicating that residues 1-12 are also likely to be helical in the bioactive conformation. Taken together, these results provide strong structural evidence that hPTH binds to its receptor in an extended helical conformation.

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The role(s) played by protein tyrosine kinases (PTKs) in the regulation of insulin secretion from pancreatic cells is not clear. We have examined the effects of glucose, the major physiological insulin secretagogue, on the tyrosine phosphorylation state of islet proteins, and assessed cell insulin secretory responses in the presence of PTK inhibitors. Under basal conditions islets contained many proteins phosphorylated on tyrosine residues, and glucose (20 mM; 5-15 min) was without demonstrable effect on the pattern of tyrosine phosphorylation, in either the absence or presence of the protein tyrosine phosphatase (PTP) inhibitor, sodium pervanadate (PV). PV alone (100 µM) increased tyrosine phosphorylation of several islet proteins. The PTK inhibitors genistein (GS) and tyrphostin A47 (TA47) inhibited islet tyrosine kinase activities and glucose-, 4 ketoisocaproic acid (KIC)-and sulphonylurea-stimulated insulin release, without affecting glucose metabolism. GS and TA47 also inhibited protein serine/threonine kinase activities to a limited extent, but had no effect on Ca 2+ , cyclic AMP-or phorbol myristate acetate (PMA)-induced insulin secretion from electrically permeabilised islets. These results suggest that PTK inhibitors exert their inhibitory effects on insulin secretion proximal to Ca 2+ entry and it is proposed that they act at the site of the voltage-dependent Ca 2+ channel which regulates Ca 2+ influx into cells following nutrient-and sulphonylurea-induced depolarisation.

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