Structure Guided Discovery of Novel Pan Metallo-ß-Lactamase Inhibitors with Improved Gram-Negative Bacterial Cell Penetration.

The use of ß-lactam (BL) and ß-lactamase inhibitor combination to overcome BL antibiotic resistance has been validated through clinically approved drug products. However, unmet medical needs still exist for the treatment of infections caused by Gram-negative (GN) bacteria expressing metallo-ß-lactamases. Previously, we reported our effort to discover pan inhibitors of three main families in this class: IMP, VIM, and NDM. Herein, we describe our work to improve the GN coverage spectrum in combination with imipenem and relebactam. This was achieved through structure- and property-based optimization to tackle the GN cell penetration and efflux challenges. A significant discovery was made that inhibition of both VIM alleles, VIM-1 and VIM-2, is essential for broad GN coverage, especially against VIM-producing P. aeruginosa. In addition, pharmacokinetics and nonclinical safety profiles were investigated for select compounds. Key findings from this drug discovery campaign laid the foundation for further lead optimization toward identification of preclinical candidates.

Rapid Evolution of a Fragment-like Molecule to Pan-Metallo-Beta-Lactamase Inhibitors: Initial Leads toward Clinical Candidates.

With the emergence and rapid spreading of NDM-1 and existence of clinically relevant VIM-1 and IMP-1, discovery of pan inhibitors targeting metallo-beta-lactamases (MBLs) became critical in our battle against bacterial infection. Concurrent with our fragment and high-throughput screenings, we performed a knowledge-based search of known metallo-beta-lactamase inhibitors (MBLIs) to identify starting points for early engagement of medicinal chemistry. A class of compounds exemplified by 11, discovered earlier as B. fragilis metallo-beta-lactamase inhibitors, was selected for in silico virtual screening. From these efforts, compound 12 was identified with activity against NDM-1 only. Initial exploration on metal binding design followed by structure-guided optimization led to the discovery of a series of compounds represented by 23 with a pan MBL inhibition profile. In in vivo studies, compound 23 in combination with imipenem (IPM) robustly lowered the bacterial burden in a murine infection model and became the lead for the invention of MBLI clinical candidates.

Serendipitous discovery of aryl boronic acids as ß-lactamase inhibitors.

High throughput screening for ß-lactamase inhibitors afforded biphenyl hits such as 1. Hit confirmation and X-ray soaking experiments with Pseudomonas Aeruginosa AmpC enzyme led to the identification of an aryl boronic acid-serine complex 4, which was formed from phenyl boronic acid 8 (an impurity in compound 1) and ethylene glycol (the cryoprotectant in the soaking experiment).

[Effect of deep and shallow electroacupuncture stimulation at "Huantiao"(GB30) on expression of phosphorylated-p38 and phosphorylated-p53 proteins and apoptosis in dorsal root ganglia in sciatic nerve injury rats].

OBJECTIVE: To observe the effect of deep electroacupuncture (EA) stimulation at "Huantiao"(GB30) on hindlimb motor function and expression of p38 mitogen-activated protein kinase (p38 MAPK ) and p53 proteins in dorsal root ganglia (DRG) in rats with chronic constrictive injury (CCI) of sciatic nerve. METHODS: Forty-eight SD rats (half male and half female) were randomly divided into control, model, shallow EA (SEA) stimulation and deep EA (DEA) stimulation groups (n=12 in each group). The CCI model was constructed by implanting a silicone tube close to the sciatic nerve of the left hind limb. For DEA group and SEA group, filiform acupuncture needles were inserted into GB30 about 12-14 mm deep and 5-8 mm deep (monitored by using a high-frequency ultrasound device), respectively, followed by electrical stimulation (2 Hz/100 Hz, 1 mA) using an EA stimulator. The intervention was conducted for 15 min every time, once daily for 14 days. The sciatic nerve function index (SFI) calculated to assess the motor function status. Histopathological changes of the sciatic nerve were displayed by H.E. staining. The expression levels of phosphorylated-p38 MAPK (p-p38) and phosphorylated-tumor protein p53 (p-p53) in DRGs of L4-L5 on the affected side were observed by immunohistochemical staining. RESULTS: Following modeling, the SFI were significantly decreased (P<0.01), and the expression levels of p-p38 and p-p53 proteins of L4-L5 DRGs were considerably increased in the model group (P<0.05). After the intervention, the SFI were obviously increased, and the expression levels of p-p38 and p-p53 proteins notably down-regulated in both DEA and SEA groups relevant to the model group (P<0.01, P<0.05). The therapeutic effect of DEA was significantly superior to that of SEA in raising SFI and down-regulating expression le-vels of p-p38 and p-p53 proteins (P<0.01, P<0.05). H.E. staining showed disordered arrangement of the sciatic nerve fibers and myelin, disaggregation of the myelin and axons with deformity and vacuolation in some of them and with an increase of Schwann cells in the model group, which was relatively milder in both DEA and SEA groups. CONCLUSION: Both DEA and SEA at GB30 can obviously improve the motor function in CCI rats, which may be associated with its function in down-regulating the expression of p-p38 and p-p53 proteins in L4-L5 DRGs, restraining p38 MAPK signaling. The therapeutic effect of DEA is evidently better than that of SEA.

Freestanding Three-Dimensional CuO/NiO Core-Shell Nanowire Arrays as High-Performance Lithium-Ion Battery Anode.

We demonstrate significant improvement of CuO nanowire arrays as anode materials for lithium ion batteries by coating with thin NiO nanosheets conformally. The NiO nanosheets were designed two kinds of morphologies, which are porous and non-porous. By the NiO nanosheets coating, the major active CuO nanowires were protected from direct contact with the electrolyte to improve the surface chemical stability. Simultaneously, through the observation and comparison of TEM results of crystalline non-porous NiO nanosheets, before and after lithiation process, we clearly prove the effect of expected protection of CuO, and clarify the differences of phase transition, crystallinity change, ionic conduction and the mechanisms of the capacity decay further. Subsequently, the electrochemical performances exhibit lithiation and delithiation differences of the porous and non-porous NiO nanosheets, and confirm that the presence of the non-porous NiO coating can still effectively assist the diffusion of Li+ ions into the CuO nanowires, maintaining the advantage of high surface area, and improves the cycle performance of CuO nanowires, leading to enhanced battery capacity. Optimally, the best structure is validated to be non-porous NiO nanosheets, in contrary to the anticipated porous NiO nanosheets. In addition, considering the low cost and facile fabrication process can be realized further for practical applications.

Can We Make Small Molecules Lean? Optimization of a Highly Lipophilic TarO Inhibitor.

We describe our optimization efforts to improve the physicochemical properties, solubility, and off-target profile of 1, an inhibitor of TarO, an early stage enzyme in the biosynthetic pathway for wall teichoic acid (WTA) synthesis. Compound 1 displayed a TarO IC50 of 125 nM in an enzyme assay and possessed very high lipophilicity (clogP = 7.1) with no measurable solubility in PBS buffer. Structure-activity relationship (SAR) studies resulted in a series of compounds with improved lipophilic ligand efficiency (LLE) consistent with the reduction of clogP. From these efforts, analog 9 was selected for our initial in vivo study, which in combination with subefficacious dose of imipenem (IPM) robustly lowered the bacterial burden in a neutropenic Staphylococci murine infection model. Concurrent with our in vivo optimization effort using 9, we further improved LLE as exemplified by a much more druglike analog 26.

Benzimidazole analogs as WTA biosynthesis inhibitors targeting methicillin resistant Staphylococcus aureus.

A series of benzimidazole analogs have been synthesized to improve the profile of the previous lead compounds tarocin B and 1. The syntheses, structure-activity relationships, and selected biochemical data of these analogs are described. The optimization efforts allowed the identification of 21, a fluoro-substituted benzimidazole, exhibiting potent TarO inhibitory activity and typical profile for a wall teichoic acid (WTA) biosynthesis inhibitor. Compound 21 displayed a potent synergistic and bactericidal effect in combination with imipenem against diverse methicillin-resistant Staphylococci.

Discovery of potent wall teichoic acid early stage inhibitors.

The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current ß-lactam antibiotics and created an urgent need for novel treatment options. Using an S. aureus phenotypic screening strategy, we have identified small molecule early stage wall teichoic acid (WTA) pathway-specific inhibitors predicted to be chemically synergistic with ß-lactams. These previously disclosed inhibitors, termed tarocins, demonstrate by genetic and biochemical means inhibition of TarO, the first step in WTA biosynthesis. Tarocins demonstrate potent bactericidal synergy in combination with broad spectrum ß-lactam antibiotics across diverse clinical isolates of methicillin-resistant Staphylococci. The synthesis and structure-activity relationships (SAR) of a tarocin series will be detailed. Tarocins and other WTA inhibitors may provide a rational strategy to develop Gram-positive bactericidal ß-lactam combination agents active against methicillin-resistant Staphylococci.

TarO-specific inhibitors of wall teichoic acid biosynthesis restore ß-lactam efficacy against methicillin-resistant staphylococci.

The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current ß-lactam antibiotics and created an urgent need for new treatment options. We report an S. aureus phenotypic screening strategy involving chemical suppression of the growth inhibitory consequences of depleting late-stage wall teichoic acid biosynthesis. This enabled us to identify early-stage pathway-specific inhibitors of wall teichoic acid biosynthesis predicted to be chemically synergistic with ß-lactams. We demonstrated by genetic and biochemical means that each of the new chemical series discovered, herein named tarocin A and tarocin B, inhibited the first step in wall teichoic acid biosynthesis (TarO). Tarocins do not have intrinsic bioactivity but rather demonstrated potent bactericidal synergy in combination with broad-spectrum ß-lactam antibiotics against diverse clinical isolates of methicillin-resistant staphylococci as well as robust efficacy in a murine infection model of MRSA. Tarocins and other inhibitors of wall teichoic acid biosynthesis may provide a rational strategy to develop Gram-positive bactericidal ß-lactam combination agents active against methicillin-resistant staphylococci.

Bioavailable pyrrolo-benzo-1,4-diazines as Na(v)1.7 sodium channel blockers for the treatment of pain.

A series of pyrrolo-benzo-1,4-diazine analogs have been synthesized to improve the profile of the previous lead compound 1. The syntheses, structure-activity relationships, and selected pharmacokinetic data of these analogs are described. The optimization efforts allowed the identification of 33, a quinoline amide exhibiting potent Na(v)1.7 inhibitory activity and moderate selectivity over Na(v)1.5. Compound 33 displayed anti-nociceptive oral efficacy in a rat CFA inflammatory pain model at 100 mpk and in a rat spinal nerve ligation neuropathic pain model with an EC50 75 µM.

Discovery of pyrrolo-benzo-1,4-diazines as potent Na(v)1.7 sodium channel blockers.

A series of pyrrolo-benzo-1,4-diazine analogs have been synthesized and displayed potent Nav1.7 inhibitory activity and moderate selectivity over Nav1.5. The syntheses, structure-activity relationships, and selected pharmacokinetic data of these analogs are described. Compound 41 displayed anti-nociceptive efficacy in the rat CFA pain model at 100 mpk oral dosing.

Molecular elimination of Br2 in photodissociation of CH2BrC(O)Br at 248 nm using cavity ring-down absorption spectroscopy.

The primary elimination channel of bromine molecule in one-photon dissociation of CH(2)BrC(O)Br at 248 nm is investigated using cavity ring-down absorption spectroscopy. By means of spectral simulation, the ratio of nascent vibrational population in v = 0, 1, and 2 levels is evaluated to be 1:(0.5 ± 0.1):(0.2 ± 0.1), corresponding to a Boltzmann vibrational temperature of 581 ± 45 K. The quantum yield of the ground state Br(2) elimination reaction is determined to be 0.24 ± 0.08. With the aid of ab initio potential energy calculations, the obtained Br(2) fragments are anticipated to dissociate on the electronic ground state, yielding vibrationally hot Br(2) products. The temperature-dependence measurements support the proposed pathway via internal conversion. For comparison, the Br(2) yields are obtained analogously from CH(3)CHBrC(O)Br and (CH(3))(2)CBrC(O)Br to be 0.03 and 0.06, respectively. The trend of Br(2) yields among the three compounds is consistent with the branching ratio evaluation by Rice-Ramsperger-Kassel-Marcus method. However, the latter result for each molecule is smaller by an order of magnitude than the yield findings. A non-statistical pathway so-called roaming process might be an alternative to the Br(2) production, and its contribution might account for the underestimate of the branching ratio calculations.

Pyrazoloquinolines as PDE10A inhibitors: discovery of a tool compound.

A series of pyrazoloquinolines, possessing (hetero)arylhydroxymethyl substituents at the quinoline C-4 position were evaluated as PDE10A inhibitors. Among these, methylpyrimidyl analogue 15 was identified as having good rodent and monkey exposure, and a MED of 10 mg/kg in an in vivo model.

The discovery of potent, selective, and orally active pyrazoloquinolines as PDE10A inhibitors for the treatment of Schizophrenia.

High-throughput screening identified a series of pyrazoloquinolines as PDE10A inhibitors. The SAR development led to the discovery of compound 27 as a potent, selective, and orally active PDE10A inhibitor. Compound 27 inhibits MK-801 induced hyperactivity at 3mg/kg with an ED(50) of 4mg/kg and displays a ∼6-fold separation between the ED(50) for inhibition of MK-801 induced hyperactivity and hypolocomotion in rats.

Discovery of orally active pyrazoloquinolines as potent PDE10 inhibitors for the management of schizophrenia.

A series of pyrazoloquinoline analogs have been synthesized and shown to bind to PDE10 with high affinity. From the SAR study and our lead optimization efforts, compounds 16 and 27 were found to possess potent oral antipsychotic activity in the MK-801 induced hyperactive rat model.

Structure-activity relationships of 2,4-diphenyl-1H-imidazole analogs as CB2 receptor agonists for the treatment of chronic pain.

A series of 2,4-diphenyl-1H-imidazole analogs have been synthesized and displayed potent human CB2 agonist activity. Many of these analogs showed high functional selectivity over human CB1 receptors. The syntheses, structure-activity relationships, and selected pharmacokinetic data of these analogs are described.

Discovery of orally active 3-pyridinyl-tropane as a potent nociceptin receptor agonist for the management of cough.

A series of 3-pyridinyl-tropane analogues based on previously reported compound 1 have been synthesized and shown to bind to the nociceptin receptor with high affinity. From the SAR study and our lead optimization efforts, compound 10 was found to possess potent oral antitussive activity in the capsaicin-induced guinea pig model. The rationale for compound selection and the biological profile of the optimized lead (10) are disclosed.

Identification of 3-substituted N-benzhydryl-nortropane analogs as nociceptin receptor ligands for the management of cough and anxiety.

A series of nortropane analogs based on previously reported compound 1 have been synthesized and shown to bind to the nociceptin receptor with high affinity. The synthesis and structure-activity relationships around the C-3 nortropane substitution are described. From the SAR study and hPXR screening effort, compound 15 was identified to possess potent oral antitussive and anxiolytic-like activities in the guinea pig models.

Caryophyllenes from a fungal culture of Chrysosporium pilosum.

Four new caryophyllene derivatives, Sch 725432 (1), Sch 601253 (2), Sch 601254 (3), and Sch 725434 (4), were isolated from the fungal fermentation broth of Chrysosporium pilosum by reversed-phase HPLC purification. The structure elucidation of trioxygenated caryophyllenes 1-4 was accomplished on the basis of spectroscopic data interpretation. Sch 725434 (4) possesses a dihydrofuran-3-one ring, forming a tricyclic ring skeleton, which represents an unprecedented ring skeleton for the caryophyllene-type of sesquiterpenes. Compounds 1-4 were evaluated for their antifungal activity.