Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts no new class of antibiotic with activity against Gram-negative bacteria has been approved in over fifty ...years. Natural products and their derivatives have a key role in combating Gram-negative pathogens. Here we report chemical optimization of the arylomycins-a class of natural products with weak activity and limited spectrum-to obtain G0775, a molecule with potent, broad-spectrum activity against Gram-negative bacteria. G0775 inhibits the essential bacterial type I signal peptidase, a new antibiotic target, through an unprecedented molecular mechanism. It circumvents existing antibiotic resistance mechanisms and retains activity against contemporary multidrug-resistant Gram-negative clinical isolates in vitro and in several in vivo infection models. These findings demonstrate that optimized arylomycin analogues such as G0775 could translate into new therapies to address the growing threat of multidrug-resistant Gram-negative infections.
Histone deacetylases (HDACs) are critical in the control of gene expression, and dysregulation of their activity has been implicated in a broad range of diseases, including cancer, cardiovascular, ...and neurological diseases. HDAC inhibitors (HDACi) employing different zinc chelating functionalities such as hydroxamic acids and benzamides have shown promising results in cancer therapy. Although it has also been suggested that HDACi with increased isozyme selectivity and potency may broaden their clinical utility and minimize side effects, the translation of this idea to the clinic remains to be investigated. Moreover, a detailed understanding of how HDACi with different pharmacological properties affect biological functions in vitro and in vivo is still missing. Here, we show that a panel of benzamide-containing HDACi are slow tight-binding inhibitors with long residence times unlike the hydroxamate-containing HDACi vorinostat and trichostatin-A. Characterization of changes in H2BK5 and H4K14 acetylation following HDACi treatment in the neuroblastoma cell line SH-SY5Y revealed that the timing and magnitude of histone acetylation mirrored both the association and dissociation kinetic rates of the inhibitors. In contrast, cell viability and microarray gene expression analysis indicated that cell death induction and changes in transcriptional regulation do not correlate with the dissociation kinetic rates of the HDACi. Therefore, our study suggests that determining how the selective and kinetic inhibition properties of HDACi affect cell function will help to evaluate their therapeutic utility.
Background: The effect of HDAC inhibitor kinetic properties on biological function is currently unknown.
Results: The kinetic rate constants of HDAC inhibitors differentially affect histone acetylation, cell viability, and gene expression.
Conclusion: Evaluating HDAC inhibitor properties using histone acetylation is not predictive of their function on cellular activity.
Significance: Characterizing the biological effect of different HDAC inhibitors will help to evaluate their clinical utility.
Immunosuppressant drugs such as cyclosporin have allowed widespread organ transplantation, but their utility remains limited
by toxicities, and they are ineffective in chronic management of ...autoimmune diseases such as multiple sclerosis. In contrast,
the immune modulating drug FTY720 is efficacious in a variety of transplant and autoimmune models without inducing a generalized
immunosuppressed state and is effective in human kidney transplantation. FTY720 elicits a lymphopenia resulting from a reversible
redistribution of lymphocytes from circulation to secondary lymphoid tissues by unknown mechanisms. Using FTY720 and several
analogs, we show now that FTY720 is phosphorylated by sphingosine kinase; the phosphorylated compound is a potent agonist
at four sphingosine 1-phosphate receptors and represents the therapeutic principle in a rodent model of multiple sclerosis.
Our results suggest that FTY720, after phosphorylation, acts through sphingosine 1-phosphate signaling pathways to modulate
chemotactic responses and lymphocyte trafficking.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins bind to host mitochondrial proteins, likely inhibiting oxidative phosphorylation (OXPHOS) and stimulating glycolysis. We ...analyzed mitochondrial gene expression in nasopharyngeal and autopsy tissues from patients with coronavirus disease 2019 (COVID-19). In nasopharyngeal samples with declining viral titers, the virus blocked the transcription of a subset of nuclear DNA (nDNA)-encoded mitochondrial OXPHOS genes, induced the expression of microRNA 2392, activated HIF-1α to induce glycolysis, and activated host immune defenses including the integrated stress response. In autopsy tissues from patients with COVID-19, SARS-CoV-2 was no longer present, and mitochondrial gene transcription had recovered in the lungs. However, nDNA mitochondrial gene expression remained suppressed in autopsy tissue from the heart and, to a lesser extent, kidney, and liver, whereas mitochondrial DNA transcription was induced and host-immune defense pathways were activated. During early SARS-CoV-2 infection of hamsters with peak lung viral load, mitochondrial gene expression in the lung was minimally perturbed but was down-regulated in the cerebellum and up-regulated in the striatum even though no SARS-CoV-2 was detected in the brain. During the mid-phase SARS-CoV-2 infection of mice, mitochondrial gene expression was starting to recover in mouse lungs. These data suggest that when the viral titer first peaks, there is a systemic host response followed by viral suppression of mitochondrial gene transcription and induction of glycolysis leading to the deployment of antiviral immune defenses. Even when the virus was cleared and lung mitochondrial function had recovered, mitochondrial function in the heart, kidney, liver, and lymph nodes remained impaired, potentially leading to severe COVID-19 pathology.
Reducing production of amyloid-β (Aβ) peptide by direct inhibition of the enzymes that process amyloid precursor protein (APP) is a central therapeutic strategy for treating Alzheimer's disease. ...However, small-molecule inhibitors of the β-secretase (BACE1) and γ-secretase APP processing enzymes have shown a lack of target selectivity and poor penetrance of the blood-brain barrier (BBB). Here, we have developed a high-affinity, phage-derived human antibody that targets BACE1 (anti-BACE1) and is anti-amyloidogenic. Anti-BACE1 reduces endogenous BACE1 activity and Aβ production in human cell lines expressing APP and in cultured primary neurons. Anti-BACE1 is highly selective and does not inhibit the related enzymes BACE2 or cathepsin D. Competitive binding assays and x-ray crystallography indicate that anti-BACE1 binds noncompetitively to an exosite on BACE1 and not to the catalytic site. Systemic dosing of mice and nonhuman primates with anti-BACE1 resulted in sustained reductions in peripheral Aβ peptide concentrations. Anti-BACE1 also reduces central nervous system Aβ concentrations in mouse and monkey, consistent with a measurable uptake of antibody across the BBB. Thus, BACE1 can be targeted in a highly selective manner through passive immunization with anti-BACE1, providing a potential approach for treating Alzheimer's disease. Nevertheless, therapeutic success with anti-BACE1 will depend on improving antibody uptake into the brain.
Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery ...and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound's inhibitory activity is also dependent on the amino acid sequence and P1' character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity.
p21-activated kinase 1 (PAK1) has an important role in transducing signals in several oncogenic pathways. The concept of inhibiting this kinase has garnered significant interest over the past decade, ...particularly for targeting cancers associated with PAK1 amplification. Animal studies with the selective group I PAK (pan-PAK1, 2, 3) inhibitor G-5555 from the pyrido2,3-dpyrimidin-7-one class uncovered acute toxicity with a narrow therapeutic window. To attempt mitigating the toxicity, we introduced significant structural changes, culminating in the discovery of the potent pyridone side chain analogue G-9791. Mouse tolerability studies with this compound, other members of this series, and compounds from two structurally distinct classes revealed persistent toxicity and a correlation of minimum toxic concentrations and PAK1/2 mediated cellular potencies. Broad screening of selected PAK inhibitors revealed PAK1, 2, and 3 as the only overlapping targets. Our data suggest acute cardiovascular toxicity resulting from the inhibition of PAK2, which may be enhanced by PAK1 inhibition, and cautions against continued pursuit of pan-group I PAK inhibitors in drug discovery.
The p21-activated kinases (PAKs) play important roles in cytoskeletal organization, cellular morphogenesis, and survival and have generated significant attention as potential therapeutic targets for ...cancer. Following a high-throughput screen, we identified an aminopyrazole scaffold-based series that was optimized to yield group I selective PAK inhibitors. A structure-based design effort aimed at targeting the ribose pocket for both potency and selectivity led to much-improved group I vs II selectivity. Early lead compounds contained a basic primary amine, which was found to be a major metabolic soft spot with in vivo clearance proceeding predominantly via N-acetylation. We succeeded in identifying replacements with improved metabolic stability, leading to compounds with lower in vivo rodent clearance and excellent group I PAK selectivity.
Caspase-6 is a cysteinyl protease implicated in neurodegenerative conditions including Alzheimer's and Huntington's disease making it an attractive target for therapeutic intervention. A greater ...understanding of the role of caspase-6 in disease has been hampered by a lack of suitable cellular assays capable of specifically detecting caspase-6 activity in an intact cell environment. This is mainly due to the use of commercially available peptide substrates and inhibitors which lack the required specificity to facilitate development of this type of assay. We report here a 384-well whole-cell chemiluminescent ELISA assay that monitors the proteolytic degradation of endogenously expressed lamin A/C during the early stages of caspase-dependent apoptosis. The specificity of lamin A/C proteolysis by caspase-6 was demonstrated against recombinant caspase family members and further confirmed in genetic deletion studies. In the assay, plasma membrane integrity remained intact as assessed by release of lactate dehydrogenase from the intracellular environment and the exclusion of cell impermeable peptide inhibitors, despite the induction of an apoptotic state. The method described here is a robust tool to support drug discovery efforts targeting caspase-6 and is the first reported to specifically monitor endogenous caspase-6 activity in a cellular context.
Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts, no new antibiotic class with activity against Gram-negative bacteria has been approved in over 50 years. ...LepB inhibitors (LepBi) based on the arylomycin class of natural products are a novel class of antibiotics and function by inhibiting the bacterial type I signal peptidase (SPase) in Gram-negative bacteria. One critical aspect of LepBi development involves optimization of the membrane-anchored lipophilic portion of the molecule. We therefore developed an approach that assesses the effect of this portion on the complicated equilibria of plasma protein binding, crossing the outer membrane of Gram-negative bacteria and anchoring in the bacterial inner membrane to facilitate SPase binding. Our findings provide important insights into the development of antibacterial agents where the target is associated with the inner membrane of Gram-negative bacteria.
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