A Gram-negative-selective antibiotic that spares the gut microbiome.

Infections caused by Gram-negative pathogens are increasingly prevalent and are typically treated with broad-spectrum antibiotics, resulting in disruption of the gut microbiome and susceptibility to secondary infections1-3. There is a critical need for antibiotics that are selective both for Gram-negative bacteria over Gram-positive bacteria, as well as for pathogenic bacteria over commensal bacteria. Here we report the design and discovery of lolamicin, a Gram-negative-specific antibiotic targeting the lipoprotein transport system. Lolamicin has activity against a panel of more than 130 multidrug-resistant clinical isolates, shows efficacy in multiple mouse models of acute pneumonia and septicaemia infection, and spares the gut microbiome in mice, preventing secondary infection with Clostridioides difficile. The selective killing of pathogenic Gram-negative bacteria by lolamicin is a consequence of low sequence homology for the target in pathogenic bacteria versus commensals; this doubly selective strategy can be a blueprint for the development of other microbiome-sparing antibiotics.

Porin-independent accumulation in Pseudomonas enables antibiotic discovery.

Gram-negative antibiotic development has been hindered by a poor understanding of the types of compounds that can accumulate within these bacteria1,2. The presence of efflux pumps and substrate-specific outer-membrane porins in Pseudomonas aeruginosa renders this pathogen particularly challenging3. As a result, there are few antibiotic options for P. aeruginosa infections4 and its many porins have made the prospect of discovering general accumulation guidelines seem unlikely5. Here we assess the whole-cell accumulation of 345 diverse compounds in P. aeruginosa and Escherichia coli. Although certain positively charged compounds permeate both bacterial species, P. aeruginosa is more restrictive compared to E. coli. Computational analysis identified distinct physicochemical properties of small molecules that specifically correlate with P. aeruginosa accumulation, such as formal charge, positive polar surface area and hydrogen bond donor surface area. Mode of uptake studies revealed that most small molecules permeate P. aeruginosa using a porin-independent pathway, thus enabling discovery of general P. aeruginosa accumulation trends with important implications for future antibiotic development. Retrospective antibiotic examples confirmed these trends and these discoveries were then applied to expand the spectrum of activity of a gram-positive-only antibiotic, fusidic acid, into a version that demonstrates a dramatic improvement in antibacterial activity against P. aeruginosa. We anticipate that these discoveries will facilitate the design and development of high-permeating antipseudomonals.

Role of internal loop dynamics in antibiotic permeability of outer membrane porins.

Gram-negative bacteria pose a serious public health concern due to resistance to many antibiotics, caused by the low permeability of their outer membrane (OM). Effective antibiotics use porins in the OM to reach the interior of the cell; thus, understanding permeation properties of OM porins is instrumental to rationally develop broad-spectrum antibiotics. A functionally important feature of OM porins is undergoing open-closed transitions that modulate their transport properties. To characterize the molecular basis of these transitions, we performed an extensive set of molecular dynamics (MD) simulations of Escherichia coli OM porin OmpF. Markov-state analysis revealed that large-scale motion of an internal loop, L3, underlies the transition between energetically stable open and closed states. The conformation of L3 is controlled by H bonds between highly conserved acidic residues on the loop and basic residues on the OmpF ß-barrel. Mutation of key residues important for the loop's conformation shifts the equilibrium between open and closed states and regulates translocation of permeants (ions and antibiotics), as observed in the simulations and validated by our whole-cell accumulation assay. Notably, one mutant system G119D, which we find to favor the closed state, has been reported in clinically resistant bacterial strains. Overall, our accumulated ∼200 µs of simulation data (the wild type and mutants) along with experimental assays suggest the involvement of internal loop dynamics in permeability of OM porins and antibiotic resistance in Gram-negative bacteria.

Precise Readout of MEK1 Proteoforms upon MAPK Pathway Modulation by Individual Ion Mass Spectrometry.

The functions of proteins bearing multiple post-translational modifications (PTMs) are modulated by their modification patterns, yet precise characterization of them is difficult. MEK1 (also known as MAP2K1) is one such example that acts as a gatekeeper of the mitogen-activating protein kinase (MAPK) pathway and propagates signals via phosphorylation by upstream kinases. In principle, top-down mass spectrometry can precisely characterize whole MEK1 proteoforms, but fragmentation methods that would enable the site-specific characterization of labile modifications on 43 kDa protein ions result in overly dense tandem mass spectra. By using the charge-detection method called individual ion mass spectrometry, we demonstrate how complex mixtures of phosphoproteoforms and their fragment ions can be reproducibly handled to provide a "bird's eye" view of signaling activity through mapping proteoform landscapes in a pathway. Using this approach, the overall stoichiometry and distribution of 0-4 phosphorylations on MEK1 was determined in a cellular model of drug-resistant metastatic melanoma. This approach can be generalized to other multiply modified proteoforms, for which PTM combinations are key to their function and drug action.

Phase I study of procaspase-activating compound-1 (PAC-1) in the treatment of advanced malignancies.

BACKGROUND: Procaspase-3 (PC-3) is overexpressed in multiple tumour types and procaspase-activating compound 1 (PAC-1) directly activates PC-3 and induces apoptosis in cancer cells. This report describes the first-in-human, phase I study of PAC-1 assessing maximum tolerated dose, safety, and pharmacokinetics. METHODS: Modified-Fibonacci dose-escalation 3 + 3 design was used. PAC-1 was administered orally at 7 dose levels (DL) on days 1-21 of a 28-day cycle. Dose-limiting toxicity (DLT) was assessed during the first two cycles of therapy, and pharmacokinetics analysis was conducted on days 1 and 21 of the first cycle. Neurologic and neurocognitive function (NNCF) tests were performed throughout the study. RESULTS: Forty-eight patients were enrolled with 33 completing ≥2 cycles of therapy and evaluable for DLT. DL 7 (750 mg/day) was established as the recommended phase 2 dose, with grade 1 and 2 neurological adverse events noted, while NNCF testing showed stable neurologic and cognitive evaluations. PAC-1's t1/2 was 28.5 h after multi-dosing, and systemic drug exposures achieved predicted therapeutic concentrations. PAC-1 clinical activity was observed in patients with neuroendocrine tumour (NET) with 2/5 patients achieving durable partial response. CONCLUSIONS: PAC-1 dose at 750 mg/day was recommended for phase 2 studies. Activity of PAC-1 in treatment-refractory NET warrants further investigation. CLINICAL TRIAL REGISTRATION: Clinical Trials.gov: NCT02355535.

Predictive compound accumulation rules yield a broad-spectrum antibiotic.

Most small molecules are unable to rapidly traverse the outer membrane of Gram-negative bacteria and accumulate inside these cells, making the discovery of much-needed drugs against these pathogens challenging. Current understanding of the physicochemical properties that dictate small-molecule accumulation in Gram-negative bacteria is largely based on retrospective analyses of antibacterial agents, which suggest that polarity and molecular weight are key factors. Here we assess the ability of over 180 diverse compounds to accumulate in Escherichia coli. Computational analysis of the results reveals major differences from the retrospective studies, namely that the small molecules that are most likely to accumulate contain an amine, are amphiphilic and rigid, and have low globularity. These guidelines were then applied to convert deoxynybomycin, a natural product that is active only against Gram-positive organisms, into an antibiotic with activity against a diverse panel of multi-drug-resistant Gram-negative pathogens. We anticipate that these findings will aid in the discovery and development of antibiotics against Gram-negative bacteria.

Target-Agnostic P-Glycoprotein Assessment Yields Strategies to Evade Efflux, Leading to a BRAF Inhibitor with Intracranial Efficacy.

The blood-brain barrier (BBB) presents a major hurdle in the development of central nervous system (CNS) active therapeutics, and expression of the P-glycoprotein (P-gp) efflux transporter at the blood-brain interface further impedes BBB penetrance of most small molecules. Designing efflux liabilities out of compounds can be laborious, and there is currently no generalizable approach to directly transform periphery-limited agents to ones active in the CNS. Here, we describe a target-agnostic, prospective assessment of P-gp efflux using diverse compounds. Our results demonstrate that reducing the molecular size or appending a carboxylic acid in many cases enables evasion of P-gp efflux in cell-based experiments and in mice. These strategies were then applied to transform a periphery-limited V600EBRAF inhibitor, dabrafenib, into versions that possess potent and selective anti-cancer activity but now also evade P-gp-mediated efflux. When compared to dabrafenib, the compound developed herein (everafenib) has superior BBB penetrance and superior efficacy in an intracranial mouse model of metastatic melanoma, suggesting it as a lead candidate for the treatment of melanoma metastases to the brain and gliomas with BRAF mutation. More generally, the results described herein suggest the actionability of the trends observed in these target-agnostic efflux studies and provide guidance for the conversion of non-BBB-penetrant drugs into versions that are BBB-penetrant and efficacious.

Facilitating Compound Entry as a Means to Discover Antibiotics for Gram-Negative Bacteria.

It has been over half a century since the last class of antibiotics active against the most problematic Gram-negative bacteria was approved by the Food and Drug Administration (FDA). The major challenge with developing antibiotics to treat these infections is not drug-target engagement but rather the inability of most small molecules to traverse the Gram-negative membranes, be retained, and accumulate within the cell. Despite an abundance of lead compounds, limited understanding of the physicochemical properties needed for compound accumulation (or avoidance of efflux) in Gram-negative bacteria has precluded a generalizable approach for developing Gram-negative antibiotics. Indeed, in many instances, despite years of intensive derivatization efforts and the synthesis of hundreds of compounds aimed at building in Gram-negative activity, little or no progress has been made in expanding the spectrum of activity for many Gram-positive-only antibiotics. In this Account, we describe the discovery and successful applications of a promising strategy for enhancing the accumulation of Gram-positive-only antibiotics as a means of imbuing compounds with broad-spectrum activity.Utilizing a prospective approach examining the accumulation in Escherichia coli for more than 180 diverse compounds, we found that small molecules have an increased likelihood to accumulate in E. coli when they contain an ionizable Nitrogen, have low Three-dimensionality, and are Rigid. Implementing these guidelines, codified as the "eNTRy rules" and assisted by web application www.entry-way.org, we have facilitated compound entry and systematically built Gram-negative activity into Gram-positive-only antibiotics. Though each antibiotic will have case-specific considerations, we describe a set of important criteria to consider when selecting candidate Gram-positive-only antibiotics for conversion to Gram-negative-active versions via the eNTRy rules. As detailed herein, using this blueprint the spectrum of activity was expanded for three antibiotic classes that engage three different biological targets: DNA gyrase inhibitor 6DNM, FabI inhibitor Debio-1452, and FMN riboswitch inhibitor Ribocil C. In each scenario, the eNTRy rules guided the synthesis of key analogues predisposed to accumulate in Gram-negative bacteria leading to compounds that display antibiotic activity (minimum inhibitory concentrations (MIC) ≤8 µg mL-1) against E. coli and other Gram-negative ESKAPE pathogens. While the eNTRy rules will continue to be refined and enhanced as more accumulation data is gathered, on the basis of these collective results and on other examples not covered herein it is clear that the eNTRy rules are actionable for the development of novel broad-spectrum antibiotics from Gram-positive-only compounds. By enabling the prediction of compound accumulation, the eNTRy rules should facilitate the process of discovering and developing novel antibiotics active against Gram-negative bacteria.

Synthetic Studies on Selective, Proapoptotic Isomalabaricane Triterpenoids Aided by Computational Techniques.

The isomalabaricanes comprise a large family of marine triterpenoids with fascinating structures that have been shown to be selective and potent apoptosis inducers in certain cancer cell lines. In this article, we describe the successful total syntheses of the isomalabaricanes stelletin A, stelletin E, and rhabdastrellic acid A, as well as the development of a general strategy to access other natural products within this unique family. High-throughput experimentation and computational chemistry methods were used in this endeavor. A preliminary structure-activity relationship study of stelletin A revealed the trans-syn-trans core motif of the isomalabaricanes to be critical for their cytotoxic activity.

Limonin as a Starting Point for the Construction of Compounds with High Scaffold Diversity.

Structurally complex natural products have been a fruitful source for the discovery and development of new drugs. In an effort to construct a compound collection populated by architecturally complex members with unique scaffolds, we have used the natural product limonin as a starting point. Limonin is an abundant triterpenoid natural product and, through alteration of its heptacyclic core ring system using short synthetic sequences, a collection of 98 compounds was created, including multiple members with novel ring systems. The reactions leveraged in the construction of these compounds include novel ring cleavage, rearrangements, and cyclizations, and this work is highlighted by the discovery of a novel B-ring cleavage reaction, a unique B/C-ring rearrangement, an atypical D-ring cyclization, among others. Computational analysis shows that 52 different scaffolds/ring systems were produced during the course of this work, of which 36 are unprecedented. Phenotypic screening and structure-activity relationships identified compounds with activity against a panel of cancer cell lines.

Gram-Negative Antibiotic Active Through Inhibition of an Essential Riboswitch.

Multidrug-resistant Gram-negative (GN) infections for which there are few available treatment options are increasingly common. The development of new antibiotics for these pathogens is challenging because of the inability of most small molecules to accumulate inside GN bacteria. Using recently developed predictive guidelines for compound accumulation in Escherichia coli, we have converted the antibiotic Ribocil C, which targets the flavin mononucleotide (FMN) riboswitch, from a compound lacking whole-cell activity against wild-type GN pathogens into a compound that accumulates to a high level in E. coli, is effective against Gram-negative clinical isolates, and has efficacy in mouse models of GN infections. This compound allows for the first assessment of the translational potential of FMN riboswitch binders against wild-type Gram-negative bacteria.

Re-engineering natural products to engage new biological targets.

Covering: up to 2020 Natural products have a long history in drug discovery, with their inherent biological activity often tailored by medicinal chemists to arrive at the final drug product. This process is illustrated by numerous examples, including the conversion of epothilone to ixabepilone, erythromycin to azithromycin, and lovastatin to simvastatin. However, natural products are also fruitful starting points for the creation of complex and diverse compounds, especially those that are markedly different from the parent natural product and accordingly do not retain the biological activity of the parent. The resulting products have physiochemical properties that differ considerably when compared to traditional screening collections, thus affording an opportunity to discover novel biological activity. The synthesis of new structural frameworks from natural products thus yields value-added compounds, as demonstrated in the last several years with multiple biological discoveries emerging from these collections. This Highlight details a handful of these studies, describing new compounds derived from natural products that have biological activity and cellular targets different from those evoked/engaged by the parent. Such re-engineering of natural products offers the potential for discovering compounds with interesting and unexpected biological activity.

How many human proteoforms are there?

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

Imidazotetrazines as Weighable Diazomethane Surrogates for Esterifications and Cyclopropanations.

Diazomethane is one of the most versatile reagents in organic synthesis, but its utility is limited by its hazardous nature. Although alternative methods exist to perform the unique chemistry of diazomethane, these suffer from diminished reactivity and/or correspondingly harsher conditions. Herein, we describe the repurposing of imidazotetrazines (such as temozolomide, TMZ, the standard of care for glioblastoma) for use as synthetic precursors of alkyl diazonium reagents. TMZ was employed to conduct esterifications and metal-catalyzed cyclopropanations, and results show that methyl ester formation from a wide variety of substrates is especially efficient and operationally simple. TMZ is a commercially available solid that is non-explosive and non-toxic, and should find broad utility as a replacement for diazomethane.

Enantioselective Synthesis of Isocarbostyril Alkaloids and Analogs Using Catalytic Dearomative Functionalization of Benzene.

Enantioselective total syntheses of the anticancer isocarbostyril alkaloids (+)-7-deoxypancratistatin, (+)-pancratistatin, (+)-lycoricidine, and (+)-narciclasine are described. Our strategy for accessing this unique class of natural products is based on the development of a Ni-catalyzed dearomative trans-1,2-carboamination of benzene. The effectiveness of this dearomatization approach is notable, as only two additional olefin functionalizations are needed to construct the fully decorated aminocyclitol cores of these alkaloids. Installation of the lactam ring has been achieved through several pathways and a direct interconversion between natural products was established via a late-stage C-7 cupration. Using this synthetic blueprint, we were able to produce natural products on a gram scale and provide tailored analogs with improved activity, solubility, and metabolic stability.

Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program.

Tumors have aberrant proteomes that often do not match their corresponding transcriptome profiles. One possible cause of this discrepancy is the existence of aberrant RNA modification landscapes in the so-called epitranscriptome. Here, we report that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate RNA methyltransferase for 5-methylcytosine. In this setting, NSUN5 exhibits tumor-suppressor characteristics in vivo glioma models. We also found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA that drives an overall depletion of protein synthesis, and leads to the emergence of an adaptive translational program for survival under conditions of cellular stress. Interestingly, NSUN5 epigenetic inactivation also renders these gliomas sensitive to bioactivatable substrates of the stress-related enzyme NQO1. Most importantly, NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival for this otherwise devastating disease.

Pharmacokinetics and derivation of an anticancer dosing regimen for the novel anti-cancer agent isobutyl-deoxynyboquinone (IB-DNQ), a NQO1 bioactivatable molecule, in the domestic felid species.

Isobutyl-deoxynyboquinone (IB-DNQ) is a selective substrate for NAD(P)H:quinone oxidoreductase (NQO1), an enzyme overexpressed in many solid tumors. Following activation by NQO1, IB-DNQ participates in a catalytic futile reduction/reoxidation cycle with consequent toxic reactive oxygen species generation within the tumor microenvironment. To elucidate the potential of IB-DNQ to serve as a novel anticancer agent, in vitro studies coupled with in vivo pharmacokinetic and toxicologic investigations in the domestic felid species were conducted to investigate the tractability of IB-DNQ as a translationally applicable anticancer agent. First, using feline oral squamous cell carcinoma (OSCC) as a comparative cancer model, expressions of NQO1 were characterized in not only human, but also feline OSCC tissue microarrays. Second, IB-DNQ mediated cytotoxicity in three immortalized feline OSCC cell lines were studied under dose-dependent and sequential exposure conditions. Third, the feasibility of administering IB-DNQ at doses predicted to achieve cytotoxic plasma concentrations and biologically relevant durations of exposure were investigated through pharmacokinetic and tolerability studies in healthy research felines. Intravenous administration of IB-DNQ at 1.0-2.0 mg/kg achieved peak plasma concentrations and durations of exposure reaching or exceeding predicted in vitro cytotoxic concentrations. Clinical adverse side effects including ptyalism and tachypnea exhibited during and post-IV infusion of IB-DNQ were transient and tolerable. Additionally, IB-DNQ administration did not produce acute or delayed-onset unacceptable hematologic, non-hematologic, or off-target oxidative toxicities. Collectively, the findings reported here within provide important safety and pharmacokinetic data to support the continued development of IB-DNQ as a novel anticancer strategy for NQO1 expressing cancers.

Deoxynyboquinones as NQO1-Activated Cancer Therapeutics.

One of the major goals of cancer therapy is the selective targeting of cancer cells over normal cells. Unfortunately, even with recent advances, the majority of chemotherapeutics still indiscriminately kill all rapidly dividing cells. Although these drugs are effective in certain settings, their inability to specifically target cancer results in significant dose-limiting toxicities. One way to avoid such toxicities is to target an aspect of the cancer cell that is not shared by normal cells. A potential cancer-specific target is the enzyme NAD(P)H quinone oxidoreductase 1 (NQO1). NQO1 is a 2-electron reductase responsible for the detoxification of quinones. Its expression is typically quite low in normal tissue, but it has been found to be greatly overexpressed in many types of solid tumors, including lung, breast, pancreatic, and colon cancers. This overexpression is thought to be in response to the higher oxidative stress of the cancer cell, and it is possible that NQO1 contributes to tumor progression. The overexpression of NQO1 and its correlation with poor patient outcome make it an intriguing target. Although some have explored inhibiting NQO1 as an anticancer strategy, this has generally been unsuccessful. A more promising strategy is to utilize NQO1 substrates that are activated upon reduction by NQO1. For example, in principle, reduction of a quinone can result in a hydroquinone that is a DNA alkylator, protein inhibitor, or reduction-oxidation cycler. Although there are many proposed NQO1 substrates, head-to-head assays reveal only two classes of compounds that convincingly induce cancer cell death through NQO1-mediated activation. In this Account, we describe the discovery and development of one of these compounds, the natural product deoxynyboquinone (DNQ), an excellent NQO1 substrate and anticancer agent. A modular synthesis of DNQ was developed that enabled access to the large compound quantities needed to conduct extensive mechanistic evaluations and animal experiments. During these evaluations, we found that DNQ is an outstanding NQO1 substrate that is processed much more efficiently than other putative NQO1 substrates. Importantly, its anticancer activity is strictly dependent on the overexpression of active NQO1. Using previous crystal structures of NQO1, novel DNQ derivatives were designed that are also excellent NQO1 substrates and possess properties that make them more attractive than the parent natural product for translational development. Given their selectivity, potency, outstanding pharmacokinetic properties, and the ready availability of diagnostics to assess NQO1 in patients, DNQ and its derivatives have considerable potential as personalized medicines for the treatment of cancer.