Speciation in Western North America: Lomatium as an Example of Diversification and Convergent Evolution

Species delimitations and understanding the processes that drive speciation are essential to nearly all aspects of human endeavor. Determining species boundaries traditionally used morphology. Phylogenetic analyses based on DNA sequence data provide a means to resolve species boundaries, as well as test hypotheses regarding the evolutionary processes. Numerous species radiations have occurred in Western North America. Among these are several plant groups such as Astragalus, Artemisia, and Lomatium. Recent phylogenetic analyses of Lomatium and related genera have demonstrated that many of the morphological characters used to delimit taxa have arisen multiple times and that most taxa are para- or polyphyletic. Here we examine one of the clades recovered in the Lomatium group of taxa that includes Lomatium triternatum and L. grayi. The several subspecific taxa of L. triternatum have not been recovered as monophyletic and L. grayi has a widespread habitat distribution that may indicate cryptic speciation. Previous analyses have not fully resolved phylogenetic relationships with strong support. In the present study we sample an additional four loci (three chloroplast and one nuclear ribosomal) to improve the support for evolutionary relationships across this clade, resolve species boundaries, and test hypotheses on the evolution of morphological traits

Hydroxybiphenylamide GroEL/ES Inhibitors Are Potent Antibacterials against Planktonic and Biofilm Forms of Staphylococcus aureus

We recently reported the identification of a GroEL/ES inhibitor (1, N-(4-(benzo[d]thiazol-2-ylthio)-3-chlorophenyl)-3,5-dibromo-2-hydroxybenzamide) that exhibited in vitro antibacterial effects against Staphylococcus aureus comparable to vancomycin, an antibiotic of last resort. To follow up, we have synthesized 43 compound 1 analogs to determine the most effective functional groups of the scaffold for inhibiting GroEL/ES and killing bacteria. Our results identified that the benzothiazole and hydroxyl groups are important for inhibiting GroEL/ES-mediated folding functions, with the hydroxyl essential for antibacterial effects. Several analogs exhibited >50-fold selectivity indices between antibacterial efficacy and cytotoxicity to human liver and kidney cells in cell culture. We found that MRSA was not able to easily generate acute resistance to lead inhibitors in a gain-of-resistance assay and that lead inhibitors were able to permeate through established S. aureus biofilms and maintain their bactericidal effects

HSP60/10 chaperonin systems are inhibited by a variety of approved drugs, natural products, and known bioactive molecules

All living organisms contain a unique class of molecular chaperones called 60 kDa heat shock proteins (HSP60 - also known as GroEL in bacteria). While some organisms contain more than one HSP60 or GroEL isoform, at least one isoform has always proven to be essential. Because of this, we have been investigating targeting HSP60 and GroEL chaperonin systems as an antibiotic strategy. Our initial studies focused on applying this antibiotic strategy for treating African sleeping sickness (caused by Trypanosoma brucei parasites) and drug-resistant bacterial infections (in particular Methicillin-resistant Staphylococcus aureus - MRSA). Intriguingly, during our studies we found that three known antibiotics - suramin, closantel, and rafoxanide - were potent inhibitors of bacterial GroEL and human HSP60 chaperonin systems. These findings prompted us to explore what other approved drugs, natural products, and known bioactive molecules might also inhibit HSP60 and GroEL chaperonin systems. Initial high-throughput screening of 3680 approved drugs, natural products, and known bioactives identified 161 hit inhibitors of the Escherichia coli GroEL chaperonin system (4.3% hit rate). From a purchased subset of 60 hits, 29 compounds (48%) re-confirmed as selective GroEL inhibitors in our assays, all of which were nearly equipotent against human HSP60. These findings illuminate the notion that targeting chaperonin systems might be a more common occurrence than we previously appreciated. Future studies are needed to determine if the in vivo modes of action of these approved drugs, natural products, and known bioactive molecules are related to GroEL and HSP60 inhibition

Analogs of nitrofuran antibiotics are potent GroEL/ES inhibitor pro-drugs

In two previous studies, we identified compound 1 as a moderate GroEL/ES inhibitor with weak to moderate antibacterial activity against Gram-positive and Gram-negative bacteria including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, and SM101 Escherichia coli (which has a compromised lipopolysaccharide biosynthetic pathway making bacteria more permeable to drugs). Extending from those studies, we developed two series of analogs with key substructures resembling those of known antibacterials, nitroxoline (hydroxyquinoline moiety) and nifuroxazide/nitrofurantoin (bis-cyclic-N-acylhydrazone scaffolds). Through biochemical and cell-based assays, we identified potent GroEL/ES inhibitors that selectively blocked E. faecium, S. aureus, and E. coli proliferation with low cytotoxicity to human colon and intestine cells in vitro. Initially, only the hydroxyquinoline-bearing analogs were found to be potent inhibitors in our GroEL/ES-mediated substrate refolding assays; however, subsequent testing in the presence of an E. coli nitroreductase (NfsB) in situ indicated that metabolites of the nitrofuran-bearing analogs were potent GroEL/ES inhibitor pro-drugs. Consequently, this study has identified a new target of nitrofuran-containing drugs, and is the first reported instance of such a unique class of GroEL/ES chaperonin inhibitors. The intriguing results presented herein provide impetus for expanded studies to validate inhibitor mechanisms and optimize this antibacterial class using the respective GroEL/ES chaperonin systems and nitroreductases from E. coli and the ESKAPE bacteria

Dual-targeting GroEL/ES chaperonin and protein tyrosine phosphatase B (PtpB) inhibitors: A polypharmacology strategy for treating Mycobacterium tuberculosis infections

Current treatments for Mycobacterium tuberculosis infections require long and complicated regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains, and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting molecular chaperones such as GroEL/ES (HSP60/10) chaperonin systems. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB) virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis – actively replicating bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease – which would be a significant advance to augment current therapeutics that primarily target actively replicating bacteria

Resolving the Anomaly of \u3cem\u3eLomatium anomalum\u3c/em\u3e: Discovery of a New Species in Southwestern Idaho (U.S.A.), \u3cem\u3eLomatium andrusianum\u3c/em\u3e (Apiaceae)

Apparent polyphyly within the unresolved clade of Lomatium (Apiaceae) containing L. triternatum, L. anomalum, L. thompsonii, and L. packardiae suggests conflict among current taxonomic classification schemes. To recover this clade and more clearly define species boundaries, we examined populations of L. anomalum from three geographic regions in Idaho and adjacent Oregon. Using phylogenetic, morphological, and ecological data, we conclude that the L. anomalum complex currently circumscribes multiple species. Phylogenetic analysis of the nuclear ribosomal ITS and ETS, and cpDNA rpl32-trnLUAG, rps-16 intron, trnD-trnT, ndhA intron, and psbA-trnH recovered populations from the Boise foothills as a distinct, monophyletic clade. Principal Components Analysis of 30 reproductive and vegetative characters show two distinct groups: one of Boise foothills and one of the combined Mann Creek and Camas Prairie vicinities. Principal Components Analysis of 16 soil characteristics show that soils occupied by Boise foothills populations are distinct from those occupied by Mann Creek and Camas Prairie populations. Based on phylogenetic, morphometric, and ecologic criteria, populations of what had been considered L. anomalum from the Boise foothills and vicinity are here described as a new species—Lomatium andrusianum

Exploiting the HSP60/10 chaperonin system as a chemotherapeutic target for colorectal cancer

Over the past few decades, an increasing variety of molecular chaperones have been investigated for their role in tumorigenesis and as potential chemotherapeutic targets; however, the 60 kDa Heat Shock Protein (HSP60), along with its HSP10 co-chaperone, have received little attention in this regard. In the present study, we investigated two series of our previously developed inhibitors of the bacterial homolog of HSP60/10, called GroEL/ES, for their selective cytotoxicity to cancerous over non-cancerous colorectal cells. We further developed a third “hybrid” series of analogs to identify new candidates with superior properties than the two parent scaffolds. Using a series of well-established HSP60/10 biochemical screens and cell-viability assays, we identified 24 inhibitors (14%) that exhibited > 3-fold selectivity for targeting colorectal cancer over non-cancerous cells. Notably, cell viability EC50 results correlated with the relative expression of HSP60 in the mitochondria, suggesting a potential for this HSP60-targeting chemotherapeutic strategy as emerging evidence indicates that HSP60 is up-regulated in colorectal cancer tumors. Further examination of five lead candidates indicated their ability to inhibit the clonogenicity and migration of colorectal cancer cells. These promising results are the most thorough analysis and first reported instance of HSP60/10 inhibitors being able to selectively target colorectal cancer cells and highlight the potential of the HSP60/10 chaperonin system as a viable chemotherapeutic target.Ralph W. and Grace M. Showalter Research Trust Fund24 month embargo; available online 19 April 2021This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Sulfonamido 2 arylbenzoxazole GroEL/ES inhibitors are potent antibacterials against methicillin resistant Staphylococcus aureus (MRSA)

Extending from a study we recently published examining the antitrypanosomal effects of a series of GroEL/ES inhibitors based on a pseudosymmetrical bis-sulfonamido-2-phenylbenzoxazole scaffold, here, we report the antibiotic effects of asymmetric analogs of this scaffold against a panel of bacteria known as the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). While GroEL/ES inhibitors were largely ineffective against K. pneumoniae, A. baumannii, P. aeruginosa, and E. cloacae (Gram-negative bacteria), many analogs were potent inhibitors of E. faecium and S. aureus proliferation (Gram-positive bacteria, EC50 values of the most potent analogs were in the 1–2 μM range). Furthermore, even though some compounds inhibit human HSP60/10 biochemical functions in vitro (IC50 values in the 1–10 μM range), many of these exhibited moderate to low cytotoxicity to human liver and kidney cells (CC50 values > 20 μM)