The contributions of Paul Ehrlich to pharmacology: a tribute on the occasion of the centenary of his Nobel Prize

On the centenary of Paul Ehrlich's Nobel Prize, this German researcher deserves to be remembered as a pioneer in a large number of scientific disciplines. As a result of his enthusiasm and scientific abilities, dedication, and contacts with other scientists of his time, he was able to make countless contributions in fields as diverse as histology, haematology, immunology, oncology, microbiology and pharmacology, among others. Although the Swedish award was meant to recognize the standardization of the manufacture of antidiphtheria serum, it was the discovery of arsphenamine (Salvarsan) for the treatment of syphilis which won him wider international acclaim. From a pharmacological perspective, Ehrlich's outstanding contributions include dissemination of the 'magic bullet' concept for the synthesis of antibacterials, introduction of concepts such as chemoreceptor and chemotherapy, and linking the chemical structure of compounds to their pharmacological activity. These achievements took place within the framework he established for the transition from experimental pharmacology to therapeutic pharmacology. He introduced a modern research system based on the synthesis of multiple chemical structures for pharmacological screening in animal models of disease states. These contributions were undoubtedly decisive in propitiating the wider development of antibiotics decades later. For these reasons, it is fitting to mark the 100th anniversary of the Nobel Prize awarded to this great scientist by commemorating the importance of his contributions to the advance of pharmacology.     

Demonstration of the role of cell wall homeostasis in Staphylococcus aureus growth and the action of bactericidal antibiotics

Bacterial cell wall peptidoglycan is essential, maintaining both cellular integrity and morphology, in the face of internal turgor pressure. Peptidoglycan synthesis is important, as it is targeted by cell wall antibiotics, including methicillin and vancomycin. Here, we have used the major human pathogen Staphylococcus aureus to elucidate both the cell wall dynamic processes essential for growth (life) and the bactericidal effects of cell wall antibiotics (death) based on the principle of coordinated peptidoglycan synthesis and hydrolysis. The death of S. aureus due to depletion of the essential, two-component and positive regulatory system for peptidoglycan hydrolase activity (WalKR) is prevented by addition of otherwise bactericidal cell wall antibiotics, resulting in stasis. In contrast, cell wall antibiotics kill via the activity of peptidoglycan hydrolases in the absence of concomitant synthesis. Both methicillin and vancomycin treatment lead to the appearance of perforating holes throughout the cell wall due to peptidoglycan hydrolases. Methicillin alone also results in plasmolysis and misshapen septa with the involvement of the major peptidoglycan hydrolase Atl, a process that is inhibited by vancomycin. The bactericidal effect of vancomycin involves the peptidoglycan hydrolase SagB. In the presence of cell wall antibiotics, the inhibition of peptidoglycan hydrolase activity using the inhibitor complestatin results in reduced killing, while, conversely, the deregulation of hydrolase activity via loss of wall teichoic acids increases the death rate. For S. aureus, the independent regulation of cell wall synthesis and hydrolysis can lead to cell growth, death, or stasis, with implications for the development of new control regimes for this important pathogen.

How bacteria grow and divide is a fundamental question in microbiology, where many of the essential processes involved are the targets of clinically important antibiotics. The cell wall is crucial for bacterial survival, forming the interface between the external and internal environments and maintaining internal turgor pressure (1, 2). The major cell wall structural component is peptidoglycan (PG), a polymer of glycan strands and peptide cross-links (3–5), the synthesis of which is the target of antibiotics including β-lactams and glycopeptides (6). These cell wall antibiotics inhibit the final stages of PG synthesis where building blocks are incorporated into the existing structure via the action of penicillin-binding proteins (PBPs) (6). Several mechanisms linking the action of antibiotics to the inhibition of essential processes in cell wall growth and division have been suggested, including lytic and nonlytic death, oxidative stress, and futile PG synthesis (7–12).As a single macromolecule that surrounds the cell, PG can increase in surface area to permit growth and division while maintaining cellular integrity. It has been proposed that areal PG growth occurs as a consequence of both synthesis and hydrolysis (4, 13, 14), with new material being covalently bound to the existing macrostructure and hydrolysis of existing bonds allowing expansion. This leads to a simple set of hypotheses for growth but also makes predictions as to the effects of inhibition of PG homeostasis activities, including cell wall antibiotics (Fig. 1A). The lack of either PG synthesis or hydrolysis will result in cell death because of the continued activity of the other, but the loss of both will lead to stasis.Open in a separate windowFig. 1.The role of regulation of PG hydrolases (PGHs) by WalKR in life and death. (A) Predictive model for how cell wall homeostasis governs bacterial life and death. Both cell wall synthesis and hydrolysis are required for growth, loss of either results in death, or both, cell stasis. (B–H) Effect of 10 × minimum inhibitory concentration (MIC) vancomycin for 3 h on conditional lethal strain S. aureus P_spac-walKR (without inducer; WalKR OFF) compared to the control (with inducer; WalKR ON). (B) CFU relative to T = 0; after t test with Welch''s correction: P (WalKR OFF − WalKR OFF + vancomycin, **) = 6.9 × 10⁻³. (C and D) PG synthesis and transpeptidase activity measured by ¹⁴C-GlcNAc and Atto 488 dipeptide (53) incorporation, normalized against WalKR ON. (E) Transmission electron microscopy (TEM) (scale bars, 300 nm). (F) Quantification of bacterial phenotypes (SI Appendix, Fig. S2; dark green: no septum, mid-green: incomplete septum, light green: complete septum, and yellow: growth defects). For samples shown, the number of individual cells quantified was n > 300. (G) AFM topographic images of sacculi (scale bars, 150, 300, and 300 nm; data scales [DS], 85, 200, and 85 nm, respectively, from Left to Right). (i) Insets show sacculus external architecture from Left to Right, (WalKR ON) from dashed box in panel G, (WalKR OFF) from SI Appendix, Fig. S2E, (WalKR OFF+Van) from SI Appendix, Fig. S2D, respectively (scale bars, 50 nm; DS, 30, 52, and 32 nm, respectively, from Left to Right; images were analyzed with NanoscopeAnalysis from Bruker using the default color scale). (H) Thickness distribution values for sacculi with SD (n = 5). For sample size and data reproducibility, see Materials and Methods.Staphylococcus aureus is a major human antimicrobial-resistant pathogen. As a spheroid cell with a simple growth and division cycle, it forms an excellent subject to demonstrate the basic principles underlying growth, division, and the action of antibiotics. Many organisms have multiple PBPs, but S. aureus has only four, of which PBP1 and PBP2 are essential for growth and division (15–19). S. aureus also has many PG hydrolases (PGHs), including SagB, which is involved in cell growth (20, 21). The bifunctional Atl is involved in generalized cell lysis and cell separation after septation and contains both amidase and glucosaminidase domains (22, 23). PGHs often show functional redundancy with several enzymes involved in the same process (20, 24). In S. aureus, no individual PGH alone has been shown to be required for either growth or division, but multiple PGHs are positively regulated by an essential two-component system, WalKR (25–27), further suggesting that their collective activity is required.Recently, using atomic force microscopy (AFM), we have revealed that the molecular architecture of the PG is that of an expanded hydrogel whose mature external surface is a porous open network but with an interior surface characterized by a much smoother and denser mesh of PG material (28). This provides an architectural framework from which to begin to elucidate the roles of PG synthesis and hydrolysis. Here, we have taken an integrated approach to determine the role of PG homeostasis in S. aureus growth, division, and the bactericidal action of cell wall antibiotics.     

A serum-stable RNA aptamer specific for SARS-CoV-2 neutralizes viral entry

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created an urgent need for new technologies to treat COVID-19. Here we report a 2′-fluoro protected RNA aptamer that binds with high affinity to the receptor binding domain (RBD) of SARS-CoV-2 spike protein, thereby preventing its interaction with the host receptor ACE2. A trimerized version of the RNA aptamer matching the three RBDs in each spike complex enhances binding affinity down to the low picomolar range. Binding mode and specificity for the aptamer–spike interaction is supported by biolayer interferometry, single-molecule fluorescence microscopy, and flow-induced dispersion analysis in vitro. Cell culture experiments using virus-like particles and live SARS-CoV-2 show that the aptamer and, to a larger extent, the trimeric aptamer can efficiently block viral infection at low concentration. Finally, the aptamer maintains its high binding affinity to spike from other circulating SARS-CoV-2 strains, suggesting that it could find widespread use for the detection and treatment of SARS-CoV-2 and emerging variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in 2020–2021 has launched a global quest to find new molecular tools for the detection of the virus and treatment of the potential deadly disease it causes, COVID-19. Despite the exceptional efforts worldwide for containment and unprecedented technological progress in vaccine development, the challenge to find an effective cure remains, due to the limited access to SARS-CoV-2 vaccines, particularly in developing countries, and the emergence of new viral strains that can evade immune responses and potentially compromise the efficacy of current vaccines. Therefore, it is of utmost importance to focus efforts on developing affordable and easy-to-produce antiviral molecules against SARS-CoV-2 infection.Like other coronaviruses, SARS-CoV-2 expresses a surface spike (S) glycoprotein which is composed of two domains (S1 and S2) (1) and forms a trimeric structure capable of interacting with human cells (2, 3). In particular, the receptor binding domain (RBD) located on the S1 subunit of the spike protein binds with high affinity to human angiotensin-converting enzyme 2 (ACE2) (4, 5), which, in conjunction with the associated transmembrane protease, serine 2 (TMPRSS2), facilitates viral uptake. Efforts to neutralize viral infection have therefore mainly focused on inhibiting the spike–ACE2 interaction. Antibodies (Abs) have been developed and are currently used for SARS-CoV-2 detection, and some, primarily those targeting RBD, show therapeutic potential due to their potent neutralizing effect (6–13). However, the high costs of Ab production, the use of animals to generate them, and their poor stability at ambient temperatures remain a disadvantage. Moreover, Ab immunogenicity and the risk of Ab-dependent enhancement of infection associated with Fc-containing Abs put their therapeutic potential at risk (14).V_HH Abs or nanobodies raised to the spike protein may overcome some of these drawbacks (15–20) but are more prone to immunological response (21, 22). Interesting alternatives such as de novo proteins based on the host ACE2 receptor (23) and other synthetic molecules (24) have been investigated and may, if potential immunogenicity and stability problems are solved, help develop efficient detection methods and drugs.Nucleic acid-based aptamers have gained increased attention as alternatives to Abs due to their ease of production, low immunogenicity, high thermal and chemical stability, and smaller size, while they still retain comparable target binding and specificity. Aptamers are short single-stranded oligonucleotides, developed through an in vitro selection process termed SELEX (systematic evolution of ligands by exponential enrichment), that bind with high affinity and selectivity to cognate targets (25–27). During the last few decades, a wide variety of aptamers binding to diverse biologically relevant targets (28), including viruses (29, 30), have been identified. However, selection of aptamers targeting spike protein has proven difficult. An explanation for this may be that highly glycosylated proteins such as SARS-CoV-2 spike are challenging to target with nucleic acid-based binders. Indeed, to date, there are only a few reports on DNA aptamers targeting SARS-CoV-2 spike where the authors report leading aptamers with affinities in the nanomolar range (31–35).Here we report the selection and characterization of a serum-stable RNA aptamer, RBD-PB6, that binds with nanomolar affinity to the RBD of SARS-CoV-2 spike protein and neutralizes viral infectivity. The aptamer contains 2′-fluoro pyrimidine modifications to increase its chemical stability and resistance to nucleases (36, 37), and it shows high selectivity to SARS-CoV-2 and related strains, including alpha and beta. Aptamer multimerization strongly enhances its affinity to the picomolar range as well as its SARS-CoV-2 neutralizing potency. These unique features open avenues for developing inexpensive, fast, and reliable detection platforms for SARS-CoV-2 and therapeutic application for COVID-19.     

Real‐time assessment of 13C metabolism reveals an early lactate increase in the brain of rats with acute liver failure

Intracranial hypertension is a severe complication of acute liver failure (ALF) secondary to brain edema. The pathogenesis of cerebral edema in ALF is not clear, but seems to be related to energy metabolism in which lactate may have an important role. The aim of this study was to follow the synthesis of brain lactate using a novel in vivo metabolic technology in a rat model of ALF. Time‐resolved ¹³C MRS of hyperpolarized ¹³C₁‐pyruvate was used to quantitatively follow the in vivo conversion of pyruvate to its substrates in a model of devascularized ALF in rats. Rats with ALF showed a significant increase in the lactate to pyruvate ratio from 36% to 69% during the progression of liver disease relative to rats with portocaval anastomosis. Rats with ALF also showed a significant increase in the alanine to pyruvate ratio from 72% to 95%. These increases were detectable at very early stages (6 h) when animals had no evident disease signs in their behavior (without loss of righting or corneal reflexes). This study shows the dynamic consequences of cerebral in vivo ¹³C metabolism at real time in rats with ALF. The early detection of the de novo synthesis of lactate suggests that brain lactate is involved in the physiopathology of ALF. Hyperpolarization is a potential non‐invasive technique to follow the in vivo metabolism, and both the development and optimization of ¹³C‐labeled substrates can clarify the mechanism involved in ALF. Copyright © 2014 John Wiley & Sons, Ltd.     

Outcome prognostic factors in inoperable malignant bowel obstruction

Purpose

Inoperable malignant bowel obstruction (MBO), a severe complication of peritoneal carcinomatosis, has a low desobstruction rate (30–40 %) and end-of-life decision-making is hampered by the lack of known prognostic factors. This study aimed to explore prognostic factors for desobstruction in MBO.

Methods

All patients with inoperable MBO admitted in our large oncology hospital between 2010 and 2013 were treated following a clinical protocol based on antiemetics, steroids and two antisecretories, octreotide, and hyoscine butylbromide. Two prognostic factor analyses using logistic regressions were performed, one based on data from day 1 of admission and the other on data from day 8.

Results

Forty-five patients were included. Frequency of desobstruction was 48.9 %. In the analysis of prognostic factors on day 1, MBO episodes derived from functional physiopathologic mechanisms (vs. mechanic or mixed) were more prone to resolve (p < 0.001 corrected for multiple comparisons). Considering patients alive with persistent obstruction on day 8, a better clinical condition was the variable more associated with desobstruction, but without statistical significance after correction for multiple comparisons.

Conclusions

A functional physiopathologic mechanism of MBO development may be an early prognostic factor for desobstruction. A high proportion of desobstruction was observed, suggesting that the combination of antisecretories with different mechanism of action warrants further investigation.