The influence of natural products upon drug discovery.

Azithromycin, a novel azalide antibiotic, concentrated in human and mouse polymorphonuclear leukocytes (PMNs), murine peritoneal macrophages, and mouse and rat alveolar macrophages, attaining intracellular concentrations up to 226 times the external concentration in vitro. In murine peritoneal macrophages, azithromycin achieved concentration gradients (internal to external) up to 26 times higher than erythromycin. The cellular uptake of azithromycin was dependent on temperature, viability, and pH and was decreased by 2,4-dinitrophenol. Azithromycin did not decrease phagocyte-mediated bactericidal activity or affect PMN or macrophage oxidative burst activity (H2O2 release or Nitro Blue Tetrazolium reduction, respectively). Azithromycin remained in cells for several hours, even after extracellular drug was removed. However, its release was significantly enhanced by phagocytosis of Staphylococcus aureus (82 versus 23% by 1.5 h). In vivo, 0.05 micrograms of azithromycin was found in peritoneal fluids of mice 20 h after oral treatment with a dose of 50 mg/kg. Following caseinate-induced PMN infiltration, there was a sixfold increase in peritoneal cavity azithromycin to 0.32 micrograms, most of which was intracellular. Therefore, the uptake, transport, and later release of azithromycin by these cells demonstrate that phagocytes may deliver active drug to sites of infection.

https://aac.asm.org/content/aac/33/3/277.full.pdf

In vitro and in vivo uptake of azithromycin (CP-62,993) by phagocytic cells: possible mechanism of delivery and release at sites of infection.

Recently, it was shown that a significant number of erythromycin-resistant Streptococcus pneumoniae and Streptococcus pyogenes strains contain a determinant that mediates resistance via a putative efflux pump. The gene encoding the erythromycin-resistant determinant was cloned and sequenced from three strains of S. pneumoniae bearing the M phenotype (macrolide resistant but clindamycin and streptogramin B susceptible). The DNA sequences of mefE were nearly identical, with only 2-nucleotide differences between genes from any two strains. When the mefE sequences were compared to the mefA sequence from S. pyogenes, the two genes were found to be closely related (90% identity). Strains of S. pneumoniae were constructed to confirm that mefE is necessary to confer erythromycin resistance and to explore the substrate specificity of the pump; no substrates other than 14- and 15-membered macrolides were identified.

mefE Is Necessary for the Erythromycin-Resistant M Phenotype in Streptococcus pneumoniae

New directions in antibacterial research.

The chemical modification of structurally complex fermentation products, a process known as semisynthesis, has been an important tool in the discovery and manufacture of antibiotics for the treatment of various infectious diseases. However, many of the therapeutics obtained in this way are no longer effective, because bacterial resistance to these compounds has developed. Here we present a practical, fully synthetic route to macrolide antibiotics by the convergent assembly of simple chemical building blocks, enabling the synthesis of diverse structures not accessible by traditional semisynthetic approaches. More than 300 new macrolide antibiotic candidates, as well as the clinical candidate solithromycin, have been synthesized using our convergent approach. Evaluation of these compounds against a panel of pathogenic bacteria revealed that the majority of these structures had antibiotic activity, some efficacious against strains resistant to macrolides in current use. The chemistry we describe here provides a platform for the discovery of new macrolide antibiotics and may also serve as the basis for their manufacture.

/pdf/a-platform-for-the-discovery-of-new-macrolide-antibiotics-381hnma6jd.pdf

A platform for the discovery of new macrolide antibiotics

A series of erythromycin A-derived semisynthetic antibiotics, featuring incorporation of a basic nitrogen atom into a ring expanded (15-membered) macrocyclic lactone, have been prepared and biologically evaluated. Semisynthetic modifications focused upon (1) varied substitution at the macrocyclic ring nitrogen and (2) epimerization or amine substitution at the C-4" hydroxyl site within the cladinose sugar. In general, the new azalides exhibit improved Gram-negative potency, expanding the spectrum of erythromycin A to fully include Haemophilus influenzae and Neisseria gonorrhoeae. When compared to erythromycin A, the azalides exhibit substantially increased half-life and area-under-the-curve values in all species studied. The overall in vitro/in vitro performance of N-methyl, C-4" epimers 3a and 9; and C-4" amine 11 identify these compounds as the most interesting erythromycin A-superior agents. Compound 3a has been advanced to clinical study.

Synthesis, in vitro and in vivo activity of novel 9-deoxo-9a-AZA-9a-homoerythromycin A derivatives; a new class of macrolide antibiotics, the azalides.


 Background: TP317 is a novel, highly stable chelate salt of resolvin E1 (RvE1), a pro-resolution mediator that stimulates myeloid cell phagocytosis of tumor debris and attenuates pro-tumoral inflammation through activation of the GPCR, ChemR23 (Sulciner et al., 2018, J. Exp. Med). We hypothesized that RvE1 shifts the immunosuppressive tumor microenvironment (TME) to an immunogenic state, thus offering combination potential with immune checkpoint inhibitors (ICI) in ICI-resistant and ICI-sensitive tumors.
 Methods: Subcutaneous murine models of lung (LLC), melanoma (B16F10), and pancreatic (Panc02; KPC) tumors were used to investigate TP-317 monotherapy and combinations with ICI. Treatment was initiated when tumors reached 125-240 mm3.
 Results: In the LLC model (N=5/group), TP317 (0.75 µg QD) inhibited tumor growth (1419 ± 266 mm3) compared to placebo (2348 ± 542 mm3; p=0.068) and anti-PD1 (200 µg IP, Q3D; 3015 ± 730; p=0.078). TP317 + anti-PD1 dual therapy was superior to placebo and anti-PD1 alone (893 ± 166 mm3; p<0.05). In the B16F10 model (N=8/group), TP317 (7.5 µg Q6D) was efficacious compared to placebo (787 ± 367 vs 1964 ± 208 mm3; p<0.01) and comparable to the ICI dual therapy of anti-PD1 + anti-CTLA4 (100 µg 1st dose, 200 µg IP Q3D up to 4 doses). Triple therapy with TP317 + anti-PD1 + anti-CTLA4 was superior to ICI dual therapy (340 ± 91 vs 757 ± 260 mm3; p<0.05). In the Panc02 model (N=8/group), TP317 (0.75 µg Q7D) was efficacious compared to placebo (1148 ± 178 vs 1992 ±165 mm3; p<0.001) and comparable to anti-PD1. TP317 + anti-PD1 dual therapy demonstrated significant anti-tumor activity compared to anti-PD1 alone (329 ± 72 vs 1446 ± 305 mm3; p<0.01). In KRAS-mutant KPC tumors (N=10/group), TP317 (7.5 µg Q6D) demonstrated significant anti-tumor activity compared to placebo (500 ± 110 vs 1314 ± 106 mm3; p<0.001) and was comparable to anti-PD1, while TP-317 + anti-PD1 was superior to anti-PD1 alone (353 ± 82 vs 710 ± 106 mm3; p<0.01). In Panc02 and KPC models, TP317’s anti-tumor efficacy was attenuated by CD8+ T cell or NK cell depletion. Consistent with the depletion study results, RNAseq analysis with cell-type deconvolution showed that TP317 enhanced CD8 and NK cell associated programs in Panc02 and B16F10 tumors, and also promoted macrophage, dendritic cell, B cell and antigen presentation functions in the TME.
 Conclusions: TP317 monotherapy and ICI combinations significantly inhibited tumor growth in various murine models of cancer. The effects on various immune cell functions and the surprising efficacy of short half-life RvE1 (<2 hours) dosed weekly suggests that TP317 is reprogramming the TME to an immunogenic state. In summary, TP317, an RvE1 drug with a high therapeutic index, has potent single agent efficacy and offers a novel approach in combination with ICI to treat ICI-resistant and ICI-sensitive tumors.
 Citation Format: Franciele C. Kipper, Eva Rothenberger, Abigail Kelly, Michael Gillespie, Ahmed Attaya, Diane R. Bielenberg, Sui Huang, Lance Pflieger, Frank Sciavolino, Aaron Mathias, Wayne Klohs, John Parkinson, Gary Mathias, Dipak Panigrahy. TP317, a first-in-class resolvin E1 small molecule, potentiates the efficacy of immune checkpoint (ICI) inhibitors in ICI-resistant and ICI-sensitive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1135.

Abstract 1135: TP317, a first-in-class resolvin E1 small molecule, potentiates the efficacy of immune checkpoint (ICI) inhibitors in ICI-resistant and ICI-sensitive tumors

Frank C. Sciavolino

Papers

Synthesis, in vitro and in vivo activity of novel 9-deoxo-9a-AZA-9a-homoerythromycin A derivatives; a new class of macrolide antibiotics, the azalides.

Abstract 1135: TP317, a first-in-class resolvin E1 small molecule, potentiates the efficacy of immune checkpoint (ICI) inhibitors in ICI-resistant and ICI-sensitive tumors

Synthesis andBiological Activities of4"-Deoxy-4"-Sulf onamido- Oleandomycin Derivatives