Plasmodium falciparum Resistance to a Lead Benzoxaborole Due to Blocked Compound Activation and Altered Ubiquitination or Sumoylation.

New antimalarial drugs are needed. The benzoxaborole AN13762 showed excellent activity against cultured Plasmodium falciparum, against fresh Ugandan P. falciparum isolates, and in murine malaria models. To gain mechanistic insights, we selected in vitro for P. falciparum isolates resistant to AN13762. In all of 11 independent selections with 100 to 200 nM AN13762, the 50% inhibitory concentration (IC50) increased from 18-118 nM to 180-890 nM, and whole-genome sequencing of resistant parasites demonstrated mutations in prodrug activation and resistance esterase (PfPARE). The introduction of PfPARE mutations led to a similar level of resistance, and recombinant PfPARE hydrolyzed AN13762 to the benzoxaborole AN10248, which has activity similar to that of AN13762 but for which selection of resistance was not readily achieved. Parasites further selected with micromolar concentrations of AN13762 developed higher-level resistance (IC50, 1.9 to 5.0 μM), and sequencing revealed additional mutations in any of 5 genes, 4 of which were associated with ubiquitination/sumoylation enzyme cascades; the introduction of one of these mutations, in SUMO-activating enzyme subunit 2, led to a similar level of resistance. The other gene mutated in highly resistant parasites encodes the P. falciparum cleavage and specificity factor homolog PfCPSF3, previously identified as the antimalarial target of another benzoxaborole. Parasites selected for resistance to AN13762 were cross-resistant with a close analog, AN13956, but not with standard antimalarials, AN10248, or other benzoxaboroles known to have different P. falciparum targets. Thus, AN13762 appears to have a novel mechanism of antimalarial action and multiple mechanisms of resistance, including loss of function of PfPARE preventing activation to AN10248, followed by alterations in ubiquitination/sumoylation pathways or PfCPSF3.IMPORTANCE Benzoxaboroles are under study as potential new drugs to treat malaria. One benzoxaborole, AN13762, has potent activity and promising features, but its mechanisms of action and resistance are unknown. To gain insights into these mechanisms, we cultured malaria parasites with nonlethal concentrations of AN13762 and generated parasites with varied levels of resistance. Parasites with low-level resistance had mutations in PfPARE, which processes AN13762 into an active metabolite; PfPARE mutations prevented this processing. Parasites with high-level resistance had mutations in any of a number of enzymes, mostly those involved in stress responses. Parasites selected for AN13762 resistance were not resistant to other antimalarials, suggesting novel mechanisms of action and resistance for AN13762, a valuable feature for a new class of antimalarial drugs

Topical Treatment for Cutaneous Leishmaniasis: Dermato-Pharmacokinetic Lead Optimization of Benzoxaboroles.

Cutaneous leishmaniasis (CL) is caused by several species of the protozoan parasite Leishmania, affecting an estimated 10 million people worldwide. Previously reported strategies for the development of topical CL treatments have focused primarily on drug permeation and formulation optimization as the means to increase treatment efficacy. Our approach aims to identify compounds with antileishmanial activity and properties consistent with topical administration. Of the test compounds, five benzoxaboroles showed potent activity (50% effective concentration [EC50] < 5 μM) against intracellular amastigotes of at least one Leishmania species and acceptable activity (20 μM < EC50 < 30 μM) against two more species. Benzoxaborole compounds were further prioritized on the basis of the in vitro evaluation of progression criteria related to skin permeation, such as the partition coefficient and solubility. An MDCKII-hMDR1 cell assay showed overall good permeability and no significant interaction with the P-glycoprotein transporter for all substrates except LSH002 and LSH031. The benzoxaboroles were degraded, to some extent, by skin enzymes but had stability superior to that of para-hydroxybenzoate compounds, which are known skin esterase substrates. Evaluation of permeation through reconstructed human epidermis showed LSH002 to be the most permeant, followed by LSH003 and LSH001. Skin disposition studies following finite drug formulation application to mouse skin demonstrated the highest permeation for LSH001, followed by LSH003 and LSH002, with a significantly larger amount of LSH001 than the other compounds being retained in skin. Finally, the efficacy of the leads (LSH001, LSH002, and LSH003) against Leishmania major was tested in vivo LSH001 suppressed lesion growth upon topical application, and LSH003 reduced the lesion size following oral administration

In vivo efficacy of the boron-pleuromutilin AN11251 against Wolbachia of the rodent filarial nematode Litomosoides sigmodontis

The elimination of filarial diseases such as onchocerciasis and lymphatic filariasis is hampered by the lack of a macrofilaricidal – adult worm killing – drug. In the present study, we tested the in vivo efficacy of AN11251, a boron-pleuromutilin that targets endosymbiotic Wolbachia bacteria from filarial nematodes and compared its efficacy to doxycycline and rifampicin. Doxycycline and rifampicin that were previously shown to deplete Wolbachia endosymbionts leading to a permanent sterilization of the female adult filariae and adult worm death in human clinical studies. Twice-daily oral treatment of Litomosoides sigmodontis-infected mice with 200 mg/kg AN11251 for 10 days achieved a Wolbachia depletion > 99.9% in the adult worms, exceeding the Wolbachia reduction by 10-day treatments with bioequivalent human doses of doxycycline and a similar reduction as high-dose rifampicin (35 mg/kg). Wolbachia reductions of > 99% were also accomplished by 14 days of oral AN11251 at a lower twice-daily dose (50 mg/kg) or once-per-day 200 mg/kg AN11251 treatments. The combinations tested of AN11251 with doxycycline had no clear beneficial impact on Wolbachia depletion, achieving a > 97% Wolbachia reduction with 7 days of treatment. These results indicate that AN11251 is superior to doxycycline and comparable to high-dose rifampicin in the L. sigmodontis mouse model, allowing treatment regimens as short as 10-14 days. Therefore, AN11251 is represents a promising pre-clinical candidate that was identified in the L. sigmodontis model, and could be further evaluated and developed as potential clinical candidate for human lymphatic filariasis and onchocerciasis.  AUTHOR SUMMARY Onchocerciasis and lymphatic filariasis are human filarial tropical diseases, which can cause blindness and severe dermatitis (onchocerciasis) or lymphedema and hydrocele (lymphatic filariasis). Current strategies to eliminate these diseases include the mass drug administration (MDA) of drugs that target the progeny of the filariae, the microfilariae, and temporarily inhibit filarial embryogenesis and, therefore, the transmission of the disease. However, MDA has several limitations that delay the goal of elimination including the lack of a drug with a short term regimen and a potent macrofilaricidal effect. As an alternative approach, the antibiotic doxycycline has been proven to be effective in depleting Wolbachia endosymbionts from adult filariae, which then leads to permanent sterilization and death of the adult worms. Due to contraindications for doxycycline and prolonged treatment regimen of at least 4 weeks, there is an urgent need for new anti-filarial drugs with an improved safety profile and shorter regimens. The current study demonstrates that the boron-pleuromutilin derivative AN11251 provides an excellent in vivo anti-Wolbachia depletion in the Litomosoides sigmodontis filarial mouse model that is superior to doxycycline and comparable to rifampicin, allowing for regimens as short as 10-14 days. Combination with doxycycline for 7 days had no significant beneficial effect on efficacy, achieving Wolbachia reductions of more than 97%. Therefore, AN11251 shows potent anti-Wolbachia activity in the L. sigmodontis mouse model and may also present an alternative pre-clinical candidate for filariasis treatment

SCYX-7158, an Orally-Active Benzoxaborole for the Treatment of Stage 2 Human African Trypanosomiasis

Human African trypanosomiasis (HAT) is caused by infection with the parasite Trypanosoma brucei and is an important public health problem in sub-Saharan Africa. New, safe, and effective drugs are urgently needed to treat HAT, particularly stage 2 disease where the parasite infects the brain. Existing therapies for HAT have poor safety profiles, difficult treatment regimens, limited effectiveness, and a high cost of goods. Through an integrated drug discovery project, we have discovered and optimized a novel class of boron-containing small molecules, benzoxaboroles, to deliver SCYX-7158, an orally active preclinical drug candidate. SCYX-7158 cured mice infected with T. brucei, both in the blood and in the brain. Extensive pharmacokinetic characterization of SCYX-7158 in rodents and non-human primates supports the potential of this drug candidate for progression to IND-enabling studies in advance of clinical trials for stage 2 HAT

From the Test Tube to the Treatment Room Fundamentals of Boron-containing Compounds and their Relevance to Dermatology

The development of new drug classes and novel molecules that are brought to the marketplace has been a formidable challenge, especially for dermatologic drugs. The relative absence of new classes of antimicrobial agents is also readily apparent. Several barriers account for slow drug development, including regulatory changes, added study requirements, commercial pressures to bring drugs to market quickly by developing new generations of established compounds, and the greater potential for failure and higher financial risk when researching new drug classes. In addition, the return on investment is usually much lower with dermatologic drugs as compared to the potential revenue from “blockbuster” drugs for cardiovascular or gastrointestinal disease, hypercholesterolemia, and mood disorders. Nevertheless, some researchers are investigating new therapeutic platforms, one of which is boron-containing compounds. Boron-containing compounds offer a wide variety of potential applications in dermatology due to their unique physical and chemical properties, with several in formal phases of development. Tavaborole, a benzoxaborole compound, has been submitted to the United States Food and Drug Administration for approval for treatment of onychomycosis. This article provides a thorough overview of the history of boron-based compounds in medicine, their scientific rationale, physiochemical and pharmacologic properties, and modes of actions including therapeutic targets. A section dedicated to boron-based compounds in development for treatment of various skin disorders is also included