Subtilisin proteases, found in all organisms, are enzymes important in the post-translational steps of protein processing. In Leishmania major and L. donovani, this enzyme has been described as essential to their survival; however, few compounds that target subtilisin have been investigated for their potential as an antileishmanial drug. In this study, we first show, by electron microscopy and flow cytometry, that subtilisin has broad localization throughout the cytoplasm and membrane of the parasite in the promastigote form with foci in the flagellar pocket. Through in silico analysis, the similarity between subtilisin of different Leishmania species and that of humans were determined, and based on molecular docking, we evaluated the interaction capacity of a serine protease inhibitor against both life cycle forms of Leishmania. The selected inhibitor, known as PF-429242, has already been used against the dengue virus, arenaviruses, and the hepatitis C virus. Moreover, it proved to have antilipogenic activity in a mouse model and caused hypolipidemia in human cells in vitro. Here, PF-429242 significantly inhibited the growth of L. amazonensis promastigotes of four different strains (IC₅₀ values = 3.07 ± 0.20; 0.83 ± 0.12; 2.02 ± 0.27 and 5.83 ± 1.2 µM against LTB0016, PH8, Josefa and LV78 strains) whilst having low toxicity in the host macrophages (CC₅₀ = 170.30 µM). We detected by flow cytometry that there is a greater expression of subtilisin in the amastigote form; however, PF-429242 had a low effect against this intracellular form with an IC50 of >100 µM for intracellular amastigotes, as well as against axenic amastigotes (94.12 ± 2.8 µM for the LV78 strain). In conclusion, even though PF-429242 does not affect the intracellular forms, this drug will serve as a tool to explore pharmacological and potentially leishmanicidal targets.

Current treatment available for leishmaniasis is fraught with numerous problems, so the search for new treatment alternatives for leishmaniasis is urgent and necessary. The proteasome has been selected as a promising target. Marizomib is a proteasome inhibitor and has shown antitumor effects, with ongoing clinical tests. In this work, we aimed to evaluate the in vitro and in vivo effects of Marizomib on Leishmania amazonensis and Leishmania infantum . Interestingly, Marizomib was not effective against promastigote forms of L. amazonensis and L. infantum in vitro but showed a significant effect against intracellular amastigotes of L. amazonensis and L. infantum , showing selectivity for the parasite when compared to the host cell. Furthermore, through transmission electron microscopy, it was possible to show that Marizomib induces extensive ultrastructural changes in amastigotes of L. amazonensis and L. infantum , such as the appearance of many vacuoles in the parasite cytoplasm and mitochondrial swelling. Marizomib was also shown to be effective in a murine model of cutaneous leishmaniasis, with a reduction in the size of lesions and parasite load in the footpads and draining lymph nodes of animals infected with L. amazonensis . It is also effective in a model of visceral leishmaniasis, with a reduction in parasite load in the spleen and liver of animals infected with L. infantum . Importantly, Marizomib, in the treatment regimens used, did not cause renal and hepatic acute toxicity to infected animals. These results highlight the antileishmanial potential of Marizomib, encouraging us to conduct preclinical tests in other animal models, as well as clinical trials.

Background/Objectives: The total lysate of Leishmania amazonensis (LaAg) is one of the most extensively studied vaccine formulations against leishmaniasis. Despite demonstrating safety and immunogenicity when administered intramuscularly, LaAg has failed to show efficacy in clinical trials and, in some cases, has even been associated with an enhanced susceptibility to infection. Adjuvants, which are molecules or compounds added to antigens to enhance the immunogenicity or modulate the immune response, are frequently employed in vaccine studies. This study aimed to evaluate different adjuvants to improve the protective efficacy of LaAg in L.amazonensis infection using a BALB/c mouse model. Methods: BALB/c mice were immunized with LaAg in combination with various adjuvants. The delayed-type hypersensitivity (DTH) test was assessed by measuring the infected paw and was used to evaluate the immunogenicity and to determine the most effective adjuvant. The immune response was analyzed through flow cytometry, focusing on cytokine production, immune cell recruitment and lesion size, alongside the control of parasite load at the infection site. The expression levels of iNOS and TGF-β were quantified using RT-qPCR, while IgG1, IgG2a and IgE antibody levels were determined via ELISA. Results: Among the adjuvants tested, only saponin (SAP) elicited a significant DTH response following LaAg challenge. SAP enhanced the immunogenicity of LaAg, as evidenced by increased IFN-γ-producing CD4⁺ and CD8⁺ T cells in the draining lymph nodes at 18 h post-challenge. Additionally, SAP facilitated the recruitment of lymphocytes, macrophages, neutrophils and eosinophils to the infection site. Conclusions: The LaAg + SAP combination conferred partial protection, as demonstrated by a reduction in lesion size and the partial control of parasite load. In conclusion, the addition of SAP as an adjuvant to LaAg effectively modulates the immune response, enhancing the vaccine’s protective efficacy. These findings provide valuable insights into the development of improved vaccines against L.amazonensis infection.

PAHO have urged member countries to recognize ethnicity as a structural deter-minant of health, emphasizing the need for evidence-based public policies to address inequalities, however, studies that systematically explore the epidemiological profile of neglected tropical diseases from this perspective remain scarce. In this sense, this study aimed to analyze the prevalence and mortality rates of ACL across the Brazilian na-tional territory, stratified by ethnic-racial groups (Brown, Black, Asian, and Indige-nous; corresponding to the Brazilian categories pardo, preto, amarelo, and indígena) and study temporal trends and spatial distribution of these indicators by states and macro-regional levels, as well as to assess the potential impact of incomplete race/color information on the observed patterns. Data was extracted from the SINAN, accessed through DATASUS, in the period from 2016 to 2025. It was observed pronounced temporal fluctuations, with a significant reduction in the number of cases from 2017 to 2025 and marked spatial heterogeneity in the distribution of ACL across Brazil, with a clear concentration in North, Central-West and Northeast and inland regions. In addi-tion, ACL prevalence is higher among Asian and Indigenous populations, mortality disproportionately affects Brown and Indigenous groups, highlighting the indigenous population's high vulnerability to morbidity and mortality from ACL. It is also worth noting that it was observed consistent patterns of territorial and ethnic-racial inequal-ity in both the burden and severity of ACL in Brazil. This work is very important be-cause identify disparities in disease burden and outcomes, contributing to a more comprehensive understanding of ACL-related vulnerabilities in Brazil.

The Pan American Health Organization (PAHO) recognizes ethnicity as a structural determinant of health, yet ethnic–racial analyses of neglected tropical diseases remain scarce. This study investigated cumulative notification and mortality rates of American cutaneous leishmaniasis (ACL) in Brazil from 2016 to 2025 according to the official Brazilian race/color categories. Data were obtained from the Notifiable Diseases Information System (SINAN/DATASUS), and temporal and spatial patterns were analyzed. ACL showed marked geographic heterogeneity, with the highest cumulative notification rates concentrated in the North and Central-West regions. Indigenous and Asian populations presented the highest cumulative notification rates, whereas mortality rates were highest among Indigenous populations and remained elevated among Brown populations in several settings. Spatial clusters were concentrated mainly in the Brazilian Amazon, reinforcing the persistence of territorial inequalities in ACL distribution. Overall, the findings reveal substantial race/color and geographic disparities in both ACL occurrence and mortality in Brazil, underscoring the need for more equitable surveillance, prevention, and control strategies.