Biology subjects

Milanes, J. E.

Publications and source records attributed to Milanes, J. E..

3 recordsLinked to original sources

bioRxiv2024

Enolase inhibitors as therapeutic leads for Naegleria fowleri infection

Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate that these agents are potent inhibitors of N. fowleri ENO (NfENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC50 value of 0.14 {+/-} 0.04 {micro}M) that was toxic to trophozoites (EC50 value of 0.21 {+/-} 0.02 {micro}M) while the reported CC50 was >300 {micro}M. Molecular docking simulation revealed that HEX binds strongly to the active site of NfENO with a binding affinity of -8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of NfENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the conclusion of the experiment, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. Brains of six of the eight survivors were positive for amoebae, suggesting the agent at the tested dose suppressed, but did not eliminate, infection. These findings suggest that HEX is a promising lead for the treatment of PAM.

biochemistry↗

bioRxiv2018

Enzymatic and structural characterization of the Naegleria fowleri glucokinase

Infection with the free-living amoeba Naegleria fowleri leads to life-threatening primary amoebic meningoencephalitis. Efficacious treatment options for these infections are limited and the mortality rate is very high (~98%). Parasite metabolism may provide suitable targets for therapeutic design. Like most other organisms, glucose metabolism is critical for parasite viability, being required for growth in culture. The genome of the parasite encodes a single glucose phosphorylating enzyme, a glucokinase (Glck). The products of this enzyme are required for both glycolysis and the pentose phosphate pathway. The N. fowleri Glck (NfGlck) shares limited (25%) amino acid identity with the mammalian host enzyme (HsGlck), suggesting that parasite-specific inhibitors with anti-amoeba activity could be generated. Following heterologous expression, NfGlck was found to have a limited hexose substrate range, with greatest activity observed with glucose. The enzyme had apparent Km values of 42.5 {+/-} 7.3 M and 141.6 {+/-} 9.9 M for glucose and ATP, respectively. The NfGlck structure was determined and refined to 2.2 [A] resolution, revealing that the enzyme shares greatest structural similarity with the Trypanosoma cruzi Glck. These similarities include binding modes and binding environments for substrates. To identify inhibitors of NfGlck, we screened a small collection of inhibitors of glucose phosphorylating enzymes and identified several small molecules with IC50 values < 1 M that may prove useful as hit chemotypes for further lead and therapeutic development against N. fowleri.

biochemistry↗

bioRxiv2018

Glucose signaling is important for nutrient adaptation during differentiation of pleomorphic African trypanosomes

The African trypanosome has evolved mechanisms to adapt to changes in nutrient availability that occur during its lifecycle. During transition from mammalian blood to insect vector gut, parasites experience a rapid reduction in environmental glucose. Here we describe how pleomorphic parasites respond to glucose depletion with a focus on parasite changes in energy metabolism and growth. Long slender bloodstream form parasites are rapidly killed as glucose concentrations fall, while the short stumpy bloodstream form parasites persist to differentiate into the insect stage procyclic form parasite. The rate of differentiation was slower than that triggered by other cues but reached physiological rates when combined with cold shock. Both differentiation and growth of resulting procyclic form parasites were inhibited by glucose and its non-metabolizable analogs in a concentration dependent manner. Procyclic form parasites differentiated from short stumpy form parasites in glucose depleted medium significantly upregulated gene expression of amino acid metabolic pathway components when compared to procyclic forms generated by cis-aconitate treatment. Additionally, growth of these parasite was inhibited by the presence of either glucose or 6-deoxyglucose. In summary, glucose transitions from the primary metabolite of the blood stage infection to a negative regulator of cell development and growth in the insect vector, suggesting that the hexose is not only a key metabolic agent but is also an important signaling molecule.\n\nAuthor SummaryAs the African trypanosome, Trypanosoma brucei, completes its lifecycle, it encounters many different environments. Adaptation to these environments includes modulation of metabolic pathways to parallel the availability of nutrients. Here, we describe how the blood-dwelling lifecycle stages of the African trypanosome, which consume glucose to meet their nutritional needs, respond differently to culture in the near absence of glucose. The proliferative long slender parasites rapidly die, while the non-dividing short stumpy remains viable and undergoes differentiation to the next lifecycle stage, the procyclic form parasite. Interestingly a sugar analog that cannot be used as an energy source inhibited the process. Furthermore, the growth of procyclic form parasite that resulted from the event was inhibited by glucose, a behavior that is similar to that of parasites isolated from tsetse flies. Our findings suggest that glucose sensing serves as an important modulator of nutrient adaptation in the parasite.

microbiology↗