Diabetic nephropathy remains an area of high unmet medical need, with current therapies that slow down, but do not prevent, the progression of disease. A reduced phosphorylation state of adenosine monophosphate-activated protein kinase (AMPK) has been correlated with diminished kidney function in both humans and animal models of renal disease. Here, we describe the identification of novel, potent, small molecule activators of AMPK that selectively activate AMPK heterotrimers containing the β1 subunit. After confirming that human and rodent kidney predominately express AMPK β1, we explore the effects of pharmacological activation of AMPK in the ZSF1 rat model of diabetic nephropathy. Chronic administration of these direct activators elevates the phosphorylation of AMPK in the kidney, without impacting blood glucose levels, and reduces the progression of proteinuria to a greater degree than the current standard of care, angiotensin-converting enzyme inhibitor ramipril. Further analyses of urine biomarkers and kidney tissue gene expression reveal AMPK activation leads to the modulation of multiple pathways implicated in kidney injury, including cellular hypertrophy, fibrosis, and oxidative stress. These results support the need for further investigation into the potential beneficial effects of AMPK activation in kidney disease.
Branched chain amino acid (BCAA) catabolic impairments have been implicated in several diseases. Branched chain ketoacid dehydrogenase (BCKDH) controls the rate limiting step in BCAA degradation, the activity of which is inhibited by BCKDH kinase (BDK)-mediated phosphorylation. Screening efforts to discover BDK inhibitors led to identification of thiophene PF-07208254, which improved cardiometabolic endpoints in mice. Structure-activity relationship studies led to identification of a thiazole series of BDK inhibitors; however, these inhibitors did not improve metabolism in mice upon chronic administration. While the thiophenes demonstrated sustained branched chain ketoacid (BCKA) lowering and reduced BDK protein levels, the thiazoles increased BCKAs and BDK protein levels. Thiazoles increased BDK proximity to BCKDH-E2, whereas thiophenes reduced BDK proximity to BCKDH-E2, which may promote BDK degradation. Thus, we describe two BDK inhibitor series that possess differing attributes regarding BDK degradation or stabilization and provide a mechanistic understanding of the desirable features of an effective BDK inhibitor.
The anteromedial portal technique for drilling of the femoral tunnel during anterior cruciate ligament (ACL) reconstruction has been advocated by many surgeons as allowing improved access to the anatomical footprint. Furthermore, suspensory fixation of soft tissue grafts has become popularized because of complications associated with cross-pin fixation. Concerns regarding the use of both have recently arisen.To raise awareness of the increased risk of graft failure when using the anteromedial portal technique with suspensory femoral fixation during ACL reconstruction.Cohort study; Level of evidence, 3.From November 1998 to August 2012, a total of 465 primary ACL reconstructions were performed using quadrupled hamstring autograft tendons, with drilling of the femoral tunnel performed via the transtibial portal. Graft fixation on the femur was achieved with cross-pin fixation, while interference screw fixation was used on the tibia. From September 2012 to October 2013, there were 69 reconstructions performed through an anteromedial portal. While there was no change in graft choice, a change was made to using suspensory femoral fixation. No other surgical or postoperative rehabilitation changes were made.During the 14-year period in which ACL reconstructions were performed via the transtibial portal and with cross-pin fixation, 2 graft failures (0.4% failure rate) were reported. After switching to the anteromedial portal with suspensory fixation, 7 graft failures (10.1% failure rate) were reported over a 13-month period. These were 5 male and 2 female patients, with a mean age of 18.8 years-all elite athletes. The same surgical technique was used in all patients, and all patients had at least an 8 mm-diameter graft. Patients were cleared to return to sport at an average of 8.4 months postoperatively, after completing functional performance tests. Of the 7 patients, 6 sustained a rerupture of the graft within 2 weeks of returning to full competition. The final patient sustained a rerupture 10 months after being cleared to play.Compared with the transtibial technique with cross-pin graft fixation, there is an increased risk of graft failure when performing autologous hamstring ACL reconstructions using the anteromedial portal technique with cortical suspensory fixation.
The catastrophic devastation from recent natural calamities in the Philippines such as Typhoon Yolanda and Central Visayas earthquake in 2013 had made disaster preparedness a primary concern in the country. Prompted by the critical need to use science to save lives, this study developed Science of Survival (SOS) pamphlets titled When the Wind Rages and Water Rises: A Science of Survival Pamphlet for Typhoon, Flashflood, Storm Surge and Tsunami and When the Earth Moves: A Science of Survival Pamphlet for Earthquakes and Their Aftermath (Liquefaction, Fire, Landslide and Tsunami). The study used the` developmental research design consisting of three phases: needs and context analysis phase, design, development and formative evaluation phase, and semi-summative evaluation phase. By carefully documenting the iterative process of analysis, design, evaluation and revision, insights were sought with regard to the development of pamphlets that provide useful and scientifically accurate information about surviving natural calamities such as typhoons and earthquakes. Experts from government agencies involved in disaster risk reduction and management, science experts in the university, and students who were victims of major disasters reviewed and evaluated the pamphlets. The results of the semi-summative quantitative evaluation showed that both pamphlets are highly acceptable as supplementary resource materials on disaster response and management.
Optimization of the pharmacokinetic (PK) properties of a series of activators of adenosine monophosphate-activated protein kinase (AMPK) is described. Derivatives of the previously described 5-aryl-indole-3-carboxylic acid clinical candidate (1) were examined with the goal of reducing glucuronidation rate and minimizing renal excretion. Compounds 10 (PF-06679142) and 14 (PF-06685249) exhibited robust activation of AMPK in rat kidneys as well as desirable oral absorption, low plasma clearance, and negligible renal clearance in preclinical species. A correlation of in vivo renal clearance in rats with in vitro uptake by human and rat renal organic anion transporters (human OAT/rat Oat) was identified. Variation of polar functional groups was critical to mitigate active renal clearance mediated by the Oat3 transporter. Modification of either the 6-chloroindole core to a 4,6-difluoroindole or the 5-phenyl substituent to a substituted 5-(3-pyridyl) group provided improved metabolic stability while minimizing propensity for active transport by OAT3.
Inhibition of acetyl-CoA carboxylases (ACCs), a crucial enzyme for fatty acid metabolism, has been shown to promote fatty acid oxidation and reduce body fat in animal models. Therefore, ACCs are attractive targets for structure-based inhibitor design, particularly the carboxyltransferase (CT) domain, which is the primary site for inhibitor interaction. We have cloned, expressed, and purified the CT domain of human ACC2 using baculovirus-mediated insect cell expression system. However, attempts to crystallize the human ACC2 CT domain have not been successful in our hands. Hence, we have been using the available crystal structure of yeast CT domain to design human ACC inhibitors. Unfortunately, as the selectivity of the lead series has increased against the full-length human enzyme, the potency against the yeast enzyme has decreased significantly. This loss of potency against the yeast enzyme correlated with a complete lack of binding of the human-specific compounds to crystals of the yeast CT domain. Here, we address this problem by converting nine key active site residues of the yeast CT domain to the corresponding human residues. The resulting humanized yeast ACC-CT (yCT-H9) protein exhibits biochemical and biophysical properties closer to the human CT domain and binding to human specific compounds. We report high resolution crystal structures of yCT-H9 complexed with inhibitors that show a preference for the human CT domain. These structures offer insights that explain the species selectivity of ACC inhibitors and may guide future drug design programs.
Abstract Background Efforts on a global scale for combating malaria have achieved substantial progress over the past twenty years. Two Central American nations have accomplished their goal of eliminating malaria: El Salvador and Belize. Honduras has decreased the incidence of malaria and now reports fewer than 4,000 malaria cases annually, aspiring to reach elimination by 2030. To accomplish this goal, it is essential to assess the existing strategies employed for malaria control and to address the task of incorporating novel intervention strategies to identify asymptomatic reservoirs. Methods A survey for detecting asymptomatic cases was carried out in the community of Kaukira, in Gracias a Dios, Honduras, considered the focus of malaria transmission during 2023. Asymptomatic community members were recruited as participants, malaria screening was performed through a rapid diagnostic test in situ, and a blood sample was collected on filter paper. Highly sensitive molecular assays based on photo-induced electron transfer PCR (PET-PCR) were performed to detect the two species of Plasmodium circulating in Honduras, Plasmodium vivax and Plasmodium falciparum . In addition, the identification of the parasite species was verified by amplifying three genetic markers (Pvmsp3α, Pvmsp3ß, and Pfmsp1). Results A total of 138 participants were recruited, mostly adult women. All individuals tested negative on the rapid diagnostic test. Positive results for malaria were detected by PET-PCR in 17 samples (12.3%). Most samples were amplified with a Ct value between 37 and 42, indicating very low parasitaemias. Out of the 17 samples, 15 of them also showed amplification in the species assays. There were nine cases of P. falciparum infections and seven cases of P. vivax infections that were further confirmed by nested PCR (nPCR) of Pvmsp3 and Pfmsp1. Parasitaemias ranged from 100 p/µL to less than 0.25 p/µL. One sample showed mixed infection. Conclusions The existence of asymptomatic malaria reservoirs in Honduras can contribute to disease transmission and poses a challenge that may hinder elimination efforts, requiring public health authorities to modify surveillance strategies to identify the disease and treat this population accordingly.
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