thymidylate kinase
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Author(s):  
Junmei Hu Frisk ◽  
Gunnar Pejler ◽  
Staffan Eriksson ◽  
Liya Wang

2021 ◽  
Vol 14 (10) ◽  
pp. 1027
Author(s):  
Danielle R. Garcia ◽  
Felipe R. Souza ◽  
Ana P. Guimarães ◽  
Martin Valis ◽  
Zbyšek Pavelek ◽  
...  

Continuing the work developed by our research group, in the present manuscript, we performed a theoretical study of 10 new structures derived from the antivirals cidofovir and ribavirin, as inhibitor prototypes for the enzyme thymidylate kinase from Variola virus (VarTMPK). The proposed structures were subjected to docking calculations, molecular dynamics simulations, and free energy calculations, using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method, inside the active sites of VarTMPK and human TMPK (HssTMPK). The docking and molecular dynamic studies pointed to structures 2, 3, 4, 6, and 9 as more selective towards VarTMPK. In addition, the free energy data calculated through the MM-PBSA method, corroborated these results. This suggests that these compounds are potential selective inhibitors of VarTMPK and, thus, can be considered as template molecules to be synthesized and experimentally evaluated against smallpox.


Author(s):  
Lijun Song ◽  
Romain Merceron ◽  
Fabian Hulpia ◽  
Ainhoa Lucía ◽  
Begoña Gracia ◽  
...  

2021 ◽  
Author(s):  
Elizabeth T. Montaño ◽  
Jason F. Nideffer ◽  
Joseph Sugie ◽  
Eray Enustun ◽  
Adam B. Shapiro ◽  
...  

In this study, we sought to determine if an in vivo assay for studying antibiotic mechanisms of action could provide insight into the activity of compounds that may inhibit multiple targets. Thus, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules against E. coli and classified them according to their in vivo mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite structural similarity to related PAINS. A genetic analysis of resistant mutants revealed thymidylate kinase (essential for DNA synthesis) as an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by in vitro activity assays as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the lowest IC 50 in vitro and MIC in vivo displayed the greatest specificity for inhibition of the DNA replication pathway, despite containing a rhodamine moiety. While it’s thought that PAINS cannot be developed as antibiotics, this work showcases novel inhibitors of E. coli thymidylate kinase. But perhaps more importantly, this work highlights the utility of bacterial cytological profiling for studying the in vivo specificity of antibiotics and demonstrates that BCP can identify multiple pathways that are inhibited by an individual molecule. Importance: We demonstrate that bacterial cytological profiling is a powerful tool for directing antibiotic discovery efforts because it can be used to determine the specificity of an antibiotic's in vivo mechanism of action. By assaying analogs of PAINS, molecules that are notoriously intractable and non-specific, we (surprisingly) identify molecules with specific activity against E. coli thymidylate kinase. This suggests that structural modifications to PAINS can confer stronger inhibition by targeting a specific cellular pathway. While in vitro inhibition assays are susceptible to false positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific in vivo activity.


2021 ◽  
Author(s):  
Elizabeth T. Montaño ◽  
Jason F. Nideffer ◽  
Joseph Sugie ◽  
Eray Enustun ◽  
Adam B. Shapiro ◽  
...  

AbstractIn this study, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules inhibiting the growth of E. coli and classified them according to their in vivo mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite their structural similarity to related PAINS. A genetic analysis of resistant mutants revealed that thymidylate kinase (an enzyme essential for DNA synthesis) is an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by assays of in vitro activity as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the lowest IC50in vitro and MIC in vivo displayed the greatest specificity for the inhibition of DNA replication in E. coli, despite containing a rhodamine moiety. While it’s generally thought that PAINS cannot be developed as antibiotics, this work highlights the utility of bacterial cytological profiling for studying the in vivo specificity of antibiotics, and it showcases novel inhibitors of E. coli thymidylate kinase.ImportanceWe demonstrate that bacterial cytological profiling is a powerful tool for directing antibiotic discovery efforts because it can be used to determine the specificity of an antibiotic’s in vivo mechanism of action. By assaying analogs of PAINS, molecules that are notoriously intractable and non-specific, we (surprisingly) identify molecules with specific activity against E. coli thymidylate kinase. This suggests that structural modifications to PAINS can confer stronger inhibition by targeting a specific cellular pathway. While in vitro inhibition assays are susceptible to false positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific in vivo activity.


2020 ◽  
Vol 206 ◽  
pp. 112659
Author(s):  
Yanlin Jian ◽  
Romain Merceron ◽  
Steven De Munck ◽  
He Eun Forbes ◽  
Fabian Hulpia ◽  
...  

2020 ◽  
Author(s):  
Janet Peace Babirye ◽  
Charles Drago Kato ◽  
Fredrick Lutwama ◽  
Carol Musubika ◽  
Rose Nabatanzi ◽  
...  

Abstract Diagnosis of pulmonary tuberculosis (PTB) in context of HIV infection remains challenging due 25 to the paucibacillary nature of the disease. Mycobacterium tuberculosis thymidylate kinase 26 (TMKmt) has been validated as a novel biomarker with high detection limits for PTB in sputum. 27 We aimed to clinically test TMKmt as a diagnostic biomarker for pulmonary tuberculosis among 28 HIV positive individuals at Makerere University Joint AIDS Program Immuno Suppression 29 Syndrome clinic. 30 Methods 31 A total of 120 participants with presumptive PTB were enrolled in a cross- sectional study 32 between January and September 2018. Venous blood and expectorated spot sputum was obtained 33 from 116 consenting participants. Tuberculosis culture was performed on sputum as the gold 34 standard to confirm PTB status while direct ELISAs were performed on sputum and serum to 35 determine the level of TMKmt antigen. Sensitivity, specificity and receiver operator 36 characteristic curves were used to assess the precision of TMKmt in diagnosing PTB. 37 Results 38 Of the 116 participants, only 22 (19%) were PTB positive based on either Lowenstein Jensen or 39 Mycobacterial growth inhibition tube `culture. The mean sputum TMKmt levels were 40 significantly higher in PTB positive individuals (4.889 ± 0.135) ng/ml as compared to PTB 41 negative individuals (4.303 ± 0.07295) ng/ml (t-test, P<0.0005). At a cut off value of > 4.683 42 ng/ml, the test had a sensitivity of 73% (95% CI, 49.78% - 89.27%) and a specificity of 72% 43 (95% CI, 62.15% - 81.07%). These results were supported by a receiver operator analysis which 44 showed an area under the curve of 0.75 (95% CI, 0.63 – O.86). 3 45 Conclusion 46 Sputum TMKmt is a potential diagnostic biomarker for active pulmonary tuberculosis. This 47 forms an alternate test that would replace smear microscopy and overcome the current TB 48 diagnostic challenges.


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