Linezolid pharmacokinetics in MDR-TB : a systematic review, meta-analysis and Monte Carlo simulation

This work was supported by the Wellcome Trust (grant numbers 109129/Z/15/Z to JM and 105620/Z/14/Z to DS and MC).Objectives The oxazolidinone linezolid is an effective component of drug - resistant TB treatment, but use is limited by toxicity and the optimum dose is uncertain . Current strategies are not informed by clinical pharmacokinetic/pharmacodynamic (PK/PD) data, we aimed to aimed to address this gap. Methods We defined linezolid PK/PD targets for efficacy; free area under the time - concentration curve: minimum inhibitory concentration ratio (ƒAUC0-24:M IC) >119m g/L/hr and safety; free minimum concentration (Cmin) <1.38mg/L . We extracted individual - level linezolid PK data from existing studies on TB patients and performed meta - analysis; producing summary estimates of ƒAUC0-24 and ƒCmin for published doses . Combining these with a published MIC distribution, we performed Monte Carlo simulations of target attainment. Results The efficacy target was attained in all simulated individuals at 300mg q12h and 600mg q12h , but only 20.7% missed the safety target at 300mg q12h versus 98.5% at 600mg q12h . Although suggesting 300mg q12h should be used preferentially, these data were reliant on a single centre . Efficacy and safety targets were missed by 41.0% and 24.2% respectively at 300mg q24h , and 44.5% and 27.5% at 600mg q24h . However, the confounding effect of between study heterogeneity on target attainment for q24h regimens was considerable. Conclusions 300mg q12h linezolid dosing may retain the efficacy of the 600mg q12h licensed dosing with improved safety. Data to evaluate commonly used 300mg q24h and 600mg q24h doses is limited. Comprehensive, prospectively obtained PK/PD data for linezolid doses in drug - resistant TB treatment are required.Publisher PDFPeer reviewe

AtomNet-Aided OTUD7B Inhibitor Discovery and Validation

Protein deubiquitinases play critical pathophysiological roles in cancer. Among all deubiquitinases, an oncogenic function for OTUD7B has been established in genetic NSCLC murine models. However, few deubiquitinase inhibitors have been developed due to technical challenges. Here, we report a putative small molecule OTUD7B inhibitor obtained from an AI-aided screen of a 4 million compound library. We validated the effects of the OTUD7B inhibitor (7Bi) in reducing Akt-pS473 signals in multiple NSCLC and HEK293 cells by blocking OTUD7B-governed GβL deubiquitination in cells, as well as inhibiting OTUD7B-mediated cleavage of K11-linked di-ub in an in vitro enzyme assay. Furthermore, we report in leukemia cells, either genetic depletion or 7Bi-mediated pharmacological inhibition of OTUD7B reduces Akt-pS473 via inhibiting the OTUD7B/GβL signaling axis. Together, our study identifies the first putative OTUD7B inhibitor showing activities both in cells and in vitro, with promising applications as a therapeutic agent in treating cancer with OTUD7B overexpression

In silico APC/C substrate discovery reveals cell cycle-dependent degradation of UHRF1 and other chromatin regulators

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease. Copyright

Crystal structure of the Arabidopsis SPIRAL2 C-terminal domain reveals a p80-Katanin-like domain.

Epidermal cells of dark-grown plant seedlings reorient their cortical microtubule arrays in response to blue light from a net lateral orientation to a net longitudinal orientation with respect to the long axis of cells. The molecular mechanism underlying this microtubule array reorientation involves katanin, a microtubule severing enzyme, and a plant-specific microtubule associated protein called SPIRAL2. Katanin preferentially severs longitudinal microtubules, generating seeds that amplify the longitudinal array. Upon severing, SPIRAL2 binds nascent microtubule minus ends and limits their dynamics, thereby stabilizing the longitudinal array while the lateral array undergoes net depolymerization. To date, no experimental structural information is available for SPIRAL2 to help inform its mechanism. To gain insight into SPIRAL2 structure and function, we determined a 1.8 Å resolution crystal structure of the Arabidopsis thaliana SPIRAL2 C-terminal domain. The domain is composed of seven core α-helices, arranged in an α-solenoid. Amino-acid sequence conservation maps primarily to one face of the domain involving helices α1, α3, α5, and an extended loop, the α6-α7 loop. The domain fold is similar to, yet structurally distinct from the C-terminal domain of Ge-1 (an mRNA decapping complex factor involved in P-body localization) and, surprisingly, the C-terminal domain of the katanin p80 regulatory subunit. The katanin p80 C-terminal domain heterodimerizes with the MIT domain of the katanin p60 catalytic subunit, and in metazoans, binds the microtubule minus-end factors CAMSAP3 and ASPM. Structural analysis predicts that SPIRAL2 does not engage katanin p60 in a mode homologous to katanin p80. The SPIRAL2 structure highlights an interesting evolutionary convergence of domain architecture and microtubule minus-end localization between SPIRAL2 and katanin complexes, and establishes a foundation upon which structure-function analysis can be conducted to elucidate the role of this domain in the regulation of plant microtubule arrays

AtomNet-Aided OTUD7B Inhibitor Discovery and Validation

Protein deubiquitinases play critical pathophysiological roles in cancer. Among all deubiquitinases, an oncogenic function for OTUD7B has been established in genetic NSCLC murine models. However, few deubiquitinase inhibitors have been developed due to technical challenges. Here, we report a putative small molecule OTUD7B inhibitor obtained from an AI-aided screen of a 4 million compound library. We validated the effects of the OTUD7B inhibitor (7Bi) in reducing Akt-pS473 signals in multiple NSCLC and HEK293 cells by blocking OTUD7B-governed GβL deubiquitination in cells, as well as inhibiting OTUD7B-mediated cleavage of K11-linked di-ub in an in vitro enzyme assay. Furthermore, we report in leukemia cells, either genetic depletion or 7Bi-mediated pharmacological inhibition of OTUD7B reduces Akt-pS473 via inhibiting the OTUD7B/GβL signaling axis. Together, our study identifies the first putative OTUD7B inhibitor showing activities both in cells and in vitro, with promising applications as a therapeutic agent in treating cancer with OTUD7B overexpression

Functional conservation and divergence of the helix-turn-helix motif of E2 ubiquitin-conjugating enzymes

Polyubiquitination by E2 and E3 enzymes is crucial to cell cycle control, epigenetic regulation, and development. The hallmark of the E2 family is the ubiquitin (Ub)-conjugating (UBC) domain that forms a dynamic thioester conjugate with ubiquitin (E2~Ub). Numerous studies have focused on E2 surfaces, such as the N-terminal and crossover helices, that directly interact with an E3 or the conjugated ubiquitin to stabilize the active, closed state of the E2~Ub. However, it remains unclear how other E2 surfaces regulate ubiquitin transfer. Here, we demonstrate the helix–turn–helix (HTH) motif of the UBC tunes the intrinsic polyubiquitination activity through distinct functions in different E2s. Interestingly, the E2HTH motif is repurposed in UBE2S and UBE2R2 to interact with the conjugated or acceptor ubiquitin, respectively, modulating ubiquitin transfer. Furthermore, we propose that Anaphase-Promoting Complex/Cyclosome binding to the UBE2SHTH reduces the conformational space of the flexible E2~Ub, demonstrating an atypical E3-dependent activation mechanism. Altogether, we postulate the E2HTH motif evolved to provide new functionalities that can be harnessed by E3s and permits additional regulation to facilitate specific E2-E3-mediated polyubiquitination

In silico APC/C substrate discovery reveals cell cycle-dependent degradation of UHRF1 and other chromatin regulators.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease