TCID

TCID is a potent, selective and cell-permeable inhibitor of UCHL3 (ubiquitin carboxyl-terminal esterase L3 (ubiquitin thiolesterase)) with IC50 of 0.6 μm. It is also a 125 fold less potent inhibitor of UCH-L1 (IC50 = 75 μm). [1]

UCHL3 belongs to the deubiquitinating enzyme family that removes ubiquitin from polypeptides. It processes ubiquitin precursors and ubiquitinated proteins to regulate cellular ubiquitin levels.

GlyT2 ubiquitination was diminished by inhibition of UCHL3 with TCID (10 μm) and inhibition of UCHL1 with LDN-57444 (10 μm) in brainstem and spinal cord primary neurons, and the effect was more significant when cell was treated with the inhibitors for a prolonged period. [2] In transfected MDCK cells, TCID (10 μm for 30 mins) was not able to affect the UCH-L1 inhibitor promoted accumulation of YFP-GLT-1 in intracellular vesicles. [3]

References:
[1] Liu Y, Lashuel HA, Choi S, Xing X, Case A, Ni J, Yeh LA, Cuny GD, Stein RL, Lansbury PT Jr.  Discovery of inhibitors that elucidate the role of UCH-L1 activity in the H1299 lung cancer cell line.  Chem Biol. 2003 Sep;10(9):837-46.
[2] de Juan-Sanz J, Núñez E, López-Corcuera B, Aragón C.  Constitutive endocytosis and turnover of the neuronal glycine transporter GlyT2 is dependent on ubiquitination of a C-terminal lysine cluster.  PLoS One. 2013;8(3):e58863.
[3] Martínez-Villarreal J, García Tardón N, Ibáñez I, Giménez C, Zafra F.  Cell surface turnover of the glutamate transporter GLT-1 is mediated by ubiquitination/deubiquitination.  Glia. 2012 Sep;60(9):1356-65.

Treatment with Thalidomide and Cyclophosphamide (TCID) is Superior to Vincristine (VID) and to Vinorelbine (VRID) Regimens in Patients with Refractory or Recurrent Multiple Myeloma.[Pubmed:23730012]

Indian J Hematol Blood Transfus. 2012 Jun;28(2):67-76.

Treatment of relapsed or refractory multiple myeloma remains a challenge and novel treatment regimen are required. Here, a matched pair analysis was performed comparing TCID (thalidomide, cyclophosphamide, idarubicin, dexamethasone) treatment to the treatment of patients with VID (vincristine, idarubicin, dexamethasone) or with VRID (vinorelbine, idarubicin, dexamethasone) for relapsed or refractory multiple myeloma. In total, 197 patients were enrolled in multicenter trials. After matching for important prognostic variables 46 matched-pairs (total of 138 patients) could be analysed with regard to survival, toxicity and efficacy. Interestingly, a significant improvement of overall response rate (ORR) for TCID treatment compared to VID and VRID was found. In addition, TCID treatment also led to a significantly higher overall survival (OS) as well as progression-free survival (PFS) compared to VID and VRID. In conclusion, TCID treatment appears to be superior to VRID and VID treatment in patients with progressive or refractory myeloma.

Virus quantitation by transmission electron microscopy, TCID(5)(0), and the role of timing virus harvesting: a case study of three animal viruses.[Pubmed:23603437]

J Virol Methods. 2013 Aug;191(2):136-40.

Quantitation of viruses is practised widely in both basic and applied virology. Infectious titration in cell cultures, the most common approach to it, is quite labour-intensive and alternative protocols are therefore sought. One of the alternatives is transmission electron microscope (TEM) quantitation using latex particles at a known concentration as a reference for counting virus particles. If virus TCID(5)(0) is determined in parallel, the ratio of infectious to non-infectious virus particles may be established. This study employs such an approach to compute the number of virus particles and TCID(5)(0), and establish their correlation for three viruses: Canine adenovirus 1 (CAdV-1), Feline calicivirus (FCV) and Bovine herpesvirus 1 (BoHV-1). Each of the viruses was grown in five replicates until complete cytopathology was recorded (time 0), then frozen. They were thawed, filter-sterilised and left for additional periods of 16, 32 and 48 h at 37 degrees C. At each time point, the infectious ability of the virus was characterised by TCID50 and the number of virions quantified by TEM, in order to evaluate the influence of timing on virus harvest. The virus particle count determined by TEM did not change for any of the viruses throughout the experiment. The relationship between virus particle counts with TCID(5)(0) at time 0 showed good linearity response; their ratio was almost constant. The virus particle-to-TCID(5)(0) ratio varied between 146 and 426 (mean+/-SD: 282+/-103) for CAdV-1, between 36 and 79 (57+/-18) for FCV and between 110 and 249 (167+/-53) for BoHV-1. The proportion of non-infectious particles did not change throughout the experiment for either CAdV-1 or BoHV-1. However, a decrease in virus infectious ability disclosed by TCID(5)(0) indicated that the fraction of non-infectious particles in FCV increased 300,000 times when time 0 and 48 h were compared. The quantitation of viruses with TEM is a simple and rapid protocol for virus quantitation but account must be taken of the type of virus and harvesting time as virus counts need not necessarily correlate with virus infectious ability.

Calculating HIV-1 infectious titre using a virtual TCID(50) method.[Pubmed:19020816]

Methods Mol Biol. 2009;485:27-35.

Studies of HIV-1 replication kinetics and fitness require an accurate determination of the level of infectious HIV-1 present in virus stocks. The standard technique for measuring the level of replication-competent infectious virus in culture supernatants or patient samples is the tissue culture dose for 50% infectivity (TCID(50)), which provides an accurate assessment of the level of infectious HIV-1. However, it is a time-consuming technique which typically takes two or more weeks to complete and requires PHA-stimulated PBMC from HIV-1 seronegative donors or an appropriate cell line. Thus rapid, cell-free surrogate measures for TCID(50) are desirable. Here, we introduce the virtual TCID(50) technique: a new cell-free method estimating a surrogate of infectious titer by comparing the reverse transcriptase activity in virus stock to that of reference viruses with a known TCID(50) value. We have demonstrated that the virtual TCID(50) obtained through this technique is comparable to the actual infectious TCID(50). This method greatly simplifies the process of accurate HIV-1 titration and is particularly beneficial for studies which require titration of large number of HIV-1 isolates.

Constitutive endocytosis and turnover of the neuronal glycine transporter GlyT2 is dependent on ubiquitination of a C-terminal lysine cluster.[Pubmed:23484054]

PLoS One. 2013;8(3):e58863.

Inhibitory glycinergic neurotransmission is terminated by sodium and chloride-dependent plasma membrane glycine transporters (GlyTs). The mainly glial glycine transporter GlyT1 is primarily responsible for the completion of inhibitory neurotransmission and the neuronal glycine transporter GlyT2 mediates the reuptake of the neurotransmitter that is used to refill synaptic vesicles in the terminal, a fundamental role in the physiology and pathology of glycinergic neurotransmission. Indeed, inhibitory glycinergic neurotransmission is modulated by the exocytosis and endocytosis of GlyT2. We previously reported that constitutive and Protein Kinase C (PKC)-regulated endocytosis of GlyT2 is mediated by clathrin and that PKC accelerates GlyT2 endocytosis by increasing its ubiquitination. However, the role of ubiquitination in the constitutive endocytosis and turnover of this protein remains unexplored. Here, we show that ubiquitination of a C-terminus four lysine cluster of GlyT2 is required for constitutive endocytosis, sorting into the slow recycling pathway and turnover of the transporter. Ubiquitination negatively modulates the turnover of GlyT2, such that increased ubiquitination driven by PKC activation accelerates transporter degradation rate shortening its half-life while decreased ubiquitination increases transporter stability. Finally, ubiquitination of GlyT2 in neurons is highly responsive to the free pool of ubiquitin, suggesting that the deubiquitinating enzyme (DUB) ubiquitin C-terminal hydrolase-L1 (UCHL1), as the major regulator of neuronal ubiquitin homeostasis, indirectly modulates the turnover of GlyT2. Our results contribute to the elucidation of the mechanisms underlying the dynamic trafficking of this important neuronal protein which has pathological relevance since mutations in the GlyT2 gene (SLC6A5) are the second most common cause of human hyperekplexia.

Discovery of inhibitors that elucidate the role of UCH-L1 activity in the H1299 lung cancer cell line.[Pubmed:14522054]

Chem Biol. 2003 Sep;10(9):837-46.

Neuronal ubiquitin C-terminal hydrolase (UCH-L1) has been linked to Parkinson's disease (PD), the progression of certain nonneuronal tumors, and neuropathic pain. Certain lung tumor-derived cell lines express UCH-L1 but it is not expressed in normal lung tissue, suggesting that this enzyme plays a role in tumor progression, either as a trigger or as a response. Small-molecule inhibitors of UCH-L1 would be helpful in distinguishing between these scenarios. By utilizing high-throughput screening (HTS) to find inhibitors and traditional medicinal chemistry to optimize their affinity and specificity, we have identified a class of isatin O-acyl oximes that selectively inhibit UCH-L1 as compared to its systemic isoform, UCH-L3. Three representatives of this class (30, 50, 51) have IC(50) values of 0.80-0.94 micro M for UCH-L1 and 17-25 micro M for UCH-L3. The K(i) of 30 toward UCH-L1 is 0.40 micro M and inhibition is reversible, competitive, and active site directed. Two isatin oxime inhibitors increased proliferation of the H1299 lung tumor cell line but had no effect on a lung tumor line that does not express UCH-L1. Inhibition of UCH-L1 expression in the H1299 cell line using RNAi had a similar proproliferative effect, suggesting that the UCH-L1 enzymatic activity is antiproliferative and that UCH-L1 expression may be a response to tumor growth. The molecular mechanism of this response remains to be determined.

TCID (4,5,6,7-Tetrachloroindan-1,3-dione) is a potent and selective neuronal ubiquitin C-terminal hydrolase (UCH-L3) inhibitor with an IC50 of 0.6 μM. TCID diminishes glycine transporter GlyT2 ubiquitination in brainstem and spinal cord primary neurons.

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