Eric Kong

IM-2/11-AP
Introduction​: Treatments of Castration-Resistant Prostate Cancer Cells (CRPC) typically

consist of targeting androgen receptors. This is called Androgen deprivation therapy (ADT) and

it is mainly for advanced Prostate Cancer. ADT is designed to disrupt the androgen receptor

(AR)

pathway such as the full-length androgen receptor’s (AR-FL), complete with an intact ligand-

binding domain (LBD). Prostate tumors that progress despite first-line ADT (e.g., LHRH

analogs), generally termed castration-resistant prostate cancer (CRPC), frequently show

continued androgen receptor signaling driven by intratumoral androgens as well as elevated

levels of AR-FL. In response to the importance of ligand-driven AR-FL signaling in CRPCs, a

number of clinically effective endocrine therapies targeting AR-LBD were recently developed to

treat patients with CRPCs (E.g abiraterone, MDV3100). Despite these treatments, the majority of

patients experience reactivated androgen receptor signaling. It was recently identified that

Androgen receptor splice variants that lack the functional LBD (AR-Vs) were responsible for the

persisting condition conveying adaptivity of CRPC (E.g. AR-V7, ARV567ES). AR-Vs originate

from androgen receptor transcripts with insertions of cryptic exons downstream of the coding

sequences for androgen receptor DNA-binding domain or due to the absence of exons coding for

AR-LBD. Due to the absence of ligand-binding domains, Castration-Resistant Prostate Cancer

cells require the use of specific inhibitory compounds that target other factors besides the

androgen receptors. Triptolide, a compound found in the Chinese medicinal plant, Thunder God

Vine, can be used to inhibit specifically the transcription aspect of Castration-Resistant Prostate

Cancer Cells.
 Eric Kong
IM-2/11-AP
Background: ​Triptolide, a compound found in the Thunder God Vine, a Chinese medicinal

plant, has been shown to have possible anticancer and immunosuppressive activities. It is an

irreversible inhibitor of eukaryotic transcription through covalent modification of XPB, a subunit

of the general transcription factor TFIIH. Cys342 of XPB specifically was identified as the

residue that undergoes covalent modification by the epoxide group of triptolide. Mutation of

Cys342 of XPB to threonine, an amino acid that is used in the biosynthesis of proteins, conferred

resistance to triptolide on the mutant protein. Replacement of the endogenous wild-type XPB

with the Cys342Thr mutant in a cell line (HEK293T) rendered it completely resistant to

triptolide, thus validating XPB as the physiologically relevant target of triptolide. Together, these

results deepen the understanding of the interaction between triptolide and XPB and have

implications for the future development of new analogues of triptolide as leads for anticancer and

immunosuppressive drugs.

Control 1​
: Triptolide, a compound found in the Thunder God Vine, a Chinese medicinal plant,

has been shown to have possible anticancer and immunosuppressive activities. It is an

irreversible inhibitor of eukaryotic transcription through covalent modification of XPB, a subunit

of the general transcription factor TFIIH. Cys342 of XPB specifically was identified as the

residue that undergoes covalent modification by the epoxide group of triptolide. Mutation of

Cys342 of XPB to threonine, an amino acid that is used in the biosynthesis of proteins, conferred

resistance to triptolide on the mutant protein. Replacement of the endogenous wild-type XPB

with the Cys342Thr mutant in a cell line (HEK293T) rendered it completely resistant to

triptolide, thus validating XPB as the physiologically relevant target of triptolide. Together, these

results deepen the understanding of the interaction between triptolide and XPB and have
 Eric Kong
IM-2/11-AP
implications for the future development of new analogues of triptolide as leads for anticancer and

immunosuppressive drugs including inhibition of the activity of a number of unrelated

transcription factors and global inhibition of mRNA synthesis. By using a systematic top-down

approach with the inhibitory effect of triptolide on RNA synthesis as the starting point, it was

identified the Xeroderma Pigmentosum B (XPB)/ERCC3 subunit of TFIIH was the new

molecular target of triptolide. We showed that triptolide forms a covalent complex with XPB and

inhibits its DNA-dependent ATPase activity without affecting its DNA helicase activity. A

number of putative cellular targets of triptolide have been reported to date. Among them are the

calcium channel polycystin-2, the membrane protease ADAM10, the dCTP pyrophosphatase

(DCTPP

Several variants of triptolide have been developed as potential anticancer and

immunosuppressive drug leads. They include PG490-88 and WilGraf which help in treating graft

rejection after organ transplantation, LLDT8 is for treating rheumatoid arthritis and Minnelide

for treating cancer. Among these alternatives, Minnelide is currently undergoing Phase I clinical

trial for cancer. All of these analogues still contain the compound triptolide in clinical

development and maintain the intact core structure of triptolide. However, Triptolide is still a

very toxic substance and requires an effective delivery system that minimizes the toxic effect and

maintains low-risk.1), and the kinase-regulating protein TAB1. [The Researchers] assessed the

cellular activity as well as the ability of each of the four analogues to covalently bind to XPB by

using a “binding-dialysis-activity recovery” sequence. All four analogues suffered from a

significant loss in activity for inhibition of cell proliferation and the ATPase activity of TFIIH,

albeit to different degrees (Sweeney, Christopher J., et al. 3).

 Eric Kong
IM-2/11-AP
Triptolide is a very toxic substance by itself. Its clinical uses have been limited by its

toxicity and low-water solubility that is typically engaged in the delivery process. However, a

conjugate has been formed that binds Triptolide to glucose. Prostate Cancer cells have a higher

metabolic rate of activity because they consistently grow and reproduce faster than typical cells.

Because of the increased metabolic activity, they develop an overexpression of glucose

transporters so by conjugating the Triptolide with glucose, it can increase the effectiveness and

absorption of glucose.

Despite its great promise as an anticancer drug lead, attempts to develop triptolide and its

synthetic derivatives over the past few decades have not succeeded. Several generations of

clinical candidates have failed except for Minnelide, the latest triptolide derivative that has

entered a phase I human clinical trial. The major obstacles for triptolide to becoming a clinically

useful drug include its general toxicity and lack of water solubility (Titov, D. V. 3).

Control 2​: Triptolide is a very toxic substance by itself. Its clinical uses have been limited by its

toxicity and low-water solubility that is typically engaged in the delivery process. However, a

conjugate has been formed that binds Triptolide to glucose. Prostate Cancer cells have a higher

metabolic rate of activity because they consistently grow and reproduce faster than typical cells.

Because of the increased metabolic activity, they develop an overexpression of glucose

transporters so by conjugating the Triptolide with glucose, it can increase the effectiveness and

absorption of glucose.

Despite its great promise as an anticancer drug lead, attempts to develop triptolide and its

synthetic derivatives over the past few decades have not succeeded. Several generations of

clinical candidates have failed except for Minnelide, the latest triptolide derivative that has
 Eric Kong
IM-2/11-AP
entered a phase I human clinical trial. The major obstacles for triptolide to becoming a clinically

useful drug include its general toxicity and lack of water solubility (Titov, D. V. 3). One strategy

to reduce the toxicity of triptolide is to deliver it selectively to tumor cells over their normal

counterparts. A unique characteristic of tumor cells is that they have a much higher demand for

glucose than normal cells partially owing to the Warburg effect. Consequently, most cancer cells

overexpress one or more isoforms of glucose transporters (GLUTs), particularly GLUT1 and

GLUT3, to sustain their growth and survival. The overexpression of GLUTs in tumor cells has

been exploited to target tumor cells selectively by conjugating cytotoxic drugs to glucose.

Glucose conjugates have been made for a number of cytotoxic drugs such as ifosfamide

and taxol and have been shown to have lower toxicity and higher tumor cell selectivity in vitro

(Talk about different delivery systems). We designed and synthesized five glutriptolide

derivatives, compounds 2–6 (Figure 1). We chose the C14 hydroxy group as the site of linkage to

glucose as it is the most reactive functional group in triptolide and can undergo facile chemical

modification. Whereas there are multiple sites in glucose that can be used to connect to

triptolide, the C1 hydroxy group in glucose has been successfully used to form active conjugates

with a number of known drugs (He, Q., et al. 3).

Control 3​
: To test Triptolide’s effect on different cells and particular genes, different cell lines

were created with the use of CRISPR method. Certain genes were knocked out (Cell KO) and

were created to isolate certain genes and analyze the effect of various concentrations of

Triptolide on the transcription aspect. Next, cells were cultured over a set amount of time with

the presence of the drug. To isolate the genes, an RNA extraction process was implemented.
 Eric Kong
IM-2/11-AP
Once the RNA was extracted, RNA transcription was used to create a strand of DNA to be

analyzed. Celltiter glo was then implemented to analyze the luminescence given off the

Carbamoyl phosphate synthetase I (CPS1), a ligase enzyme located in the mitochondria involved

in the production of urea (RNA extraction, Reverse DNA transcription, luminescence, Cell

Culturing, cell KO with CRISPR)

CONTROL 3 INCOMPLETE UNTIL DATA COLLECTION