Rational Design and Synthesis of Novel Kinase Inhibitors against RET, PDHK, FAK, FLT3, ACK1-GCK and BRAF

Injae Shin 고려대학교 KU-KIST융합대학원 2022 국내박사

Kinase is an enzyme that transfers a phosphate from ATP to a substrate protein and induces signal transduction through the phosphate. It is involved in and regulates cell cycle, growth, proliferation, differentiation, development and metabolism. Kinase inhibitors have started from imatinib, for treatment of chronic myeloid leukemia (CML), launched by Novartis in 2000, and 73 kinase inhibitors have been approved by the FDA as of 2022. However, it is necessary to develop inhibitors based on the role of kinases that have not yet been revealed and their association with diseases, as only less than 10% of the target kinases among the 580 known kinases have been developed as therapeutic agents. In this thesis, it deals with design and synthesis of selective kinase inhibitors (RET, PDHK) in chapter 1-2 and multi-targeted kinase inhibitors (FAK, FLT3, ACK1/GCK, BRAF) in chapter 3-6 for anticancer treatment. Selective kinase inhibitors may be disadvantageous in terms of efficacy, but have advantageous features in terms of toxicity, and conversely, multi-target kinase inhibitors may be disadvantageous in terms of toxicity, but have advantageous features in terms of activity. In chapter 1, I reported highly selective RET inhibitors with high potency against not only RET-wt but also RET-V804M gatekeeper mutant. This study started from our previous selective and potent RET inhibitor (1) having 3-(isoxazol-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amines moiety. Compound 1 was shown to have a high RET kinase inhibitory activity, but it displayed poor metabolic stability in mouse liver microsomes. Through structural modification of each part of the RET inhibitor (1), compound 17l of 5-aminopyrazole-4-carboxamide scaffold was derived. Moreover, metabolic stability was significantly improved compared to the previous compound (1). 17l displays not only improved metabolic stability but also improved kinase selectivity and inhibited only RET out of 369 kinases by more than 90% (1 μM of 17l). This study is about highly selective and potent RET inhibitors against RET-V804M gatekeer mutatnt and RET-wt before FDA-approval of pralsetinib and selpercatinib (highly potent against RET-V804M), providing insight into the design and synthesis of specific RET inhibitors. In chapter 2, I reported highly selective PDHK inhibitors with high potency against PDHK1/2/3/4 and with anti-proliferation activity on prostate cancer and lung cancer cells. This study started from reported crystal structures of a PDHK inhibitor VER-246608 on PDHK2. Thirty compounds were designed and synthesized through structural modification of VER-246608 at chloromethylpyrimidine (R1) and difluorobenzylacetamide (R2) moieties, and compounds 29n and 29t, having higher potency on from PDHK1/2/4 relative to VER-246608, were derived. 29t selectively inhibited only PDHK1/2/3/4 out of 336 kinases by more than 50% (1 μM of 29t) and 29n had superior anti-proliferation activity compared to compound VER-246608 in prostate cancer (DU145) and lung cancer (H1299) cells. This study is about highly selective and potent PDHK inhibitors, providing insight into the the design and synthesis of specific PDHK inhibitors as potential anticancer agents. In chapter 3, I reported dual FAK, FLT3 inhibitors possessing high potency and anti-proliferation activities on triple-negative breast cancer (TNBC) and acute myeloid leukemia (AML) cells. A novel thieno[3,2-d]pyrimidine scaffold capable of maintaining hydrophobic interactions through thiophene was derived from the diaminopyrimidine scaffold of representative FAK inhibitors. Forty compounds having a thieno[3,2-d]pyrimidine scaffold were designed and synthesized, and optimization was carried out through structure-activity relationship studies in R1 (head group) and R2 (right wing part) positions. A representative compound 56 had excellent inhibitory activities against FAK and FLT3 mutations (ITD, ITD-D835Y, D835Y, ITD-F691L, ITD-F691L), and strongly suppressed proliferation of TNBC and AML cells. Furthermore, 56 had favorable metabolic stability and little or no inhibition effects on CYPs. This study provides insight into the design and synthesis of FAK, FLT3 dual inhibitors as potential anticancer agents. In chapter 4, a structure-activity relationship study of GNF-7 derivatives was conducted for the purpose of treating acute myeloid leukemia (AML) with NRAS mutations. This study started based on the results of previous studies that GNF-7, a mul-targeted kinase inhibitor was effective for the treatment of AML with NRAS mutation through dual inhibition of ACK1/GCK. Twentynine GNF-7 derivatives were designed and synthesized, and optimization was conducted through structure-activity relationship studies in three parts of R1 (head group), R2 (tail group) and R3 (scaffold methyl group) in GNF-7. Compounds 88d and 91i, having superior inhibitory and anti-proliferative activities compared to GNF-7, were derived, and compound 88k having enhanced DMPK (PK, metabolic stability, CYP) compared to GNF-7 was also derived. Through the co-crystal structure of 88d on ACK1, it was explained why inhibitory activity of 88d was superior against ACK1 compared to GNF-7. This study provides insight into the design and synthesis of ACK1/GCK dual inhibitors to overcome AML with NRAS mutations. In chapter 5, I reported that GNF-7, a multitarget-kinase inhibitor, is capable of blocking the mutated RAS signaling pathway. This study started based on the results of previous studies that GNF-7 strongly suppressed proliferation of NRAS-G12D Ba/F3 cell line. Fourteen GNF-7 derivatives were designed and synthesized, and optimization was carried out through structure-activity relationship studies at the R1 (head group) position of GNF-7. GNF-7 and its derivatives had anti-proliferactive ativities not only on NRAS-G12D and -G12V Ba/F3 cells but also on cancer cells harboring mutated KRAS (G12C, G12D, G12V, G13D, and Q61L). Compounds 105 and 109, having superior anti-proliferactive ativities in Ba/F3 cells transformed with mtRAS and cancer cells harboring mtRAS compared to GNF-7, were derived. Furthermore, 105 and 109 are capable of blocking mtRAS downstream signaling pathway (MAPK and PI3K/AKT) through inhibition of phospho-MEK/ERK (MAPK signaling) and -AKT (PI3K/AKT signaling) in cancer cells harboring mtRAS. This study reported that GNF-7, a multi-target-kinase inhibitor has capability of inhibiting mtRAS-signaling pathway, which causes 30% of all cancers and has a very high medical unmet need. In chapter 6, I reported that GNF-7, a multi-target-kinase inhibitor, has a therapeutic effect on melanoma harboring BRAF class I/II/III mutations. This study started based on the results of previous studies that GNF-7 strongly suppressed cancer cells harboring BRAF class II/III mutations insensitive to vemurafenib. Seven GNF-7 derivatives were designed and synthesized, and optimization was carried out through structure-activity relationship studies at the R1 (head group) position of GNF-7. Compounds 106 displayed enhanced anti-proliferative activities compared with GNF-7 against melanoma cells harboring BRAF class I/II/III mutations. Furthermore, 106 substantially inhibited activation of MEK/ERK (MAPK signaling) and AKT (PI3K/AKT signaling). This study reported that GNF-7, a multi-target-kinase inhibitor is capable of overcoming vemurefenib resistant melanoma harboring pan-class (class I/II/III) BRAF mutations. I rationally designed and synthesized the novel kinase inhibitor against RET, PDHK, FAK, FLT3, ACK1-GCK, BRAF by identifying binding mode of known inhibitor to target kinases based on the X-ray co-crystal structures in chapter 1-6. Structure-activity relationship (SAR) studies were conducted to validate rational design and to derive potent inhibitors. Sructure-property relationship (SPR) studies were also conducted to derive inhibitors having better drug properties. Based on these studies, I discovered novel inhibitors having superior not only activities but also drug properties compared to known inhibitors. Taken together, this thesis provides insight into the design and synthesis of novel kinase inhibitors against new kinase targets as well as RET, PDHK, FAK, FLT3, ACK1-GCK, BRAF.

천연자원으로부터 serine/threonine protein kinase inhibitor의 탐색 및 분리

梁惠英 明知大學校 大學院 2005 국내석사

Signal transduction system은 세포의 생명현상을 유지하는데 가장 중요한 기구중 하나이며, 여기에는 많은 protein kinase가 작용한다. 따라서 protein kinase inhibitor의 발견은 학문의 발달에 기여하고 의약품의 개발에 적용할 수 있을 것으로 기대되어 매우 중요하게 여겨진다. 본 연구에서는 방선균 내에 작용하는 eukaryotic-type protein kinases에 대해 연구하고, serine/threonine protein kinase의 활성을 저해하는 noble compounds를 발견하려는 일환으로, 천연자원으로부터 protein kinase inhibitor를 분리하기로 하였다. 한편, 인산화 저해 활성을 검정하기 위해 FITC-labelled peptide를 사용한 nonradioisotope phosphorylation assay system 및 S. griseus를 사용한 cell based assay system을 적용하여 실험하였다. 330여종의 식물 추출물을 대상으로 nonradioisotope phosphorylation assay를 실시한 결과, 강진향이 IRS-2 peptide의 인산화를 강하게 저해하는 것으로 검출되었다. 또한 강진향을 S. geiseus IFO13350의 agar plate에 적용하였을 때, 형태적 분화를 저해시키는 것으로 관찰되었으며, 이는 강진향 추출물이 방선균의 형태적 분화에 작용하는 serine/threonine protein kinases를 저해하여 나타난 결과로 여겨진다. Kinase inhibitor를 분리하기 위해 강진향 추출물을 용매 추출 및 여러 단계의 chromatography에 적용하였으며, 최종적으로 KJH-P1, P2, P3, P4, P5, P6, P7을 분리해 내었다. 분리한 KJH-compounds는 다양한 assay 방법을 적용하여 활성을 검정하였으며, 그 결과 방선균의 형태적 분화를 저해하는 강력한 kinase inhibitor 인 KJH-P2를, 항암효과를 나타내는 Akt kinase inhibitor인 KJH-P3를 각각 검출하게 되었다. Signal transduction system is one of the most important tools to maintain the life, and many protein kinases are involved in cellular signal transduction system. Finding a protein kinase inhibitor seems to be very valuable because it can be used for studying cell biology and applicable for pharmaceuticals. Many kinds of herbs were collected and screened for the chemicals that can act as an inhibitor of protein kinase. Nonradioisotope phosphorylation assay system using FITC-labelled peptide and cell based assay system using S. griseus IFO13350 were introduced for kinase assay. Among the 330 kinds of herb extracts, Dalbergia odorifera gave the strongest inhibitory activity for the kination of IRS-2 peptide. When the Dalbergia odorifera extract was applied on the plate of S. geiseus IFO13350, the morphological differentiation was also severely inhibited, which suggested that many protein kinases of Streptomyces that are involved in morphological differentiation are inhibited by the extract. To isolate the inhibitor, the Dalbergia odorifera extract were applied to solvent extraction and to several steps of chromatography. And finally KJH-P1, P2, P3, P4, P5, P6, P7 were seperated. Seperated KJH-compounds were tested their acitivity by applying various assay. In conclusion, KJH-P2, which is a powerful kinase inhibitor inhibiting the morphological differenciation of S.griseus and KJH-P3, which is a Akt kinase inhibitor acting as a anti-cancer were isolated.

Characterization of Ecdysteroid, cAMP, and cAMP-dependent protein kinase activity and kinase inhibitor activity in the diapausing larvae of overwintering chestnut weevil, Curculio dentipes : 밤바구미(Curculio dentipes) 월동 중 휴면유충의 Ecdysteroid의 농도 변화와 cAMP, cAMP-depende

류진수 Graduate School of Kon-Kuk University 1995 국내박사

밤바구미(Curculio dentipes) 월동중 휴면유충의 발생에 따른 cyclic AMP와 ecdysone 및 20-hydroxyecdysone의 농도를 High Performance Liquid Chromatography (HPLC)를 이용하여 조사하고, cyclic AMP-dependent protein kinase의 활성도를 측정하여 이들의 기능적 상관관계를 연구하였으며, cyclic AMP-dependent protein kinase와 cAMP-dependent protein kinase inhibitor를 부분정제하여 Liquid Scintillation counter(LSC)를 이용하여 그들의 활성도와 저해능을 비교 연구하였다. Ecdysone 농도는 pre-diapausing larvae(19.81㎍/g)에서 최저치를 나타내었고, diapausing larvae(29.97㎍/g)에서 최대를 보였으며, post-diapausing larvae에서 25.71㎍/g로 약간 감소하였다. 그러나 20-hydroxyecdysone은 pre-diapausing larvae(29.29㎍/g)에서 최고의 농도를 보인 후, diapausing larvae(5.05㎍/g)에서 급격히 감소하였고, post-diapausing larvae에서 다시 상승하는 양상을 보여 서로 역관계를 나타내었다. Cyclic AMP와 cyclic AMP-dependent protein kinase의 활성도는 pre-diapausing larvae(0.57μM/g, 9.09 unit/mg)에서 최고치를 나타내었고, diapausing larvae(0.14 μM/g, 4.16 unit/mg)에서는 급격히 감소하였으며, post-diapausing larvae(0.29 μM/g, 7.52 unit/mg)에서 다시 상승하는 경향을 보였다. DEAE-cellulose fractionate를 통하여 정제된 cAMP-dependent protein kinase의 in vitro 실험에서, cAMP-dependent protein kinase는 저농도의 cAMP에 의해서도 활성화를 이루며, 외생기질에 대한 선호도는 slightly lysine rich histone f_(2b), histone type Ⅱ, histone f_(2a) 의 순서로 나타났다. 2가 양이온의 cAMP-dependent protein kinase에 대한 활성화 효과는 5mM의 Mg^(2+)을 첨가했을 때 최적을 나타냈고, 반면에 Ca^(2+)는 저해효과를 나타냄이 확인되었다. 5% TCA precipitate를 이용하여 측정한 cAMP-dependent protein kinase inhibitor의 저해 활성도는 pre-diapausing larvae에서 0.6802 unit/mg을 보인후, diapausing larvae에서 4.61 unit/mg으로 급격히 상승하였고, post-diapausing larvae에서 0.6560 unit/mg로 다시 감소하여 cAMP-dependent protein kinase와 역관계로 나타났다. 이상의 결과로 ecdysteroids, cAMP 및 cAMP-dependent protein kinase의 농도 변화가 휴면 발생동안에 유사한 경향으로 나타나, 이들 생리활성물질이 서로 상호작용을 통해 곤충의 생존 적응형태인 휴면에 영향을 미치는 것으로 생각된다. 또한 cAMP-dependent protein kinase inhibitor의 저해활성도가 cAMP-dependent protein kinase의 활성도와 역관계를 보여 cAMP-dependent protein kinase inhibitor는 효과적으로 저해능을 나타내었다. 곤충의 생존 적응형태인 휴면에서 ecdysteroid 호르몬은 휴면의 개시와 종결에 관여하는데, 이는 제2차 전달자인 cAMP의 활성화에 영향을 미치는 것으로 이해할 수 있으며, 활성화된 cAMP의 효과는 cAMP-dependent protein kinase에 의해 중재되는데, 이러한 kinase는 inhibitor와 상호작용 함으로서 그농도가 조절되는 것으로 생각된다. The concentration of ecdysteroid and cyclic AMP(cAMP) of whole body, activity of cyclic AMP-dependent protein kinase(PKA) and inhibition activity of cAMP-dependent protein kinase inhibitor(PKI) in partial purification were measured in chestnut weevil, Curculio dentipes, according to the pre-diapausing, diapausing, and post-diapausing larvae respectively. Ecdysteroid titers, cAMP concentration and enzyme activities were measured by High Performance Liquid Chromatography and Liquid Scinti1lation Counter, and the simultaneous changes of three components during development was also examined. The concentration of ecdysone was found to be the minimum(19.81μg/g) at pre-diapausing larvae and the maximum(29.97μg/g) at diapausing larvae, and it decreased to 25.71μg/g at post-diapausing larvae, and then rotatively kept in high level. But the amount of 20-hydroxy-ecdysone was highest at pre-diapausing larvae(29.29μg/g) and minimum(5.05μg/g) at diapausing larvae, but it increased to the 12.53μg/g at post-diapausing larvae. The cAMP concentration was increased to the maximum at the pre-diapausing larvae(0.57 μM/g), but it decreased at the diapausing larvae(0.14 μM/g). At the post-diapausing larvae, it increased again(0.29 μM/g). The activity of cAMP-dependent protein kinase was shown the highest at the pre-diapausing larvae(9.09 unit/mg) and decreased at the diapausing larvae(4.16 unit /mg) in DEAE-cellulose fraction from the purification steps, wherase it increased again at post-diapausing larvae(7.52 unit/mg). As the results of in vitro experiment with purified cAMP-dependent protein kinase by DEAE-cellulose chromatography, the enzyme was activated by low concentration of cAMP, and preferred mostly slightly lysine rich histone f_(2b), then histone type Ⅱ and histone f_(2a) as exogenouse substrates. The optimal effect of cAMP on the activation of cAMP-dependent protein kinase was observed in the presence of 5mM Mg^(2+). But Ca^(2+) inhibited the activation of cAMP-dependent protein kinase by cAMP. The inhibition activity of cAMP-dependent protein kinase inhibitor in the pre-diapausing larvae indicated 0.6802 unit/mg. After that, it sharply increased and its maximum activity reached at diapausing larvae with 4.61 unit/mg. At the post-diapausing larvae, its inhibition activity declined to 0.6560 unit/mg. This results indicated reverse relationships between cAMP- dependent protein kinase inhibitor and cAMP-dependent protein kinase and cAMP-dependent protein kinase inhibitor inhibited effectively cAMP-dependent protein kinase activity. Therefore, it is suggested that cAMP and cAMP-dependent protein kinase were interplayed with ecdysone, regarding that the changes in cAMP, cAMP-dependent protein kinase and ecdysone concentration were shown to be correlated during the development of the chestnut weevil, Curculio dentipes.

Biological Assessment and Action Mechanism Studies of Novel Kinase Inhibitors against ALK1, ACK1-GCK, PDHK and FAK-FLT3

Hanna Cho 고려대학교 KU-KIST융합대학원 2020 국내박사

Kinases have been important drug targets in the past 20 years against a variety of human diseases including cancer, inflammatory, neurodegenerative, metabolic and cardiovascular diseases. To date, 48 kinase inhibitors have been approved by FDA, and 43 of them are used in the treatment of cancer. In this thesis, we evaluated and explored novel kinase inhibitors targeting ALK1, ACK1-GCK, PDHK and FAK-FLT3 in terms of their action mechanism in angiogenesis and cancer. Also, we found a new target of the existing drug, AZD4547 as a program of drug repurposing. In part 1, a reversible version of L-783277 was explored as a selective and potent ALK1 inhibitor. Our study showed that 1a possesses great kinase selectivity against a panel of 342 kinases and more potent activity against ALK1 than L-783277. Compound 1a suppresses the BMP9-induced Smad1/5 pathway by mainly inhibiting ALK1 in C2C12 cells. To the best of our knowledge, 1a is the first selective ALK1 inhibitor. Furthermore, 1a promotes angiogenesis in both endothelial tube formation and microfluidic chip-based 3D angiogenesis assays, suggesting that 1a could be a lead compound for therapeutic angiogenesis agents. For overriding NRAS mutant-driven AML, in part 2, GNF-7 derivatives were evaluated. 2a, 2c and 2e, which possess single-digit nanomolar inhibitory activity against both ACK1 and GCK strongly suppress proliferation of mtNRAS expressing AML cells via apoptosis and AKT/mTOR signaling blockade. Moreover, 2d prolonged life-span of Ba/F3-NRAS-G12D injected mice and significantly delayed tumor growth of OCI-AML3 xenograft model without causing the prominent level of toxicity found with GNF-7. Taken together, this study provides insight into the design of novel ACK1 and GCK dual inhibitors. Pyruvate dehydrogenase kinases (PDHKs) promote abnormal respiration in cancer cells. In part 3, 3e containing unique structural features was explored as a selective PDHK inhibitor. 3e induces apoptosis of cancer cells via mitochondrial dysfunction, suppresses tumorigenesis and displays a synergistic effect on satraplatin suppression of cancer cell proliferation. Focal adhesion kinase (FAK) is over-expressed in highly invasive and metastatic cancer. To develop a novel FAK inhibitor, in part 4, we assess the biological function of thieno[3,2-d]pyrimidine scaffold derivative 4 exhibiting great potency against FLT3 as well as FAK. In the in vivo studies using MDA-MB-231 inoculated xenograft and orthotopic mouse model, 4 demonstrated a good bioavailability and significant suppression of the tumor growth and metastasis without appreciable toxicity. These data collectively support the potential of 4 to be an effective treatment for cancer. As a drug re-purposing program, in part 5, we designed to find a new target of AZD4547. Through the cancer cells profiling with in-house cancer cell panel, we found that AZD4547 shows anti-proliferation activity on KM12 harboring TPM3-NTRK1 fusion gene (GI50= 100 nM). In vitro biochemical assay reveals that AZD4547 virtually exhibits 18.7 nM, 22.6 nM and 2.9 nM of IC50 against TRKA, B and C respectively. In cellular context, AZD4547 shows anti-tumor activity on KM12 by downregulating the TRKA/B signaling (AKT, PLCg) as well as MAPK target genes (DUSP6, CCND1, ETV1) and E2F1. In addition, AZD4547 delays tumor growth in KM12 xenograft model. Together, we demonstrate a potential application of AZD4547 for NTRK fusion driven cancers.

Design, synthesis and screening of new pyrazole derivatives as kinase inhibitor

박병선 경희대학교 대학원 2010 국내석사

Cancer is generated by excessive expression of extracellular signal-regulated kinase. Principally, it is generated by somatic point mutation of RAS and RAF as a signal transfer factor in ERK signaling pathway. V599E mutation of B-Raf gene in particular has higher frequency relative to other mutations. This mutation activates ERK signaling pathway excessively and this excessive activation was observed in various malignant tumors such as metastatic melanoma. We have designed, synthesized and evaluated a number of pyrazole derivatives as inhibitors for B-Raf kinase. Each of the pyrazole derivatives (1a-d) was obtained by 11 steps of reaction in a moderate yield (56~64%). All the newly synthesized compounds were screened using A375P melanoma cell line in vitro. But the synthesized new compounds generally did not show good activity. Accordingly, compound 1d was selected for screening over a panel of 45 different kinases in order to investigate its kinase inhibitory profile. The pyrazole compound 1d was tested initially at a single dose concentration of 10 uM. At this concentration, a 94% inhibition of the enzymatic activity of ROS1 kinase was observed, while the inhibition in activity was below 30% in all of the other kinases. The pyrazole compound 1d was further tested in a 10-dose IC50 mode and showed an IC50 value of 199 nM for ROS1 kinase. ROS1 protein is a receptor tyrosine kinase that has been reported mainly in malignant glioblastoma, and until now, there is no selective inhibitor for this kinase. The pyrazole compound 1d can be used as a promising lead for the development of new selective inhibitors for ROS1 kinase, and it may open the way for new selective therapeutics for glioblastoma. 암은 세포의 신호전달경로 내에서 Extracellular signal-regulated kinase (ERK)의 과도한 발현에 의해 발생하게 되는데, 주로 신호전달 요소 중에 하나인 Ras, Raf의 돌연변이에 의해서 발생하게 된다. 특히, B-Raf gene의 돌연변이 중에 하나인 B-Raf V599E는 다른 돌연변이에 비하여 매우 높은 빈도로 발생한다. 이러한 돌연변이는 ERK 신호전달경로를 과도하게 활성화시키게 되고, 이런 과도한 활성화는 전이성 흑색종과 같은 악성 종양의 주요 원인이 된다. 따라서 B-Raf kinase의 활성을 저해할 수 있는 저해제로써 다양한 피라졸 유도체들을 설계, 합성하고 합성한 모든 화합물들은 A375P cell line을 이용하여 생물학적 활성을 측정하였다. 각각의 피라졸 유도체들 (1a-d)은 평균 (56~64%)의 yield로 11 step의 반응을 통해 얻었다. 합성한 화합물들에 대해 활성도를 측정한 결과, A375P cell에 대해 좋지 않은 활성 저해능을 보였다. 따라서 합성한 화합물 중에 1d 화합물을 선택하여 45가지의 발암과 관련이 있는 kinase에 대해 screening을 실시하였고, 그 중에서 오직 ROS1 kinase에 대해서만 선택적으로 94%의 높은 활성 저해능을 갖는 것이 관찰되었고, IC50 값을 측정한 결과 199nM의 IC50값을 확인할 수 있었다. ROS1 protein은 receptor tyrosine kinase의 하나로써 ROS1의 과도한 발현은 악성 뇌종양인 교모세포종의 주요 원인으로 알려져 있고, 현재까지 ROS1 kinase의 활성을 선택적으로 저해할 수 있는 저해제는 존재하지 않는다. 이번 실험을 통해 합성한 1d 화합물은 ROS1 kinase를 선택적으로 저해할 수 새로운 저해제로써, glioblastoma를 치료할 수 있는 새로운 표적 치료제로써의 길을 열 것으로 기대된다.

Design, synthesis and biological evaluation of new 4-aryloxy-5-(substituted amino)pyrimidines and quinazolinedione derivatives as potential anticancer kinase inhibitors

백소라 경희대학교 대학원 2017 국내석사

The aims from this study are to design and synthesize new hit antiproliferative compounds with novel scaffolds able to inhibit the oncogenic epidermal growth factor receptor (EGFR) and DDR1 kinase. Regarding EGFR study, fourteen 4-aryloxy-5-aminopyrimidine scaffold-derived compounds were designed, synthesized and evaluated as hit anticancer EGFR inhibitors. 4-Aryloxy-5-benzamidopyrimidines inhibited EGFR with IC50 1.05~5.37 μM. Cell-based assay of the most potent EGFR inhibitor hit (1.8a) confirmed its cytotoxicity against different cancerous cells. Cell-based evaluation of 4-aryloxy-5-(thio)ureidopyrimidines in conjunction with compare analysis suggested them as antiproliferative hits possibly associated with sex-hormones dependent/related pathways. Molecular docking study provided a plausible explanation of incapability of 4-aryloxy-5-(thio)ureidopyrimidines to inhibit EGFR and suggested a reasonable binding mode of 4-aryloxy-5-benzamidopyrimidines which provides a basis to develop more effective optimized ligands. Regarding DDR1 kinase study, fifteen quinazolinedione scaffold-derived compounds were designed, synthesized and evaluated as potential DDR1 inhibitors. Eight compounds exhibited very weak inhibitory activity for DDR1 kinase namely 2.6a, 2.6d, 2.6e, 2.6f, 2.7c, 2.7d, 2.7g and 2.8a. Three compounds showed weak inhibitory activity namely 2.6b, 2.7e and 2.7f. Moderate activity has been exerted with compounds 2.6c, 2.7a and 2.7b with percent inhibition of 29.18%, 27.53% and 22.61%, respectively. Surprisingly, compound 2.6g showed the highest inhibitory activity over DDR1 kinase with %inhibition of 70.20%. Structure activity relationship (SAR) study has been carried out and suggested substituent position affects the inhibitory effect. In addition, kinase selectivity panel for the most active compound 2.6g has been performed. The result showed compound 2.6g selectively inhibits DDR1 kinase. 이 연구의 목적은 종양을 형성하는 표피성장인자수용체(EGFR)과 DDR1 인산화효소를 저해할 수 있는 새로운 골격을 가지는 항증식성 화합물들을 디자인하고 합성하는데 있다. EGFR 연구와 관련하여, 14개의 4-aryloxy-5-aminopyrimidine 골격을 가지는 화합물들이 디자인되고 합성되었으며, 새로운 EGFR 저해제로서 평가되었다. 4-Aryloxy-5-bezamidopyrimidine 유도체들은 EGFR을 IC50 1.05~5.37 μM 농도로 저해하였다. 가장 강력한 새로운 EGFR 저해제인 1.8a 화합물의 Cell line assay를 통해 각기 다른 암세포에 대한 1.8a 화합물의 세포독성을 확인했다. 비교분석과 함께4-aryloxy-5-(thio)ureidopyrimidine 유도체들의 세포주 분석은 이 유도체들을 성호르몬 의존적/연관된 경로와 관련이 있는 가능한 항증식성 화합물로서 제안했다. Molecular docking 연구를 통해, 4-aryloxy-5-(thio)ureidopyrimidine이 EGFR을 저해할 수 없는 이유와 더 효과적으로 최적화된 리간드를 개발하기 위한 토대를 제공하는 4-aryloxy-5-(thio)ureidopyrimidine의 타당한 결합모드를 알 수 있다. DDR1 인산화 효소 연구와 관련하여, 15개의 quinazolinedione 골격을 가지는 화합물들이 디자인되고 합성되었으며 잠재적인 DDR1 저해제로서 평가되었다. 8개 화합물(2.6a, 2.6d, 2.6e, 2.6f, 2.7c, 2.7d, 2.7g 그리고2.8a)은 DDR1 인산화효소에 대해 아주 약한 저해 효과를 나타냈으며 3개 화합물(2.6b, 2.7e 그리고2.7f)은 8개의 화합물보다는 강한 저해 효과를 나타냈지만 이 또한 약학 저해 효과를 나타냈다. 또 다른 3개 화합물(2.6c, 2.7a 그리고2.7b) 은 DDR1 인산화효소에 대해 각각 29.18%, 27.53% 그리고 22.61%의 저해 효과를 나타냈다. 놀랍게도, 화합물2.6g는 DDR1 인산화효소에 대해 70.20%의 가장 높은 저해 효과를 나타냈다. 합성된 화합물에 대한DDR1 인산화 효소 저해 효과 결과를 토대로 구조활성관계(SAR) 연구가 수행 되었으며, 이를 통해 치환기의 위치와 수가 DDR1에 대한 저해 효과에 크게 영향을 미치는 것으로 나타났다. 화합물 2.6g에 대한 키나아제 패널테스트가 수행되었고 이를 통해 화합물 2.6g는 DDR1을 선택적으로 저해한다는 것을 알 수 있다.

Biological Evaluation of Novel Targeted Inhibitors Against SbnE, RAF1, and BRAF Class I/II/III Mutants to Overcome Drug Resistance

Kim, Nam Kyoung 고려대학교 KU-KIST융합대학원 2023 국내박사

Pim kinase에 저해 효능을 보이는 (Z)-3-((1H-imidazol-5-yl)methylene)indolin-2-one 계열의 화합물 합성

강지희 계명대학교 대학원 2017 국내석사

The Pim kinases are serine/threonine kinases encoded by the genes Pim-1, Pim-2 and Pim-3 and are involved in many cellular phenomena such as cell growth, proliferation and apoptosis. Pim kinases regulate a number of signaling pathways that are fundamental to tumorigenesis. Overexpression of Pim kinases has been shown in hematological malignancies and solid cancers. The structural feature of interest is the presence of a proline residue (Pro123) in the hinge region of Pim kinases, not generally present in other serine/threonine kinase hinges. Such unique features can be exploited to derive selectivity over other kinases. In this study, a novel series of (Z)-3-((1H-imidazol-5-yl)methylene)indolin-2-one derivatives were designed and synthesized to find new type of scaffold for Pim kinases inhibitors. Compounds were designed to interact with the ε-amino group of Lys67, the carboxylate group of Asp128 as well as the backbone carbonyl group of Glu121 in an ATP binding pocket of Pim-1 kinase. Synthesized compounds demonstrated enzyme inhibitory activities and structure-activity relationship (SAR). The most potent compound, 12i, exhibited inhibitory activities against Pim-3 kinase with a 0.073 µM IC50 value. As a result, a novel scaffold for the development of Pim kinase inhibitors was found through synthesis and biological evaluation. Pim kinase는 Pim-1, Pim-2, Pim-3 유전자로 암호화된 세린/트레오닌 kinase로, 세포 성장, 증식 및 세포 사멸과 같은 많은 세포 현상에 관여한다. Pim kinase는 종양 형성에 기본이 되는 다수의 신호전달경로를 조절하며, 과다 발현된 Pim kinase는 혈액암과 고형암에서 관찰된다. Pim kinase의 흥미로운 구조적 특징은 다른 세린/트레오닌 kinase와는 달리 hinge 부위에 proline 잔기(Pro123)를 가지는 것이다. 이러한 특징을 통해 다른 kinase들과 달리 선택성을 유도할 수 있다. 본 연구에서는 Pim kinase 저해제의 새로운 형태의 모핵을 발굴하기 위해 (Z)-3-((1H-imidazol-5-yl)methylene)indolin-2-one 유도체를 고안하고 합성하였다. 합성한 화합물들은 Pim-1 kinase ATP 결합자리의 Lys67의 ε-아미노기, Asp128의 카복실레이트 이온과 Glu121의 backbone 카보닐기와 상호작용할 것으로 고안되었다. 합성한 화합물들은 효소 활성 저해능과 구조-활성상관관계(SAR)를 보였다. 그 중 가장 저해능이 뛰어난 화합물 12i는 Pim-3 kinase에 대하여 0.073 µM의 IC50값을 나타내었다. 본 연구 결과, 합성과 생물학적 평가를 통해 Pim kinase 저해제 개발에 사용될 수 있는 새로운 모핵구조가 발굴되었다.

Multiple protein kinase 저해제로서 4-aryl-thieno[1,4]diazepin-2-one 유도체의 설계와 합성

이정헌 한양대학교 2023 국내박사

Discovery of new therapeutic agents using extracellular vesicles based sepsis drug screening system

서지혜 포항공과대학교 일반대학원 2015 국내석사

Sepsis is a systemic inflammatory response syndrome triggered by an infection, with the symptoms such as hypothermia, tachypnea and leukopenia. Secretion of pro-inflammatory cytokines like TNF-α is induced in septic environment, and especially, TNF-α, a master regulator, excessively activates the host immune system by triggering inflammatory cascades. This massive activation provokes the successive intravascular coagulopathy and multiple organ failure eventually. Due to this severe consequence of sepsis, its mortality rate reaches up to 30%, which increases the social cost of treatment. Though antibiotics treatment is usually used to treat sepsis, it can even exacerbate the status of disease by the activation of host immune system with increased amount of released bacterial cell wall components. Because there is no commercial and specific drug for sepsis, new therapeutic agents are urgently required. To discover new therapeutic agents, I used the extracellular vesicles (EVs)-based in vitro screening system. The EVs derived from Escherichia coli were treated to Raw264.7 to induce the secretion of TNF-α. The 80 different kinds of kinase inhibitors were screened the ability to reduce TNF-α secretion in the system. From the screening, Damnacanthal was selected for the potent drug candidate, which is previously known as an inhibitor of p56 Lck. Moreover, Damnacathal showed the inhibitory effects on TNF-α secretion induced by various single PAMPs in vitro and on the infiltration of immune cells in skin inflammation model induced by EVs in vivo. To verify the mechanism of anti-inflammatory function of Damnacanthal, the inhibitory effects on TNF-α secretion was verified in Src family kinase inhibitors such as PP1, PP2 and PP3 (negative control for PP2); PP1 and PP2 also reduced the TNF-α secretion induced by EVs. Then, the kinase inhibitor activities of Damnacanthal, PP1 and PP2 were assessed to confirm the relationship between the suppression of TNF-α secretion and the inhibition of Src family kinase. As a result, PP1 and PP2 inhibited the phosphorylation (Tyr416) of Src family kinase, but Damnacanthal did not inhibit the phosphorylation despite the existence of Hck, Lck and c-Src in Raw264.7 cell. These indicated that the anti-inflammatory effects of Damnacanthal are not dependent on the inhibition of Src family kinase activity. As an alternative, I focused on the anti-inflammatory cytokines, especially interleukin-10, which is known to suppress the production and release of TNF-α. Since Damnacanthal induced the secretion of interleukin-10 in the EV-based screening system, the inhibitory effects on TNF-α secretion by Damnacanthal seem to be mediated by interleukin-10. Therefore, the inhibitory ability of Damnacanthal for TNF-α secretion, which reduces the damages on cells or tissues, can be used to treat sepsis. 패혈증 (sepsis)는 감염에 의해 전신에 심각한 염증 반응이 나타나는 질병이다. 저체온증, 빈호흡, 백혈구 감소 등의 증상을 동반하며, TNF-α 와 같은 염증성 사이토카인 분비를 촉진 시켜 면역 체계를 과도하게 활성화하고, 이는 혈관 내 혈액 응고 장애를 야기시켜, 마침내 복합 장기 부전을 유발시킨다. 이러한 패혈증 환자의 사망률은 30%에 이르고, 이로 인한 사회적 비용 역시 크게 증가하고 있다. 이를 치료하기 위해 흔히 사용되는 항생제 치료의 경우 오히려 세균의 구성 성분들을 혈액 내로 퍼지게 하여 패혈증을 더욱 악화시킬 수 있으며, 이 밖에 다른 치료제의 개발이 시급한 상황이다. 이러한 패혈증의 새로운 약물을 발굴하기 위해 Raw264.7 세포에 세균 유래 Extracellular vesicle을 처리하여 TNF-α 분비를 유도하는 screening system을 이용하였다. Extracellular vesicles-based screening system에서 80 종의 Kinase inhibitor가 EV로 유도된 TNF-α 분비를 저해하는 정도를 확인하였고, 그 결과 p56 Lck의 기능을 저해한다고 알려진 Damnacanthal이 치료 약물 후보로 선정되었다. Damnacanthal의 항 염증 효과를 확인하는 실험에서 Damnacanthal은 다양한 single PAMP로 인해 유도된 TNF-α 분비를 억제하였고, EV로 피부에 유도된 염증에서 패혈증의 증상 중 하나인 면역 세포의 조직 내 침입을 저해하는 효과를 보였다. Damnacanthal이 대식세포에서 TNF-α 분비를 저해하는 항 염증 작용의 기전을 알아보고자 Damnacanthal과 같이 Src family kinase를 저해하는 PP1, PP2와 PP2의 대조군으로 사용되는 PP3에 대해 TNF-α 분비 저해 능력을 살펴 본 결과, PP1과 PP2 역시 TNF-α 분비를 저해하였다. Src family kinase 억제가 TNF-α 분비 저해 능력과 관계가 있는지 확인하기 위해 Damnacanthal, PP1, PP2가 Src family kinase 활성화를 억제하는 지 여부를 확인하였다. Hck, Lck, c-Src과 같은 Src family kinase 가 Raw264.7 세포에 존재하는 것을 확인하였으나, Damnacanthal의 경우 Src family kinase의 인산화 (활성화)를 저해하지 못해, Damnacanthal의 TNF-α 분비 저해 능력은 Src family kinase 비활성화에 의존적이지 않다는 결론을 내릴 수 있었다. TNF-α와 같은 염증성 사이토카인의 생성과 분비를 억제한다고 알려진 항 염증성 사이토카인 IL-10에 주목하여, EV-based screening system에서 Damnacanthal이 IL-10 생성을 유도하는지 여부를 확인한 결과, Damnacanthal은 대식세포에서 IL-10 생성 및 분비를 촉진하는 것으로 확인되었다. 이로써 Damnacanthal의 TNF-α 분비 저해 능력은 IL-10을 통한 항 염증성 반응에 의해 매개되는 것으로 보이며, TNF-α 분비를 저해하여 면역 체계가 과도하게 활성화되는 것을 막는 Damnacanthal은 패혈증의 치료 약물로 사용될 수 있을 것으로 보인다.