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Following the identification of thieno[2,3-d]pyrimidine-based selective and potent inhibitors of MCL-1, we explored the effect of core swapping at different levels of advancement. During hit-to-lead optimization, X-ray-guided S-N replacement in the core provided a new vector, whose exploration led to the opening of the so-called deep-S2 pocket of MCL-1. Unfortunately, the occupation of this region led to a plateau in affinity and had to be abandoned. As the project approached selection of a clinical candidate, a series of core swap analogues were also prepared. The affinity and cellular activity of these compounds showed a significant dependence on the core structure. In certain cases, we also observed an increased and accelerated epimerization of the atropoisomers. The most potent core replacement analogues showed considerable in vivo PD response. One compound was progressed into efficacy studies and inhibited tumor growth.

Inhibitors of leucine-rich repeat kinase 2 (LRRK2) and mutants, such as G2019S, have potential utility in Parkinson's disease treatment. Fragment hit-derived pyrrolo[2,3-d]pyrimidines underwent optimization using X-ray structures of LRRK2 kinase domain surrogates, based on checkpoint kinase 1 (CHK1) and a CHK1 10-point mutant. (2R)-2-Methylpyrrolidin-1-yl derivative 18 (LRRK2 G2019S cKi 0.7 nM, LE 0.66) was identified, with increased potency consistent with an X-ray structure of 18/CHK1 10-pt. mutant showing the 2-methyl substituent proximal to Ala147 (Ala2016 in LRRK2). Further structure-guided elaboration of 18 gave the 2-[(1,3-dimethyl-1H-pyrazol-4-yl)amino] derivative 32. Optimization of 32 afforded diastereomeric oxolan-3-yl derivatives 44 and 45, which demonstrated a favorable in vitro PK profile, although they displayed species disconnects in the in vivo PK profile, and a propensity for P-gp- and/or BCRP-mediated efflux in a mouse model. Compounds 44 and 45 demonstrated high potency and exquisite selectivity for LRRK2 and utility as chemical probes for the study of LRRK2 inhibition.

The serine/threonine kinase DYRK1A has been implicated in regulation of a variety of cellular processes associated with cancer progression, including cell cycle control, DNA damage repair, protection from apoptosis, cell differentiation, and metastasis. In addition, elevated-level DYRK1A activity has been associated with increased severity of symptoms in Down's syndrome. A selective inhibitor of DYRK1A could therefore be of therapeutic benefit. We have used fragment and structure-based discovery methods to identify a highly selective, well-tolerated, brain-penetrant DYRK1A inhibitor which showed in vivo activity in a tumor model. The inhibitor provides a useful tool compound for further exploration of the effect of DYRK1A inhibition in models of disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2), such as G2019S, are associated with an increased risk of developing Parkinson’s disease. Surrogates for the LRRK2 kinase domain based on checkpoint kinase 1 (CHK1) mutants were designed, expressed in insect cells infected with baculovirus, purified, and crystallized. X-ray structures of the surrogates complexed with known LRRK2 inhibitors rationalized compound potency and selectivity. The CHK1 10-point mutant was preferred, following assessment of surrogate binding affinity with LRRK2 inhibitors. Fragment hit-derived arylpyrrolo[2,3-b]pyridine LRRK2 inhibitors underwent structure-guided optimization using this crystallographic surrogate. LRRK2-pSer935 HEK293 IC50 data for 22 were consistent with binding to Ala2016 in LRRK2 (equivalent to Ala147 in CHK1 10-point mutant structure). Compound 22 was shown to be potent, moderately selective, orally available, and brain-penetrant in wild-type mice, and confirmation of target engagement was demonstrated, with LR...

We describe our work to establish structure- and fragment-based drug discovery to identify small molecules that inhibit the anti-apoptotic activity of the proteins Mcl-1 and Bcl-2. This identified hit series of compounds, some of which were subsequently optimized to clinical candidates in trials for treating various cancers. Many protein constructs were designed to identify protein with suitable properties for different biophysical assays and structural methods. Fragment screening using ligand-observed NMR experiments identified several series of compounds for each protein. The series were assessed for their potential for subsequent optimization using 1H and 15N heteronuclear single-quantum correlation NMR, surface plasmon resonance, and isothermal titration calorimetry measurements to characterize and validate binding. Crystal structures could not be determined for the early hits, so NMR methods were developed to provide models of compound binding to guide compound optimization. For Mcl-1, a benzodioxane/benzoxazine series was optimized to a Kd of 40 μM before a thienopyrimidine hit series was identified which subsequently led to the lead series from which the clinical candidate S 64315 (MIK 665) was identified. For Bcl-2, the fragment-derived series were difficult to progress, and a compound derived from a published tetrahydroquinone compound was taken forward as the hit from which the clinical candidate (S 55746) was obtained. For both the proteins, the work to establish a portfolio of assays gave confidence for identification of compounds suitable for optimization.

Avoidance of apoptosis is critical for the development and sustained growth of tumours. The pro-survival protein myeloid cell leukemia 1 (MCL1) is overexpressed in many cancers, but the development of small molecules targeting this protein that are amenable for clinical testing has been challenging. Here we describe S63845, a small molecule that specifically binds with high affinity to the BH3-binding groove of MCL1. Our mechanistic studies demonstrate that S63845 potently kills MCL1-dependent cancer cells, including multiple myeloma, leukaemia and lymphoma cells, by activating the BAX/BAK-dependent mitochondrial apoptotic pathway. In vivo, S63845 shows potent anti-tumour activity with an acceptable safety margin as a single agent in several cancers. Moreover, MCL1 inhibition, either alone or in combination with other anti-cancer drugs, proved effective against several solid cancer-derived cell lines. These results point towards MCL1 as a target for the treatment of a wide range of tumours.

International audience; Escape from apoptosis is one of the major hallmarks of cancer cells. The B-cell Lymphoma 2 (BCL-2) gene family encodes pro-apoptotic and anti-apoptotic proteins that are key regulators of the apoptotic process. Overexpression of the pro-survival member BCL-2 is a well-established mechanism contributing to oncogenesis and chemoresistance in several cancers, including lymphoma and leukemia. Thus, BCL-2 has become an attractive target for therapeutic strategy in cancer, as demonstrated by the recent approval of ABT-199 (Venclexta™) in relapsed or refractory Chronic Lymphocytic Leukemia with 17p deletion. Here, we describe a novel orally bioavailable BCL-2 selective and potent inhibitor called S55746 (also known as BCL201). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile demonstrates no significant binding to MCL-1, BFL-1 (BCL2A1/A1) and poor affinity for BCL-XL. Accordingly, S55746 has no cytotoxic activity on BCL-XL-dependent cells, such as platelets. In a panel of hematological cell lines, S55746 induces hallmarks of apoptosis including externalization of phosphatidylserine, caspase-3 activation and PARP cleavage. Ex vivo, S55746 induces apoptosis in the low nanomolar range in primary Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma patient samples. Finally, S55746 administered by oral route daily in mice demonstrated robust anti-tumor efficacy in two hematological xenograft models with no weight lost and no change in behavior. Taken together, these data demonstrate that S55746 is a novel, welltolerated BH3-mimetic targeting selectively and potently the BCL-2 protein.

Mcl-1 is highly expressed in a variety of human cancers (including those of hematopoietic and lymphoid origin) and is exploited by cancer cells to evade cell death and to develop resistance to diverse chemotherapeutic agents. We disclose, for the first time, the structure of S64315 (also named MIK665) a highly potent and selective inhibitor of Mcl-1 with improved potency over its predecessor S63845 (Kotschy et al, Nature, 2016). S64315/MIK665 is currently in phase 1 in AML (Acute Myeloid Leukemia) and MDS (Myelodysplastic Syndrome) (EudraCT 2016-003768-38, NCT 02979366) and in MM (Multiple Myeloma) and lymphoma (NCT02992483). A fragment-based, structure-guided drug discovery effort led to the identification of S64315/MIK665 that binds to human Mcl-1 with a sub-nanomolar affinity (Ki 0.048 nM) and selectively over other anti-apoptotic Bcl-2 family members. It has similar affinity for human, rat, dog and monkey Mcl-1 but about a ten-fold lower affinity for mouse Mcl-1. S64315/MIK665 causes dose-dependent activation of the intrinsic apoptosis pathway in a Bax/Bak-dependent manner, as measured by increased caspase activity and cleaved PARP. S64315/MIK665 shows strong cell killing activity in a diverse panel of human hematological tumor cell lines, including AML, lymphoma and MM. The activity profile of S64315/MIK665 is distinct from that of venetoclax, a selective Bcl2 inhibitor. In vivo, S64315 as single agent demonstrated potent and dose-dependent apoptotic and antitumor response after intravenous administration in several human hematological tumor models grafted in immuno-compromised mice and rats. Complete regression of established tumors, at well tolerated doses, was achieved using different intravenous dosing regimens in rats as well as in mice. Finally, dual BH3-mimetic targeting approach combining S64315/MIK665 with BCL2 inhibitors showed strong and durable antitumor responses in several hematological tumor models both in vitro and in vivo. Citation Format: Ana Leticia Maragno, Prakash Mistry, András Kotschy, Zoltán Szlavik, James Murray, James Davidson, Gaëtane Le Toumelin-Braizat, Maïa Chanrion, Alain Bruno, Audrey Claperon, Heiko Maacke, Erick Morris, Youzhen Wang, Alix Derreal, Márton Csekei, Attila Paczal, Zoltán Szabo, Szabolcs Sipos, Agnes Proszenyak, Balázs Balint, Allan Surgenor, Pawel Dokurno, Natalia Matassova, Ijen Chen, Gaëlle Lysiak-Auvity, Anne-Marie Girard, Fabienne Grave, Frédéric Colland, Ensar Halilovic, Olivier Geneste. S64315 (MIK665) is a potent and selective Mcl1 inhibitor with strong antitumor activity across a diverse range of hematologic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4482.

The anti-apoptotic function of the 70 kDa family of heat shock proteins and their role in cancer is well documented. Dual targeting of Hsc70 and Hsp70 with siRNA induces proteasome-dependent degradation of Hsp90 client proteins and extensive tumor specific apoptosis as well as the potentiation of tumor cell apoptosis following pharmacological Hsp90 inhibition.We have previously described the discovery and synthesis of novel adenosine-derived inhibitors of the 70 kDa family of heat shock proteins; the first inhibitors described to target the ATPase binding domain. The in vitro activity of VER-155008 was evaluated in HCT116, HT29, BT474 and MDA-MB-468 carcinoma cell lines. Cell proliferation, cell apoptosis and caspase 3/7 activity was determined for VER-155008 in the absence or presence of small molecule Hsp90 inhibitors.VER-155008 inhibited the proliferation of human breast and colon cancer cell lines with GI(50)s in the range 5.3-14.4 microM, and induced Hsp90 client protein degradation in both HCT116 and BT474 cells. As a single agent, VER-155008 induced caspase-3/7 dependent apoptosis in BT474 cells and non-caspase dependent cell death in HCT116 cells. VER-155008 potentiated the apoptotic potential of a small molecule Hsp90 inhibitor in HCT116 but not HT29 or MDA-MB-468 cells. In vivo, VER-155008 demonstrated rapid metabolism and clearance, along with tumor levels below the predicted pharmacologically active level.These data suggest that small molecule inhibitors of Hsc70/Hsp70 phenotypically mimic the cellular mode of action of a small molecule Hsp90 inhibitor and can potentiate the apoptotic potential of a small molecule Hsp90 inhibitor in certain cell lines. The factors determining whether or not cells apoptose in response to Hsp90 inhibition or the combination of Hsp90 plus Hsc70/Hsp70 inhibition remain to be determined.

Summary Localization of cyclic AMP (cAMP)-dependent protein kinase (PKA) by A kinase-anchoring proteins (AKAPs) restricts the action of this broad specificity kinase. The high-resolution crystal structures of the docking and dimerization (D/D) domain of the RIIα regulatory subunit of PKA both in the apo state and in complex with the high-affinity anchoring peptide AKAP- IS explain the molecular basis for AKAP-regulatory subunit recognition. AKAP- IS folds into an amphipathic α helix that engages an essentially preformed shallow groove on the surface of the RII dimer D/D domains. Conserved AKAP aliphatic residues dominate interactions to RII at the predominantly hydrophobic interface, whereas polar residues are important in conferring R subunit isoform specificity. Using a peptide screening approach, we have developed SuperAKAP- IS , a peptide that is 10,000-fold more selective for the RII isoform relative to RI and can be used to assess the impact of PKA isoform-selective anchoring on cAMP-responsive events inside cells.

6,8-Dihydro-6-(5,6,7,8-tetrahydro-6-me thyl-1,3-dioxolo[4,5-g]isoquinolin-5-yl)furo[3,4-e]- 1,3-benzodioxol-8-ol, C 20 H 19 NO 6 , M r =369.4, tri clinic, P1, a=10.833 (2), b=13.184 (3), c=13.375 (3) A, a=89.73 (3), b=74.52 (3), γ=71.95 (3) o , V=1744.0 (5) A 3 , Z=4, D x =1.407 g cm -3 , λ(Cu Kα)=1.54178 A, μ=8.30 cm -1 , F(000)=776, room temperature, R=0.051 for 3375 observed reflections. The relative con figuration of the hemiacetal C atom is established. The heterocyclic fragment of isoquinoline exhibits a half-chair conformation; all five-membered rings show envelope conformations

The cytoskeletal protein alpha-catenin, which shares structural similarity with vinculin, is required for cadherin-mediated cell adhesion, and functions to modulate cell adhesive strength and to link the cadherins to the actin-based cytoskeleton. Here we describe the crystal structure of a region of alpha-catenin (residues 377-633) termed the M-fragment. The M-fragment is composed of a tandem repeat of two antiparallel four-helix bundles of virtually identical architectures that are related in structure to the dimerization domain of alpha-catenin and the tail region of vinculin. These results suggest that alpha-catenin is composed of repeating antiparallel helical domains. The region of alpha-catenin previously defined as an adhesion modulation domain corresponds to the C-terminal four-helix bundle of the M-fragment, and in the crystal lattice these domains exist as dimers. Evidence for dimerization of the M-fragment of alpha-catenin in solution was detected by chemical cross-linking experiments. The tendency of the adhesion modulation domain to form dimers may explain its biological activity of promoting cell-cell adhesiveness by inducing lateral dimerization of the associated cadherin molecule.

p97, an abundant hexameric ATPase of the AAA family, is involved in homotypic membrane fusion. It is thought to disassemble SNARE complexes formed during the process of membrane fusion. Here, we report two structures: a crystal structure of the N-terminal and D1 ATPase domains of murine p97 at 2.9 A resolution, and a cryoelectron microscopy structure of full-length rat p97 at 18 A resolution. Together, these structures show that the D1 and D2 hexamers pack in a tail-to-tail arrangement, and that the N domain is flexible. A comparison with NSF D2 (ATP complex) reveals possible conformational changes induced by ATP hydrolysis. Given the D1 and D2 packing arrangement, we propose a ratchet mechanism for p97 during its ATP hydrolysis cycle.

Proteins of the Bcl-2 family are central regulators of programmed cell death. Pro-survival Bcl-2 proteins, such as Bcl-2, Bcl-xL and Mcl-1 are often over-expressed in human tumours and participate in tumour initiation, progression and chemo-resistance. Therefore drugs targeting these pro-survival Bcl-2 proteins represent a promising therapeutic approach for cancer treatment. The most advanced drug targeting this protein family is ABT-263, a potent Bcl-2 and Bcl-xL inhibitor showing anti-tumour efficacy in preclinical models of leukaemia and small cell lung carcinoma. Survival of circulating platelets has been shown to be highly Bcl-xL dependent; consequently the dose-limiting toxicity of ABT-263 is an on-target peripheral thrombocytopenia. We have used a range of biophysical methods to guide the structure-based generation of a significant number of small molecules,* which bind with high affinity (MW < 780; KD < 1 nM) to the BH3 binding groove of Bcl-2, and with high selectivity versus other members of the Bcl-2 family (selectivity > 100 fold). In cellular assays, our lead compounds efficiently displace Bax from Bcl-2 with near complete inhibition of Bcl-2 / Bax co-immunoprecipitation at 100 nM. These compounds are strong inducers of cell death in Bcl-2 dependent cellular models such as the acute myeloblastic leukaemia (AML) cell line RS4;11, affording sub-10 nM IC50's for the most potent compounds. In vivo, in agreement with their mechanism of action, these Bcl-2 selective inhibitors, given either intravenously or orally, elicit a rapid (30 min iv, and 2 hours po) and strong apoptotic response in mouse xenografts of the RS4:11 cell line. When the most potent compounds are given orally to RS4;11 xenograft-bearing mice, apoptosis in tumor cells is induced more than 15 fold (at 25 mg/kg) and more than 20 fold (at 50 mg/kg) compared to untreated mice. Importantly, in agreement with the selectivity of the compounds for Bcl-2 versus Bcl-xL, no platelet loss was observed in mice treated with our compounds, in sharp contrast to ABT-263. Finally, we observe very robust anti-tumor activity when a lead compound is given orally at 50 mg/kg and 100 mg/kg (with complete regression at 100 mg/kg) in an RS4;11 mouse xenograft model. This anti-tumor activity was similar whether the compound was dosed daily or twice a week over two weeks. Altogether our data demonstrate that highly Bcl-2 selective antagonists show anti-tumor activity and no platelet toxicity, in contrast to Bcl-2 / Bcl-xL dual inhibitors. Such compounds represent promising drug candidates for the treatment of Bcl-2 dependent malignancies such as chronic lymphocytic leukaemia (CLL) and other leukaemias and lymphomas. * Chemical structures of compounds will not be disclosed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2782. doi:1538-7445.AM2012-2782

The role of the 70 kDa heat shock protein isoforms (Hsc70 and Hsp70) in cancer development and progression through their ability to inhibit apoptosis and via their role as Hsp90 co-chaperones has been well documented. Dual targeting of Hsc70 and Hsp70 with siRNA has previously been demonstrated to induce proteasome-dependent degradation of Hsp90 client proteins and extensive tumor specific apoptosis as well as the potentiation of tumor cell apoptosis following pharmacological Hsp90 inhibition. The design and synthesis of novel adenosine-derived inhibitors of Hsp70, guided by modelling and X-ray crystallographic structures of these compounds in complex with Hsc70/BAG-1, has been described.1 These were the first inhibitors described to target the ATPase binding domain of this family of chaperones. Many of these compounds exhibited submicromolar affinity for Hsp70, were highly selective over Hsp90, and displayed in vitro activity against a variety of human tumor cell lines. We further describe the in vitro mode of action of one of the most potent analogues, VER-155008 in HCT116, HT29, BT474 and MDA-MB-468 carcinoma cell lines. Cell proliferation, cell cycle, cell apoptosis and caspase 3/7 activity was determined for VER-155008 in the absence or presence of small molecule Hsp90 inhibitors. VER-155008 inhibited the proliferation of human breast and colon cancer cell lines with GI50s in the range 5.3 to 14.4 M, and induced Hsp90 client protein degradation in both HCT116 and BT474 cells. As a single agent, VER-155008 induced caspase-3/7 dependent apoptosis in BT474 cells and non-caspase dependent cell death in HCT116 and HT29 cells. VER-155008 potentiated the apoptotic potential of the small molecule Hsp90 inhibitors VER-821602 and 17-AAG in HCT116 but not HT29 or MDA-MB-468 cells. In vivo, VER-155008 demonstrated rapid metabolism and clearance, along with tumor levels below the predicted pharmacologically active level. These data suggest that as a single agent, small molecule inhibitors of Hsc70/Hsp70 phenotypically mimic the cellular mode of action of a small molecule Hsp90 inhibitor and can potentiate the apoptotic potential of a small molecule Hsp90 inhibitor in certain cell lines. The factors determining whether or not cells apoptose in response to Hsp90 inhibition or the combination of Hsp90 plus Hsc70/Hsp70 inhibition remain to be determined. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A212.