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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5371_Библиотеки_им_академика_М_И_Перельмана
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Chemistry and Pharmacology of Drug Discovery
First-generation inhibitors
Second-generation inhibitors
Figure 2. Structures of select examples of first-generation clinically approved JAK
inhibitors 2–4 and second-generation experimental JAK family inhibitors 5–6
1.1. TYK2 Pseudokinase: The Opportunity for
Selectivity
The non-receptor tyrosine kinase 2 (TYK2) has garnered considerable interest recently due
to the pivotal role it plays in mediating signal transduction pathways downstream of the
pro-inflammatory receptors for IL-23, IL-12, and Type 1 interferons (IFNα and IFNβ), all
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Chapter 15. Deucravacitinib (Sotyktu)
important drivers of autoimmune and inflammatory diseases.7 TYK2 represents an ideal
therapeutic target due to its association with a small number of cytokine receptors relative
to the other JAKs. Genetic studies have revealed that TYK2-deficient mice are protected
from various models of experimental autoimmunity, including collagen-induced arthritis
8, 9
and experimental autoimmune encephalomyelitis.
The TYK-2 dependent pathways have
also been validated in treating human disease with antibody therapeutics. The IL-12/IL-23
antibody ustekinumab (Stelara) is currently
clinical
development underway for treatment of Crohn’s disease.
marketed for the treatment of psoriasis, with
10, 11
The anti-type 1
interferon receptor antibody anifrolumab has been reported to provide benefit for the
12
treatment of systemic lupus erythematosus (SLE).
Despite the potential of TYK2 inhibition, only modest progress has been reported
in identifying potent and highly selective small molecules that target the catalytically active
13, 14
JH1 domain.
The pseudokinase (JH2) domains of JAK family kinases have previously
been implicated to play an autoinhibitory role in regulating activation of the adjacent
15, 16
catalytic domains.
Using a chemogenic approach, the elegant work of researchers at
Bristol-Myers Squibb (BMS) led to the identification of molecules that act on TYK2 by
preventing the receptor-mediated activation of the TYK2 JH1 domain as a consequence of
binding and stabilization of the adjacent catalytically inactive JH2 domain, ultimately
blocking downstream signal transduction (Figure 3).17 It was also found that there is a
greater residue differentiation in the TYK2 pseudokinase domain from the other JAK
family members pseudokinase domains, offering the greatest opportunity to design
inhibitors that would yield both kinase and family selectivity. This breakthrough provided
the first example of TYK2 protein function inhibition through an allosteric mechanism and
set the stage for capitalizing on this novel approach to optimize potent and selective
molecules for the treatment of autoimmune and inflammatory disorders.
Figure 3. Schematic depicting prevention of receptor-mediated activation. Source:
Adapted from
17, 18
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The combined limitations of existing therapies for psoriasis and the novel
opportunity seen through TYK2 inhibition, led BMS to undertake a research program that
culminated in the successful launch of deucravacitinib, a first-in-class highly selective
allosteric TYK2 inhibitor small molecule approved by the FDA in September 2022 for
treating moderate-to-severe plaque psoriasis. The launch of the only approved TYK2
inhibitor worldwide and the first innovation in oral treatment for this disease in nearly 10
years was a defining moment. The path to approval will be discussed in the remainder of
the chapter.
Chemistry and Pharmacology of Drug Discovery
2. Pharmacology
Deucravacitinib, marketed as Sotyktu, works by stabilizing an auto-inhibitory interaction
between the regulatory (JH2) and catalytic (JH1) domains of the enzyme, thereby trapping
the kinase in its inactive state and preventing receptor mediated activation and its
downstream functions in cells.
19
Figure 4. Proposed mechanism of action of deucravacitinib. Source: 20/with permission of
Inhibition of signal transduction downstream of the IL-23 receptor is being
actively pursued as an intriguing approach to the treatment of autoimmunity and reports
Bristol-Myers Squibb Company
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strongly linking IL-23 to the pathogenesis of psoriasis have recently emerged.
the intricate mechanistic details linking TYK2 inhibition to its efficacy are not currently
known, a proposed mechanism is outlined in Figure 4.
and induces production of the proinflammatory mediator IL-17 by T helper 17 (Th17) cells.
By blocking this IL-23/Th17 axis through inhibition of TYK2 activation and its
downstream activation of STATs, gene transcription is halted, leading to a reduction in IL17 production and subsequent therapeutic effectiveness for autoimmune and chronic
inflammatory diseases.
Chapter 15. Deucravacitinib (Sotyktu)
20, 22
IL-23 is secreted by skin cells
19, 21
While
3. Structure–Activity Relationship (SAR)
3.1. Imidazopyridazine Series
In their quest to develop an orally dosed small-molecule TYK2 inhibitor, BMS focused
their efforts on an allosteric approach to target the JH2 domain. Herein, the key aspects of
the long and arduous SAR campaign that culminated in the discovery of deucravacitinib
will be highlighted.
A phenotypic screen of a large collection of kinase inhibitors using an IL-23/IFNα
stimulated reporter assay led to the identification of imidazopyridazine hit compound 7,
which displayed relatively weak affinity for the TYK2 pseudokinase but exhibited
excellent selectivity (with only 0.5% of kinases in the 386-kinase panel being inhibited by
greater than 67% at 1 μM compound concentration).
positions resulted in compound 8, that exhibited improved binding affinity and cellular
potency, was fully selective over the Janus catalytic domains and showed excellent ligand
and ligand lipophilic efficiency (LE = 0.35, LLE = 6.2) (Figure
23
Modifications at the C6 and C8
5).
Figure 5. Left, high-throughput screen hit 7 for IL-23 inhibition; Right, structure and
associated data of 8
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Chemistry and Pharmacology of Drug Discovery
The pocket proximal to the C3 amide in the TYK2 JH2 domain contains a
combination of residues that are largely unique relative to the kinome, such as the small
Ala671
residue under the “gatekeeper” (Thr687). Of note is the single residue change
preceding the activation loop (Ser 758), that alters the positions of the conserved catalytic
Lys642 and Asp759 (Figure 6). It was postulated that interactions made by the C3 amide
in this pocket, accounted for the kinome selectivity of compound 8. The co-crystal structure
revealed the two key hydrogen bond interactions that 8 makes with TYK2 JH2. One takes
place at the hinge region between the C8 methylamino NH and the carbonyl of Val690 and
between the N of the imidazopyradazine and the NH of Val690. The other occurs near the
gatekeeper involving the C3 amide carbonyl to the NH of Lys642 and to the carbonyl of
23
Glu688 mediated by a bridging water molecule.
Figure 6. Close-up of the binding site highlighting key interactions made by 8. Source:
Seeking to improve permeability, further SAR exploration, guided by these
critical structure-based insights led to 2-pyridyl substituted pyridone 9. The intramolecular
hydrogen bonds effectively masked the molecule’s polarity and consequently increased the
permeability (Figure 7).24
Modifications of the C3 amide side chain to improve metabolic stability resulted
in the enantiomeric (1R,2S)-2-fluorocyclopropyl group as the optimal substituent, leading
to a four-fold enhancement in Tyk2 JH2 affinity, cellular and human whole blood (hWB)
activities. Of note, 10 remarkably displayed >10,000-fold selectivity for Tyk2 JH2 and
Image design by Dr. Yi Li
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Chapter 15. Deucravacitinib (Sotyktu)
demonstrated in vivo proof of concept, giving confidence that a small-molecule TYK2 JH2
ligand could be developed into a therapeutic for autoimmune and inflammatory disorders.
Figure 7. Left, structure and associated in vitro data for 9; Right, structure and associated
in vitro data for 10
24
3.2. Nicotinamide Series
In parallel, BMS simultaneously conducted a second high-throughput screening (HTS)
campaign of the larger corporate compound database utilizing a scintillation proximity
assay (SPA) and identified a novel nicotinamide TYK2 JH2-binding scaffold 11 (Figure
25
8).
The SPA assay was replaced with a more sensitive homogenous time-resolved
fluorescence (HTRF) assay due to the notably higher potencies observed in this series. The
good functional potency displayed by 11 was offset by its poor kinome and family
selectivity (Figure 8). However, its good metabolic stability and high ligand efficiency
(LE = 0.30, LLE = 5.8) warranted further multiparameter optimization of potency,
selectivity, and drug-like properties and this series was prioritized over the
imidazopyridazines.
TYK2
JH2
(IC
50
,
nM)
IL-2/GM-CSF (IC50,
nM)
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Chemistry and Pharmacology of Drug Discovery
Figure 8. Structures and kinase selectivity of 11 and 12
Applying structural lessons from the imidazopyridazine series, methyl amide 12
was made (Figure 8). Binding affinity SAR was rationalized on the basis of co-crystal
structures, with the key feature being projection of the C3 amide methyl toward the
distinctive “alanine pocket” containing Ala671, an uncommon residue found in the TYK2
pseudokinase domain, but only found in 2% of the entire kinome. The methyl amide 12
retained the TYK2 JH2 affinity while imparting a remarkable improvement in both kinome
and family selectivity relative to 11. This improved family selectivity was reflected in the
cellular assays, with 12 showing no measurable activity in IL-2 (JAK 1 and JAK 3
dependent) and GM-CSF (JAK 2 dependent) control assays (Table 1).
11 0.5 15/26/24 89/37 400/210
12 1.5 >2000 790/280 >12500/>12500
The less than desirable mouse pharmacokinetics of 12 (C
AUC = 150 nM h, following a po dose of 10 mg/kg) were improved by lowering the polar
surface area (PSA) through replacement of the amide with a methyl sulfonyl group. Sulfone
13 maintained the binding affinity of 12, while being slightly more potent in the reporter
assay and a hWB assay of IFNα-induced STAT5 phosphorylation (Figure
permeability accompanied by a decrease in efflux ratio (er) led to 13 exhibiting a much
more acceptable PK profile in a 10 mg/kg mouse oral PK study (C
AUC = 9830 nM h).
JH1
TYK2/JAK1/
JAK2 (IC
, nM)
50
TYK2 dependent TYK2 independent
IL-23/IFNα (IC
,
50
nM)
Table 1. Associated in vitro data comparing 11 and 12
= 36 nM;
max
9). The boost in
= 1620 nM;
max
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Chapter 15. Deucravacitinib (Sotyktu)
An X-ray co-crystal structure of 11 bound to TYK2 JH2 confirmed the two critical
hydrogen bond interactions made by one of the sulfone oxygens and the methyl amide
carbonyl oxygen to the Lys642 residue, stabilizing the active conformation of 13.
Figure 9. Structure and associated in vitro/in vivo data for Compound 13
Expecting in vivo demethylation of the methyl amide to occur and the lack of
selectivity displayed by primary amide 11, there was concern that the anticipated primary
amide metabolite of 13 could falsify in vivo efficacy data and lead to off-target effects. The
putative primary amide metabolite of 13 was prepared and its potency and poor selectivity
profile was confirmed (14, Figure 10). An analysis of plasma samples from a mouse PK
study of 14 dosed orally and intravenously, revealed circulating primary amide metabolite
levels between 5% and 30%.
A prior report of metabolic demethylation of tertiary amides being significantly
26
diminished by the kinetic isotope effect,
prompted the preparation of trideuteromethyl
secondary amide 15 in an attempt to curtail formation of the primary amide. A mouse PK
study of 15 dosed orally and intravenously revealed circulating primary amide metabolite
levels below the lower limit of quantification (LLQ) at all time points tested (Figure
10).
While deuterium incorporation circumvented the formation of a non-selective
primary amide metabolite, 13 presented with a hERG liability (2.5 μM IC
measured in a
50
whole cell patch clamp assay). To avoid a narrow therapeutic window toward undesirable
cardiovascular effects, achieving selectivity over hERG while maintaining all the necessary
interactions, required some clever medicinal chemistry. Reducing hERG affinity can often
be accomplished by removing peripheral rings to disrupt potential π-stacking within the
27
aromatic residue-rich hERG channel,
and as such C6 amide exploration was carried out.
It turned out that replacement of the 2-aminopyridine with cyclopropylamide 16 greatly
reduced hERG channel inhibition as confirmed in the patch clamp assay (IC
> 30 μM).
50
A cocrystal structure of 13 and 16 overlaid with TYK2 JH2 revealed that both bind in an
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almost identical fashion concluding that the cyclopropyl amide in essence, serves as an
isostere for 2-aminopyridine, lending credence to the similar binding potencies observed
between 13 and 16 (Figure
Chemistry and Pharmacology of Drug Discovery
11).
Figure 10. Profiles of 13, primary metabolite 14 and trideuteromethyl analog 15
Unfortunately, the good hERG selectivity of 16 was accompanied with poor
mouse oral PK presumably due to its poor permeability (Figure
overcome by replacing
pyridazine core, which is also a strong hydrogen bond acceptor.
corresponding pyridazine-matched molecular pairs were synthesized and evaluated. The
pyridazine analogues consistently provided improved exposures without sacrificing
potency. Pyridazines 17 and 18 emerged as the most promising based on their composite
in vitro/in vivo properties (Figure
11). This limitation was
the central pyridine ring with the less basic and more lipophilic
Several pyridine- and
25
12).
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Chapter 15. Deucravacitinib (Sotyktu)
Figure 11. Left, structure and associated in vitro/in vivo data for compound 16; Right,
overlay of 13 and 16 with TYK2 JH2 showing cyclopropylamide as isostere for 2-
aminopyridine (PDB ID: 6NZH)
Figure 12. Structure and associated in vitro/in vivo data for compounds 17 and 18
Compound 18 displayed a dose-dependent response in a mouse model of IBD,
albeit with effective inhibition achieved at a high 250 mg/kg dose.
optimization campaign, the objectives of the program were to retain the key discoveries
25
Entering into the lead
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